text:
Tuesday, December 12, 2017
Germany condemns antisemitism while welcoming millions of antisemites
German authorities have strongly condemned the wave of anti-Israeli protests that have gripped Berlin for three consecutive days in response to Donald Trump's move to recognize Jerusalem as Israel's capital, saying anti-Semitism has no place in the country.
Some 2,500 people waved Palestinian flags and carried placards denouncing the US and Israel. Anti-Semitic calls were heard in Arabic and German as Israel's Star of David flag was set alight. Burning a flag on its own does not constitute a criminal offense, unless it is attached to an embassy or public institution, police said. Officers detained eleven people from the vocal crowd before releasing them after writing up criminal complaints.
Sunday's rally was Berlin's third show of anger against Trump's move since Friday. Two flags had been burned at the start of the weekend in an anti-Israel rally in front of the American Embassy on Pariser Platz, which drew around 1,500 people. The enraged crowd chanted "Death to the Jews!" and "Jews, remember Khaybar, the army of Muhammad is coming again!" According to legend, Khaybar was a Jewish-populated oasis in Saudi Arabia, which was attacked and conquered by Prophet Muhammed and his army.
The crowd also shouted 'Allahu Akbar' (Allah is great), and "in spirit and in blood we will redeem you." In addition, several people dressed in Palestinian colors also waved Hamas flags, classified by the European Union and the United States as a terrorist group. Police said they arrested ten people Friday, citing 12 criminal charges.
Support Grows for Air Force Colonel Suspended Over Religious Beliefs on Marriage
When Heather Wilson was picked to be secretary of the Air Force, she told the Senate: "Air Force policy must continue to ensure that all airmen are able to choose to practice their particular religion." Now, she has a chance to prove it.
On Wednesday, the Family Research Council's Lt. General Jerry Boykin and Travis Weber gave Wilson 77,024 reasons to reconsider the action taken against Col. Leland Bohannon.
A distinguished combat pilot, Bohannon has served his country for more than 20 years. In May, the reputation he'd built in the Air Force came crashing down when his superiors decided that the colonel's decision not to sign a "certificate of appreciation" for a same-sex spouse was enough to suspend him from duty.
Never mind that Bohannon had requested a religious accommodation. Or that he'd consulted the command chaplain and a staff judge advocate. Leaders seemed determined to make an example of the dad of five, grounding him and snuffing out any chance of promotion. Eight senators were outraged, demanding that Air Force leaders intervene. Then, more than 77,000 of you piled on, urging justice for Bohannon and others like him.
Wednesday, Boykin and Weber delivered those petitions on behalf of the Family Research Council and our friends at the American Family Association—along with a letter signed by 31 religious advocacy groups.
"In his Executive Order Promoting Free Speech and Religious Liberty issued earlier this year," the organizations write, "President Trump stated that 'it shall be the policy of the executive branch to vigorously enforce Federal law's robust protections for religious freedom,' and that people should be 'free to practice their faith without fear of discrimination or retaliation by the Federal Government.' Yet despite his impressive decades-long career with the Air Force, Col. Bohannon's life and service are about to be derailed by the violation of this fundamental principle."
Not only is there no right to a certificate of spousal appreciation, an accommodation should easily have been granted here … If the Air Force policy reflected the view of [Department of Justice] on this issue, those in Col. Bohannon's situation would have no need to be concerned for their religious liberty in the first place, and we urge the Air Force to bring its policies into line with the understanding of the DOJ in this area … In addition to correcting Air Force policy to ensure this does not happen again, we respectfully request that you reverse the complaint against Col. Bohannon and remove any unfavorable materials related to this complaint from his record.
As far as Boykin is concerned, "We not only delivered petitions, we delivered a message: We will not back down from defending the religious liberty of those in the military. The action taken against Colonel Bohannon is unacceptable, and Air Force policy must be corrected to ensure this does not happen again."
Thanks to tens of thousands of you, Secretary Wilson may be motivated to do just that.
Female Entrepreneur Says Google's 'Women in Tech' Program Ostracized Her for Being Conservative
A female tech entrepreneur alleges she faced character assassination and career sabotage by two "women in tech" groups over her conservative beliefs, including Google's Women Techmakers.
Senior software engineer and co-founder of Polyglot Programming Marlene Jaeckel says that Martin Omander, Google Developer Group program manager for North America, formally banned her from the Google Developer Group and Google Women Techmakers after complaints from a feminist activist who objected to her moderate conservative positions.
According to Jaeckel , Omander "declined to provide me with any details of the complaints against me or the rules that I'd allegedly violated."
In a Medium post published earlier this week, Jaeckel explained that the two Atlanta-based feminists who reported her to Google, local Women Who Code director Alicia Carr and Atlanta Google Women Techmakers organizer Maggie Kane, had become hostile to her after a series of disagreements over politics, and repeatedly sought to damage her career.
The publicly-stated objectives of both Women Who Code and Google Women Techmakers are, ostensibly, non-partisan. Women Who Code says their goal is to "inspire women to excel in technology careers," while Google Women Techmakers says they merely wish for "visibility, community, and resources for women in technology."
None of these organizations openly say that Republican or conservative women are excluded from their goals. Yet Jaeckel , a senior software engineer and co-founder of a tech company, says that is the reason why Carr and Kane sought to both exclude her from the groups and sabotage her career in tech.
According to Jaeckel 's account, which can be read in full at Medium, she had a falling-out with Carr over a number of issues, including her opposition to gender-segregated classes. Jaeckel says she was also banned from two other coding workshops in Atlanta because the founders "strongly objected" to her conservative political views.
Unfortunately, during the Women Who Code hackathon, it became clear to me that this event focused on marketing strategies, creativity, and the discussion of gender politics, and not on the development of technical skills. At the group presentations and award ceremony, I observed that my group of mentees were being discouraged from discussing any of the technical details of the fully-functional application they had developed in less than two days, and I expressed my frustration about it on Twitter, stating that "when you're a mentor and your mentees don't get the recognition they deserve, you go to bat."
In August 2016, Alicia reached out me via email and private Slack messages. She proposed forming a class for female coders who were interested in learning iOS development and asked me to tutor these students. I told her that I'd be glad to teach if the class also included males. She refused, stating that "I need everybody and anybody to help my Women and I'm sorry there is a gender issues [sic] but right now it [sic] about my ladies." We were unable to reach an agreement, so I declined.
In September 2016, I again crossed paths with Alicia at a monthly meeting of the Atlanta iOS Developers group. She was extremely irate over my Twitter comment and my refusal to teach women-only classes. She became loud and disruptive during the meeting and the event's organizer had to intervene repeatedly.
Despite her hostility, I still wanted to participate in Alicia's ConnectTech panel discussion. I spent weeks preparing to represent iOS developers and the "Apple way" of doing things. Alicia was, however, completely unprepared to moderate and many of the attendees were visibly disappointed. Shortly after the session, Alicia posted disparaging remarks about me on Twitter, implying that she had to "carry the iOS side" and that I failed to contribute anything to the panel discussion.
Following this incident, I had limited interaction with other women in technology groups in Atlanta until January 2017, when I decided to volunteer as a mentor for a RailsGirls and RailsBridge workshop. Within hours of signing up, both organizations banned me from their groups and events. They even enlisted the help of two young white male developers to replace me as a mentor. Although the organizers of both groups declined to provide me with a formal explanation and refused to explain why or how I had allegedly violated their codes of conduct, I later learned that they strongly objected to my conservative political views. In addition, they were also friends of Alicia.
Jaeckel later volunteered to assist the Atlanta chapter of Google Women Techmakers, which was then being organized by Maggie Kane, and says she worked to secure a venue and speakers for their event. However, after viewpoint diversity advocate James Damore was fired from Google, Jaeckel says her public support for him drew a backlash.
In mid-September 2017, Maggie contacted me and told me that Alicia, acting on behalf of Women Who Code, had sent her an email to lodge a written complaint against me and Polyglot Programming. She stated that Women Who Code refused to work with the Atlanta GDG, or attend or sponsor any of the group's events because of my involvement. Then she added that Alicia had accused me of harassing and doxxing Women Who Code members by contacting their employers to get them fired.
I was absolutely dumbfounded by these ludicrous allegations. It made no sense — I've had almost no interaction with Women Who Code's members beyond my exchanges with Alicia Carr. It's simply not in my nature to harass anyone and I've always been strongly opposed to retaliatory actions like doxxing and no-platforming.
Maggie informed me that she had forwarded Alicia's statements to Google and that she had also filed a written complaint with Google because I had "violated the codes of conduct". She even felt that it might be best if I stopped attending any GDG and Google Women Techmakers events, because members might be "triggered" by my presence.
When Jaeckel later sought to attend an event organized by Google Women Techmakers, a group of which she was still a member, she was asked to leave by Kane, who said she held views that were "very harmful to gender equality." Jaeckel also claims that Kane falsely accused her of "stalking" her, after which Jaeckel and her company were banned from a number of influential tech groups in Atlanta.
Two days later, I got an email from TechSquare Labs. Daniel had discussed the incident with Allen Nance, Paul Judge, and Rodney Sampson, the owners of the facility, and he informed me that they had collectively decided to ban me and my company from using their venue or attending any of their events because they were concerned about the "safety" of their members. I later learned from a fellow developer that Maggie had, in fact, told various people that I'd been stalking her. She also recruited a young white male developer, David Hope, to replace my partner Lance as GDG organizer and invited David to act as her co-organizer for Google Women Techmakers.
The following week, Martin Omander, GDG program manager for North America, formally banned me from the Google Developer Group and Google Women Techmakers and, again, declined to provide me with any details of the complaints against me or the rules that I'd allegedly violated.
At this point, says Jaeckel , she realized that the "women in tech" activists had become "determined to ostracize me from the tech industry and ruin the business that I've painstakingly built ."
Her story, in particular the intervention from Google's Martin Omander, bears many similarities to that of James Damore, who was fired from Google after expressing a moderate critique of the company's diversity agenda and for calling for more political tolerance. Like Damore, Jaeckel says she is now facing censure from Google and Google-backed activists over her moderate conservative politics.
Jaeckel has now taken to the legal system to fight back:
I decided that it was time to fight back. I retained renowned civil rights lawyer and GOP official Harmeet Dhillon, who sent a cease and desist letter to Women Who Code, Alicia Carr, Maggie Kane, and Google. In the letter, we demanded a full retraction of the defamatory statements about me. I also requested to have my GDG and Google Women Techmakers memberships reinstated, because I'd been unfairly banned based on false allegations and not on any actual code of conduct violations.
In a comment to Breitbart News, Maggie Kane denied the allegations made against her in Jaeckel 's story, calling them "untrue on many counts and defamatory towards me."
Kane also claims she received an email from someone who "read Marlene's article and accused me of being a criminal, which is also defamatory and untrue."
"I hope Marlene's legal counsel directs her to retract these defamatory statements and personal attacks towards me as they are causing unnecessary harm to our free and volunteer-run tech community groups here in Atlanta."
Alicia Carr, Martin Omander, and Google did not return requests for comment.
PC Grinches at Huffington Post Are Hating on 'Rudolph'
On Saturday night, CBS will rebroadcast the annual Christmas special "Rudolph the Red-Nosed Reindeer."
The seasonal favorite, narrated by Burl Ives, was first telecast in 1964, and has entertained three generations of Americans in the half-century since.
It's must-see TV for anyone who has ever sung along with Ives' Sam the Snowman character crooning "Holly, Jolly Christmas." But in an era of political correctness that has left hardly any corner of pop culture unscathed, not everyone is experiencing the Christmas cheer.
Ever the PC Grinch, the Huffington Post published a withering critique of "Rudolph" that was subsequently picked up and repeated by Yahoo "News."
HuffPo begins by renaming the stop-motion animation classic "Rudolph the Marginalized Reindeer," because the title character is teased and bullied by his peers because of his peculiar proboscis.
"Viewers are noticing the tale may not be so jolly after all and [are] sharing their observations online," it says, reposting a series of tweets critical of the beloved—by most of us, anyway—special.
One Twitter user calls it "a parable of racism and homophobia w/Santa as a bigoted, exploitative pr—." Another opines that "Santa's operation is an HR nightmare and in serious need of diversity and inclusion training."
"Rudolph's father [Donner] verbally abuses him" and forces the young reindeer to conceal his unique attribute so he can fit in with the other young reindeer, while Santa blames Donner, and Rudolph's school P.E. coach, Comet, is portrayed as a discriminatory "bully" because he won't "let Rudolph join in any reindeer games."
Rudolph has a girlfriend, Clarice, which is probably where the charge of "homophobia" comes in, inasmuch as Rudolph isn't gay.
And if that's not bad enough, "Clarice's dad is a bigot" because he's not as willing to overlook Rudolph's nonconforming nose as she is. "No doe of mine is going to be seen with a red-nosed reindeer," he harrumphs.
Meanwhile, Hermey, one of Santa's elves, is excoriated by the tyrannical elfin boss of Santa's workshop because he aspires to be a dentist (not that there's anything wrong with that), rather than make toys.
As fellow "misfits," Hermey and Rudolph become fast friends, because, as one of the Twitter posts reprinted by HuffPo notes, "It's good we don't fit in. It means we're not a–h—s."
Even the fable's happy ending, where Rudolph's unusual attribute saves the day, is cast by HuffPo in the worst possible—dare we say, Marxist—light. "In the end, Rudolph and friends learn the bitter truth," it says. "Deviation from the norm will be punished unless it's exploitable."
HuffPo might have unwittingly given Dr. Seuss' heirs the sequel to "How the Grinch Stole Christmas," but it somehow missed one decidedly un-PC thread in the storyline: It failed to criticize Mrs. Claus, who early on in the film prods Santa to eat to fatten him up before Christmas Eve.
Doesn't that qualify as "body-shaming"?
More tellingly, HuffPo's narrative conveniently fails to acknowledge that, in the end, Donner apologizes to Rudolph for being so hard on him and that the nonconforming "misfit toys" finally find loving homes.
In any case, these PC critics of "Rudolph" need to take a deep breath and get a grip, and if they must, just change the channel. (They got a Christmas fable surely more to their liking Tuesday night, when the cast of NBC's "Will & Grace" imagined traveling back in time—to 1912—for an episode, "A Gay Olde Christmas," replete with loads of double-entendre gay sex jokes.)
One can easily imagine that that other perennial Yuletide favorite, "A Charlie Brown Christmas" (which debuted in 1965, a year after "Rudolph"), will be the next target in HuffPo's and the PC left's cross-hairs. They'll likely start by faulting the climactic scene, in which Linus recites "what Christmas is all about," as too sectarian and exclusionary.
These are the same leftists, by the way, who deny there's a "war on Christmas."
"Ho-ho-ho," indeed.
Political correctness is most pervasive in universities and colleges but I rarely report the incidents concerned here as I have a separate blog for educational matters.
American "liberals" often deny being Leftists and say that they are very different from the Communist rulers of other countries. The only real difference, however, is how much power they have. In America, their power is limited by democracy. To see what they WOULD be like with more power, look at where they ARE already very powerful: in America's educational system -- particularly in the universities and colleges. They show there the same respect for free-speech and political diversity that Stalin did: None. So look to the colleges to see what the whole country would be like if "liberals" had their way. It would be a dictatorship.
For more postings from me, see TONGUE-TIED, GREENIE WATCH, EDUCATION WATCH INTERNATIONAL, AUSTRALIAN POLITICS and DISSECTING LEFTISM. My Home Pages are here or here or here. Email me (John Ray) here. Email me (John Ray) here.
No comments:
Subscribe to: Post Comments (Atom)


text:
Armonia Evolution, Armonia, Apertura's bestseller features also its "Evolution"...
As her elder sister, Edena Evolution , Armonia Evolution has a new slimmer and more elegant cabinet. The stands and spikes design has been improved, and are available in 2 different finishes : black or silver.
A High End Technology
The main differences between Armonia - our best seller model - and Armonia Evolution are the ribbon tweeter twice as big as in Armonia, a brand new cross-over, new high end components and many other details...
The gain in performance is as important as in Edena Evolution. Just discover it by yourself!
As each of our speakers, Armonia Evolution is designed and manufactured in our facility on the west coast of France (near Nantes).
Armonia Evolution is available in 5 standard finishes and with on demand high gloss or metallic paints.

subset: commoncrawl


text:
Wednesday, August 5, 2009
Moe's Disco Dance - Contest Entry!
So I tried a couple of different moves and for the past few days have been working on just this little tiny 30 second clip as an entry. It's amazing what you can learn doing something like this and I've certainly gained a mountain of knowledge from doing this minuscule clip. It seemed easy to do at first but post-production (mostly cause of my own laziness during animation) took far longer than I anticipated. Never-the-less after just two normal work days and then two sleepless nights I've completed it and I'm pretty satisfied.
Any tips / critiques about the animation are extremely welcome. If you really like it drop a vote - but I'd love a comment about anything I can do to up the animation game. Thanks for lookin'!
9 comments:
This comment has been removed by the author.ReplyDelete
woops, i thought the animation was pretty neat and demonstrated your progress well. the popping robot part didn't look as smooth as i'd like--but i don't know if that was on purpose (considering joe is an old guy) or not. the clip itself for the contest was kind of lacking creatively though... too simple--maybe you could have shown him actually put on music or something like thatReplyDelete
Haha, yeah I wanted to at least put the sound in when he walks off screen - like an tape insert but the requirement was that it be 30 seconds flat and I couldn't even fit that sound byte (3 seconds) in there. The constraints suck because the titles are kind of a must. The robot part I made him do like a tiny jiggle at each hit to imitate the bounce back that a lot of people when doing the robot. Maybe that played out badly haha. Thanks for the feedback though Pha.ReplyDelete
I'm learning that "at laaaaast" bit you posted :)
This is really clever, Tony. And you actually did something! Wooo hoo!ReplyDelete
very cool man. congrats!ReplyDelete
Hi there, thanks for dropping by my blog! Your puppet work looks awesome! I wish I had the patience for it! :) Loving your etsy comp entry, the animation looks great!ReplyDelete
Yeah Shelley haha, something actually done! Thanks for stopping by as well Rich / Steff!ReplyDelete
Vaoww Tony, Moe is very good at disco dancing!!ReplyDelete
so exciting!ReplyDelete


text:
Back in January, Amanda Bowman Gray, a mother from Utah, shared a video of her children singing "You Are My Sunshine" on Facebook, where it quickly attracted attention. Not only was it the most adorable sight as Amanda's daughter Lydia played the guitar and sang, but it was also extremely special. As Lydia sang the song over and over the again, her brother started picking up words she sang, thus learning his first words through music. Bo has a vocabulary of 12 words, with 'happy' being his first word.
"Of course, I screamed, and I brought down my husband! I teared up, I was very emotional," Amanda told PEOPLE about the first time she heard her son speak. She says that her son's progress is a proof that music therapy works as the specialist had warned her that Bo most likely would only start to speak at the age of 3 and later.
READ MORE: boredpanda.com | facebook


text: Green Circle Organization established a technology transfer center at its site to educate, train and motivate the farmers to use modern technology. This center has equipment and technologies include: rotavator, thrasher, ridger, ripper, potato planter, and other technologies. These machines are provided to the farmers for very nominal charges. This center also has laser leveler and bed shaper to assist the farmers for getting maximum yield. This center was established on May 2001, with the financial assistance of MASSEY University under the project titled Resource Conservation Dissemination (NZODA/ADAF Project).


text:
Preparation time: 15 Minutes
Cooking time: 35 Minutes
Difficulty: Easy - for beginners
Serves: 4
Dietary guidance: n g
4 - Boneless Skinless Chicken Breasts
1 - Large Onion
1 - Pepper (Green Bell, Poblano, or your own favorite pepper)
1 - Lime
1 Bottle - Lime Juice
Salt and Pepper - To Taste
1 TB - Oil
Spanish Rice:
1.5 Cup - Long Grain White Rice
3 Cups - Chicken Stock
1 Can - Diced Tomatoes with Mild Roasted Peppers, drained.
1 Clove - Garlic, Minced
1 Can - Pinto Beans
Chili Powder, Salt and Pepper - To Taste
Lime Cream Sauce:
1 Cup - Sour Cream
1/2 Cup - Cream Cheese
3 TB - Lime Juice
Zest of 1/2 Lime
1 ts - Salt
1 ts - Cumin
1/2 ts - Chili Powder
Grated Cheese
Tortilla Chips (corn or flour)
Spanish Rice (35 minutes):
Take minced garlic and rice, place into heavy bottom pot on medium heat, allow the rice to cook, dry, until it turns a light brown. Make sure to stir often so that rice does not burn. When it has turned brown, add Chicken stock, tomatoes and peppers, and garlic to the pot, cover, allow to come to a boil then reduce to simmer for 30 minutes or until rice is tender. Do not add the beans or salt and pepper until after the rice has cooked. Add beans a few minutes before serving to heat through. Taste and add salt and pepper as needed.
Chicken (20 minutes):
Slice chicken breasts into strips or medallions. Cut onion and pepper into strips. Place chicken, onion and pepper into heavy bottom skillet with oil. Cook until chicken it browned and almost cooked through. Add in 1/2 - 1 cup of Lime juice (depending on how much lime flavor you would like), the zest of 1/2 a lime, chili powder, salt and pepper to taste. Mix, cover the pan and reduce heat to simmer until liquid has reduced by half and begins to glaze chicken.
Lime Cream Sauce:
With a hand or stand up mixer, cream the sour cream and cream cheese together. Add in the lime juice, zest of lime, chili powder, salt and cumin to taste. Cream together, tasting to adjust for flavor.
Place Rice on the plate, place a small amount of cheese on the rice, place the chicken on the cheese, add some cheese on top of the chicken. Serve with cream sauce on the side for placing on the chicken and chips on the side for eating with the rice and beans.


text:
Developing Detailed Test Plans with a Test Management Tool
Budget $60 USD
Agile Testing environment and use of Agile toolchains. I will provide complete details in the chat.
22 freelancers are bidding on average $110 for this job
Hello! I am a web developer. I've read your job description with great interest. I've fully understood what you want and if you hire me, you would get the best result. I can start work immediately and can chat with yo More
Hi, I have read your requirements and ready to start immediately. I have 8 years experience in software testing. Please visit my profile for details. Recently I have successfully completed the following projects on More
Hello, Greetings of the day!!! I would like to work with you on this assignment. I have 14 years of experience on web development, deployment and project management. Below are my core skills: •ASP.Net/MVC/HTML/CSS/Java More
Hello, I can provide you the test plan . I have 4.5+ yrs of experience in manual testing. I have experience on testing web application. I'm a professional QA. I can test your application from end to end testing with a More
Hello, Pay me if you like my work else I will not ask for payment. I have more than 5+ year of experience in Testing. ISTQB Manager Level Certified ISTQB Foundation Level Certified During my tenure, I have tested lo More
Hi, I am Software QA/Tester with 6+ years of involvement in testing of Web, Mobile applications and Windows-based application testing. I have sound involvement of Mobile Application testing in Android/iOS/Windows Pho More
Having 15 years of testing experience certification in agile methodology from virginia university Handled many projects for international clients
I am working as Software Testing Professional using Agile Methodologies for the past 5 years, and can write detailed test plan for given requirement.
Dear Sir, I am interested in this opportunity. PFB my experience summary. 8 + years of Industry experience in Manual Testing , API Testing , App Testing and Test Automation; Develop & obtain approval of test plan More
Hi There, I have worked as Software quality assurance tester for over 8 years in Agile team, I have complete understanding of Agile and Scrum processes. I would like to share more details. Looking to hear back from you More
I have around 3 years of experience in Testing. - I have experience in Regression Testing, System Testing,Smoke Testing. - Good experience in automating tests of web based application. -Involved in writing Test Pla More
I have over 5 years of experience in this field and I can finish the project in 5 [login to view URL] in manual, automation, web service, mobile testing.
Hi, I expertise in software testing I have more than 2 years experience in Automation testing for both mobile and web application and making the Test plan with test management tools. I read your job description that More
Assures high quality deliverable Relevant Skills and Experience Having 8+ years of experience in software testing.I have worked with giant IT companies and worked on Manual testing, automation [login to view URL] strong k More
Hi, Lets be professional ignoring our nationalities. I have experience with testing and quality assurance. maintaining complete documentation of test cases. Let me know if you can trust a Pakistani and assign me work. More
Quality Assurance Engineer with multiple years experience in agile.


text:
INDIAN WELLS, CA, USA - 2015 BNP Paribas Open champion Simona Halep heads to the Indian Wells Tennis Garden as the top seed in singles, but unseeded in doubles alongside fellow Romanian Irina-Camelia Begu; should she take home both titles, the World No.1 will be awarded a $1 million bonus.
Halep has proven her doubles prowess in the past, finishing runner-up at the Rogers Cup with Monica Niculescu; defending champion Elena Vesnina partnered Ekaterina Makarova to defeat the Romanians that day, and are the top seeds this week in Indian Wells.
Makarova and Vesnina headline a tricky section that features Aussie sensations Daria Gavrilova and Samantha Stosur, with the winner to play one of two high-profile wildcard teams in Eugenie Bouchard and Sloane Stephens or Victoria Azarenka and Aryna Sabalenka.
Double Trouble 👯
See which @WTA stars are pairing up at the #BNPPO18: https://t.co/MUJLWopy3w pic.twitter.com/M8uU46FXK3
— BNP Paribas Open (@BNPPARIBASOPEN) March 6, 2018
Bouchard and Stephens reached the Citi Open final last summer as Stephens was making her way back to tennis following foot surgery, going on to win the US Open two months later. Azarenka has been off the tour since Wimbledon; in the meantime fellow Belarusian Sabalenka has been making waves on the WTA tour, making her first final at the Tianjin Open.
Reigning Australian Open champions Timea Babos and Kristina Mladenovic are Makarova and Vesnina's projected semifinal opponents in what would be a rematch of the championship match in Melbourne.
The bottom half of the draw boasts the reunion of Chan sisters Latisha and Hao-Ching. Latisha Chan finished the 2017 atop the WTA doubles rankings alongside the recently re-retired Martina Hingis, and began the new year with BNP Paribas WTA Finals Singapore presented by SC Global champion Andrea Sestini Hlavackova.
Sestini Hlavackova is also using Indian Wells to unveil a new partnership, pairing with Romanian veteran Niculescu; the two will open against none other than Karolina Pliskova and sister Kristyna.
The Pliskova twins last played together back in 2016, and have a 3-1 record in finals as a team.


text: Join Alpaca-Llips Farm for its first annual We Love Local, Alpaca Farm Days to be held at the farm, 156 Intervale Rd. Rutland, MA on Saturday, Sept. 23 from 9 a.m. to 3 p.m. - rain date Sunday, Sept. 24. Hand feed an alpaca, visit with them in their own environment, and learn the fleece to sweater process. The farm will have several products for sale made from alpaca fleece. Crafters will sell wares, J. Carbonneau Photography will be onsite for senior and family photos as well as photos with the alpacas/llamas, Dog and Horse Portraits by Carole Kinder-McNiff will be in attendance as will Mayer Woodcraft, and many others. Enjoy music, space for a picnic lunch (feel free to bring your own food), and more. Admission is free. To learn more visit www.alpacallipsfarm.com. Note: you cannot get to the farm from Rutland Center due to the Intervale Rd. bridge closing. Follow Rt. 56 to Rt 68, the farm is on your second left off of Rt. 68.


text: Intern at Izar Capital Group Sean is a finance major at Juniata College in Pennsylvania. He participated in TWC's Fall 2017 Academic Internship Program, where he interned at Izar Capital Group, a financial consulting firm. Stories By This Author Discovering the Past at the Holocaust Museum November 20, 2017 Three Reasons I Love My Internship at Izar Capital Group November 8, 2017 Finding Beauty At The National Portrait Gallery October 12, 2017


text:
Today is my three year wedding anniversary and the last thing I feel like doing is celebrating.
I know, I know. That sounds really harsh and I wish I didn't feel that way but I'm just trying to be honest here.
I love my wife and I am so happy that we have spent another year of our lives loving, learning and growing together but the depression I feel today is getting the better of me.
I'm just tired…
I've been working two jobs for the past three months and I am always tired. I'm usually up for my day job at a property management firm at 6:40 am and I don't get back home until around 12:30 am which means by the time I get in the house and wind down it's somewhere around 1:00 am. Now, I've heard of people who can survive, and thrive, on just four to five hours of sleep but I'm just not that person. I am exhausted.
"Why am I subjecting myself to sleep deprivation?" You ask.
Well, it's quite simple really, and the oldest reason in the book. It's because I need more money to be able to provide for my family. I won't go into the details because I believe I have gone into it all before on the website , but just in case you don't see anything about it, feel free to ask me anything you want to know.
Moving on.
I've been averaging about 70 hours a week and as you can imagine it is physically draining. Add mental health instability and you have the perfect recipe for pure insanity. Somehow I've been making it all work. Well not somehow. I know exactly how I've been making it work.
I have generalized anxiety and just one quick Google search will confirm that it is not recommended for a person who manages anxiety to consume caffeinated beverages as it can exacerbate your symptoms and possibly trigger an attack. This isn't particularly good news for someone like myself who is barely getting any sleep.
Since I don't need any help having more anxiety attacks, I've been steering clear of coffee to keep myself awake and instead, I've been relying on sugar.
In all forms.
Cakes, cookies, pies, candy, sodas, juices, carbs, you name it, I've been eating it. My main go to, double stuffed Oreo's. Oh, how I love those little chocolaty delicious bastards. So much so, that I've been clearing a whole pack of the sons of bitches in a matter of days.
When I stepped on the scale last night after another grueling sixteen hour day, the scale read 213.4. Since I was still fully clothed and had been eating about 2 hours before, I tried to dismiss the numbers and I went to bed. As soon as I laid down, I felt myself about to cry because deep down I knew the truth. I woke up this morning and weighed myself again to find that the needle had only barely moved to reflect an even 212 lbs.
Just 11 months ago I weighed 182 lbs.
I have been convincingly lying to myself, again, about the confidence I feel in my now sized 16 body. As a result, I semi-unconsciously put on another two pounds bringing my already out of line weight to a whopping 212 lbs.
212 lbs. 82 lbs over weight. I've gained 30 lbs in 11 months.
All the work I had put in to lose weight last year is now down the drain and the point was driven home more pointedly when I went to put on a special outfit I had picked out specifically for our anniversary today and my sized 14 pants just wouldn't close.
So here I sit. At work. Depressed. In desperate need of a good cry that I likely won't get today because today is supposed to be a happy occasion.
I'm struggling with life right now.
Struggling to keep face.
Struggling with maintaining energy.
Struggling with my weight.
Struggling with my mental health.
Struggling with feelings of resentment.
Struggling with depression.
Struggling with feelings of hopelessness.
I'm going to go put on my make up and slide on my new black boots. I'm get all dolled up and go to lunch with my beautiful wife and try to find a way to put myself in a festive mood because she deserves it.
Until next time…
Don't judge me, you don't know my life!


text:
Former Kenya death row prisoner gets law degree
A former death row inmate in Kenya has expressed his joy at having graduated with a law degree from the University of London. William Okumu, one of several inmates or former inmates who graduated at a ceremony at the Kamiti Maximum Security Prison in Nairobi, told the BBC that 'I think the whole world now knows that something good can come out of trash'. Okumu, who was released in May after serving a 10-year jail term for armed robbery, said the degree had changed his life. He said his motivation to pursue law was through his realisation that his sentence had been due to his lack of a proper understanding of the law. He was assisted by the African Prisons Project, a charity which helps prisoners get access to justice.


text:
- © 2008 American Society of Plant Biologists
The plant UDP-dependent glucosyltransferase (UGT) BpUGT94B1 catalyzes the synthesis of a glucuronosylated cyanidin-derived flavonoid in red daisy (Bellis perennis). The functional properties of BpUGT94B1 were investigated using protein modeling, site-directed mutagenesis, and analysis of the substrate specificity of isolated wild-type and mutated forms of BpUGT94B1. A single unique arginine residue (R25) positioned outside the conserved plant secondary product glycosyltransferase region was identified as crucial for the activity with UDP-glucuronic acid. The mutants R25S, R25G, and R25K all exhibited only 0.5% to 2.5% of wild-type activity with UDP-glucuronic acid, but showed a 3-fold increase in activity with UDP-glucose. The model of BpUGT94B1 also enabled identification of key residues in the acceptor pocket. The mutations N123A and D152A decreased the activity with cyanidin 3-O-glucoside to less than 15% of wild type. The wild-type enzyme activity toward delphinidin-3-O-glucoside was only 5% to 10% of the activity with cyanidin 3-O-glucoside. Independent point mutations of three residues positioned near the acceptor B ring were introduced to increase the activity toward delphinidin-3-O-glucoside. In all three mutant enzymes, the enzymatic activity toward both acceptors was reduced to less than 15% of wild type. The model of BpUGT94B1 allowed for correct identification of catalytically important residues, within as well as outside the plant secondary product glycosyltransferase motif, determining sugar donor and acceptor specificity.
Glycosylation of bioactive plant natural products (secondary metabolites), xenobiotics, and hormones provides a means to regulate their activity, to secure safe storage in specific cellular compartments, and to modulate their chemical properties, such as solubility and stability important for their in planta function (Jones and Vogt, 2001; Kristensen et al., 2005). The glycosylation process is catalyzed by glycosyltransferases (GTs), enzymes found in organisms across all phyla. In mammalians, glycosylation plays an important role in detoxification, e.g. by glucuronosylation of xenobiotica, while in plants glycosylation constitutes the last step in the biosynthesis of numerous plant natural products. These include major classes like terpenes, phenylpropanoids, cyanogenic glucosides, and glucosinolates. Vitus vinifera contains more than 200 different aglycones conjugated to Glc (Sefton et al., 1993, 1994), and for the flavonol quercetin alone, 300 different glycosides have been identified (Jones and Vogt, 2001). It remains an open question how the specificity of GTs is controlled. High specificity would indicate the requirement for many more enzymes than would be predicted from the genome sequencing programs, whereas a low specificity would pose problems with respect to undesired side reactions like inactivation of plant hormones (Ford, 1998; Jones et al., 1999). Further knowledge of the in vivo substrate specificity of GTs is important to understand the basis for plant plasticity with respect to natural product synthesis and adaptation to abiotic and biotic environmental stress factors.
Despite low primary sequence similarity, the secondary and tertiary structures of GTs are highly conserved. So far, the crystal structures available show that the GTs adopt one of two distinct folds, designated GT-A fold and GT-B fold, with an emerging third fold being identified as GT-A-like (http://www.cermav.cnrs.fr/glyco3d/). The GTs are currently divided into 91 families (http://www.cazy.org/fam/acc_GT.html) based on sequence identity scores (Campbell et al., 1997; Coutinho et al., 2003). Family 1 GTs contain a large number of GTs that use a UDP-activated sugar as donor in the enzymatic reaction, and these are often referred to as the UDP-dependent glucosyltransferases (UGTs; Mackenzie et al., 1997; Lim and Bowles, 2004). The family 1 UGTs constitute a large multiprotein family. They share a conserved sequence motif at the C-terminal end, the UGT-defining sequence (Mackenzie et al., 1997). In Arabidopsis (Arabidopsis thaliana), 120 UGT-encoding genes have been identified (Paquette et al., 2003), and phylogenetic analyses have shown these to group into tree clades. The major clade is plant specific and contains the Arabidopsis UGTs known to be involved in plant natural product synthesis, control of plant hormone homeostasis, and detoxification of xenobiotics (Vogt and Jones, 2000; Jones and Vogt, 2001; Paquette et al., 2003; Lim and Bowles, 2004). These plant UGTs have an expanded UGT-defining sequence, denoted the plant secondary product GT (PSPG) motif (Hughes and Hughes, 1994; Paquette et al., 2003).
New UGT sequences are obtained too fast to be accompanied by functional characterization (Coutinho et al., 2003). Bioinformatic analyses for accurate prediction of sequence features important for defining the substrate specificity of a particular UGT have not yet been developed. State of the art may be exemplified by the recent development of a program to predict acceptors for bacterial GTs glycosylating antibiotics (Kamra et al., 2005). This program uses a knowledge-based approach and relies on available biochemical and crystal structure data. This means that high homology of the query sequence with the templates is essential to achieve reliable predictions. It also underscores the need for substantial additional amounts of biochemical and structural data to enable development of bioinformatic programs with reliable predictive power. This is especially important for the GTs, as annotations based on primary structure analysis have proven difficult, and substrate specificity can be highly diverse for UGTs within groups defined by phylogenetic analyses (Richman et al., 2005; Modolo et al., 2007). The biochemical data available on UGTs enable some conclusions on UGT acceptor specificity to be drawn. Several UGTs have broad acceptor specificity as demonstrated by their ability to glycosylate a large number of structurally quite different aglycones in vitro (Vogt et al., 1997; Vogt and Jones, 2000; Hansen et al., 2003; Kramer et al., 2003; Modolo et al., 2007). Other UGTs are highly specific for a single acceptor (Fukuchi-Mizutani et al., 2003; Kramer et al., 2003). An emerging picture is that UGTs show regiospecificity toward the part of the substrate molecule harboring the site of glycosylation (Vogt et al., 1997; Kramer et al., 2003; Lim et al., 2003b, 2004; Richman et al., 2005; He et al., 2006). This would be in accordance with the broad acceptor specificity of some UGTs, as the presence of a specific structural domain to be glycosylated can be presented on otherwise differing molecules. Biochemical studies seem to support regiospecificity as a key parameter for UGT substrate specificity. At the same time it is also evident that the presence of a specific structural domain will not by itself assure activity, but should be considered in conjunction with the overall structure of the aglycone (Richman et al., 2005; He et al., 2006; Kogawa et al., 2007; Modolo et al., 2007). Several studies of biochemically described UGTs show some conservation of regiospecificity within phylogenetic groups (Vogt and Jones, 2000; Lim et al., 2003a; Morita et al., 2005; Yonekura-Sakakibara et al., 2007; Caputi et al., 2008).
UGTs recognizing several different sugar donors have been identified. These include UDP-Glc, UDP-Gal, UDP-Xyl, UDP-Rha, and UDP-GlcUA. In plant UGTs, the most common sugar donor is UDP-Glc. Several conserved residues, most of which are found in the PSPG motif of plant UGTs, interact with the sugar donor (Shao et al., 2005; Thorsoe et al., 2005; Offen et al., 2006; Li et al., 2007). The sugar donor preference of a specific UGT is often very narrow, showing little or no activity with alternative sugars. Replacement of a single amino acid residue has been shown to change the sugar specificity of Aralia cordata UGT78A2 (GaT; Kubo et al., 2004) and Medicago truncatula UGT71G1 (He et al., 2006). Similar results have been obtained from other GTs (Qasba et al., 2005). Nevertheless, no conserved amino acid residues have been identified as general determinants of sugar specificity (Ouzzine et al., 2002; Kubo et al., 2004; Modolo et al., 2007; Yonekura-Sakakibara et al., 2007).
Functional characterization of a UGT requires time-demanding biochemical studies as well as the study of site-specific mutants. Knowledge derived from crystal structures is an important tool for the rational design of mutants for such biochemical studies. So far the crystal structures of 11 family 1 GTs have been solved. Ten are listed at http://www.cermav.cnrs.fr/glyco3d/ and the structure coordinates of all 11 are available at http://www.rcsb.org/pdb/home/home.do. These GTs all adopt the GT-B fold. Obtaining GT crystals has proven difficult (Breton et al., 2006) and structure solution progresses slowly in comparison to the rate of appearance of new UGT sequences. To circumvent this bottleneck in the study of UGTs, homology modeling has proved to be a strong tool for the identification of potentially important residues (Hans et al., 2004; Thorsoe et al., 2005). The recently solved crystal structures of the first plant UGTs, MtUGT71G1 from M. truncatula (Shao et al., 2005) and VvGT1 from V. vinifera (Offen et al., 2006), essentially add confidence to the structures derived from homology modeling.
A plant UGT with affinity for the alternative sugar UDP-GlcUA (Fig. 1 ) UGT94B1 has been identified in red daisy (Bellis perennis; Sawada et al., 2005). Together with the UGT UBGAT from Scutellaria baicalensis (Nagashima et al., 2000), these are the only characterized plant UGTs using UDP-GlcUA as the sugar donor. BpUGT94B1 shows specificity for the acceptor cyanidin 3-O-glucoside, whereas delphinidin 3-O-glucoside is a poor acceptor (Fig. 1). UGT94B1 is unique both with respect to sugar donor and acceptor specificity by using the alternative sugar donor UDP-GlcUA and by forming a diglycoside (Fig. 1). To gain an understanding of the molecular structure governing acceptor as well as donor specificity of this enzyme, a model of BpUGT94B1 was constructed using coordinates of the two plant crystal structures VvGT1 (Offen et al., 2006) and MtUGT71G1 (Shao et al., 2005) as scaffolds. Residues constituting the substrate pocket in the model were identified. The significance of these residues for activity and substrate specificity was investigated by site-specific mutagenesis and by biochemical characterization of the catalytic properties of the isolated mutant proteins. A unique Arg residue (R25) situated outside the conserved PSPG motif was identified as essential for BpUGT94B1 activity with the sugar donor UDP-GlcUA. In addition, several residues forming the acceptor pocket were identified as important for the catalytic properties. Based on these findings, the conservation of primary and secondary structure in relation to substrate specificity of UGTs is discussed.
Homology Modeling of UGT94B1
Prediction of red daisy UGT94B1 sary structure identified 16 potential α-helices and 13 potential β-strands. The secondary structure predictions were used for an alignment of BpUGT94B1 with sequences of the plant enzymes MtUGT71G1 (Shao et al., 2005) and VvGT1 (Offen et al., 2006; Fig. 2 ), for which crystal structure coordinates have been published. The BpUGT94B1 amino acid sequence has approximately 26% and approximately 20% overall sequence identity to MtUGT71G1 and VvGT1, respectively, as determined by ClustalX alignment.
The structural alignment (Fig. 2) was entered into the Composer function of the Sybyl protein modeling software and divided into 17 structurally conserved regions (SCRs) and 18 loops (Fig. 2). All 18 SCRs and 13 of the 18 loops were constructed using the coordinates of the MtUGT71G1 and VvGT1 crystal structures as templates. The missing loops were loops 1, 7, 9, 11, and 18. Loop 1 corresponds to the N-terminal end and loop 18 to the C terminus. Construction of loop 1 (residues 1–9), loop 7 (residues 156–162), and loop 11 (residues 232–242) was performed by search in the GT crystal structure database. Two loops were left out of the model. Loop 9 (residues 204–208) was left out because it could not be modeled without major energy constraints. The N-terminal loop 18 was also left out, as no acceptable template for this region was found.
The model was optimized by several steps of energy minimization of side chains and loop regions (Imberty et al., 2006). Chirality and charges of all amino acids were verified. Stereochemistry of the backbone structure was verified by consultation of Ramachandran maps followed by optimization of torsion angles and energy. In the final model, no residues were in disallowed regions, and only 15 of the 423 amino acid residues in the model were in generously allowed regions. The PROCHECK (Laskowski et al., 1996) overall g factor evaluating all torsion angles and bond lengths was −0.46, indicating a high-quality model (Bhattacharya et al., 2008). The g factors for the two template structures VvGT1 and MtUGT71G1 were 0.13 and 0.24, respectively. The overall model quality was also validated by calculation of the ProSA Z score (Wiederstein and Sippl, 2007). The Z score for the BpUGT94B1 model was calculated to be −8.29. Z scores for experimentally determined x-ray structures of proteins of similar size (around 450 amino acids) lie in the range of −6 to −13. The Z score of the model was thus in the range of scores typical for proteins of similar size. For comparison the calculated Z scores for the template structures MtUGT71G1 and VvGT1 were −12.75 and −10.39, respectively. The ProSA residue score calculates local model quality. In general, positive values indicate problematic zones. Looking at the residue score for the BpUGT94B1 model, approximately 90% of the residues in the model lie between 0 to −2, as also observed for the template structures. Two short regions of the N terminus (approximately 10 amino acids) show a score of 0.5 while the linker region has a score of 1.
Docking of Sugar Donor and Acceptor into the Model
In vitro, BpUGT94B1 catalyzes the regiospecific transfer of GlcUA from UDP-GlcUA to the 2″-OH of cyanidin 3-O-β-glucoside and cyanidin 3-O-β-6″-O-malonylglucoside (Fig. 1; Sawada et al., 2005). The sugar donor and acceptor were inserted into the model by homology docking (Imberty et al., 2006). Docking of the BpUGT94B1 sugar donor UDP-GlcUA was performed guided by the coordinates of the crystal structure of MtUGT71G1 crystallized with UDP-Glc. The acceptors cyanidin 3-O-β-glucoside or cyanidin 3-O-β-6″-O-malonylglucoside were inserted guided by the position of acceptors in the crystal structures of the V. vinifera UGT VvGT1 containing the sugar acceptor kaempferol (Offen et al., 2006) and the Amycolatopsis orientalis GT GtfD crystallized with the acceptor l-vancosamine (Mulichak et al., 2004).
The VvGT1 acceptor is a flavonoid, as is the acceptor in BpUGT94B1, whereas the glycosylated position of the GftD acceptor is the 2′-OH group of a Glc residue, as is also the case in BpUGT94B1. Docking was followed by energy optimization of amino acid side chains surrounding the substrates.
Analyzing the Model
The tertiary structure of the model takes a GT-B-fold conformation with two distinct domains formed by the N- and C-terminal parts of the primary structure (Fig. 3 ). In the final model, the N-terminal domain folds into seven α-helices and a β-sheet composed of seven parallel β-strands. The C-terminal domain folds into eight α-helices, whereas the ninth predicted α-helix is positioned in the C terminus that was left out of the model. In the C-terminal domain, five of the predicted six β-strands are formed in the predicted regions and fold into a parallel β-sheet.
The N- and C-terminal domains form a deep cleft accommodating a tight binding site for the sugar acceptor and sugar donor deeply buried inside the protein (Fig. 4, A–E ). The sugar donor is positioned so that it mainly interacts with residues in the C-terminal domain, whereas the acceptor mainly interacts with residues in the N-terminal domain (Fig. 4, B–E).
Sugar Donor Interactions
In the BpUGT94B1 model, several residues are positioned so that they may interact with the activated sugar donor (Fig. 5, A and B ). The majority of these residues are situated within the PSPG motif that is highly conserved between different UGTs (BpUGT94B1 residues 315–358; Fig. 2). Nine PSPG residues are predicted to interact with the sugar donor in the BpUGT94B1 model (Fig. 5A). These nine residues possess similar positions in the crystal structure of MtUGT71G1, with five being identical and four being similar residues (Fig. 2).
In the BpUGT94B1 model, the residues Q355, D357, and Q358 are all positioned near the sugar moiety of the sugar donor (Fig. 5A). The residue V316 lies within H-bond distance to the uridine ring of the donor. The position of the uridine ring is further stabilized by aromatic ring stacking with the indole ring of W315. E341 can interact with the Rib ring of the donor. H333, S337, and S338 can form H bonds with the α-P, while S264 and maybe Q355 can interact with the β-P. Two highly conserved residues outside the PSPG motif are the residues corresponding to BpUGT94B1 H22 and D121. These are directly involved in the catalytic process and have been shown to be crucial for activity in many plant UGTs (Hans et al., 2004; Shao et al., 2005; Offen et al., 2006). In the BpUGT94B1 model these residues are positioned in a similar manner as in the MtUGT71G1 and VvGT1 crystals, favoring a similar catalytic mechanism. The conserved position of UDP-sugar donor interacting residues (PSPG motif residues) and catalytically important residues (H22 and D121) between the model and the template structures confirm a high degree of confidence of the model for conserved regions.
Less conserved residues outside the PSPG motif also exhibit interaction with the sugar donor. These residues are S143 that can H bond with the sugar moiety and is paralleled by a Thr residue in MtUGT71G1, S21 that interacts with the β-P, and the positively charged R25 that is positioned near the negatively charged carboxylate group of the GlcUA (Fig. 5B).
Sugar Acceptor Interactions
The residues in the BpUGT94B1 model that form the acceptor pocket are mainly positioned in the N-terminal domain. A hydrophobic environment is created by the residues W17, P88, P89, F122, L148, L151, P174, I187, Y186, and F356. Additional residues forming part of the acceptor pocket are T92, D121, N123, D152, K175, N176, Q355, and D357. An analysis of the model following docking of the acceptor cyanidin 3-O-glucoside shows that the acceptor is held in place by a tight fit of the surrounding amino acid residues. Stabilizing interactions are also envisioned between specific amino acid residues and the acceptor. The residues N123 and T92 are positioned close to the sugar part of the acceptor (Fig. 5A), and the acceptor B ring can be stabilized by H-bond formation to D152 (Fig. 5C). The A and C rings of the cyanidin moiety can be stabilized by aromatic ring stacking with the side chain of F356.
The possibility to dock cyanidin 3-O-6″-O-malonylglucoside (Fig. 1) as an alternative acceptor was also investigated. Despite the tight fit of the enzyme around the smaller acceptor cyanidin 3-O-glucoside, it was possible to accommodate the acceptor cyanidin 3-O-6″-O-malonylglucoside within the acceptor pocket with favorable energy. This is accomplished when the malonic acid is bent toward the aglycone, which is also the energetically favored configuration of the molecule (Fig. 4, D and E).
Test of the Model by Site-Directed Mutagenesis Activity of UGT94B1
To study the substrate specificity and enzyme kinetic properties of BpUGT94B1, a full-length cDNA clone encoding the enzyme was obtained from petals of red daisy. An N-terminal His-tag was added for heterologous expression in Escherichia coli and subsequent affinity purification (Fig. 6 ). The three-dimensional structure of UGTs suggests that N-terminal His-tagging would position the tag on the surface of the protein far from the active site, i.e. at a position that would not be expected to interfere with protein folding and activity (Fig. 3).
The isolated His-tagged enzyme was highly specific with respect to the sugar donor UDP-GlcUA. Activity with other activated sugars such as UDP-Glc, UDP-Gal, and UDP-Xyl was very low. The activity with UDP-Glc was <0.5% of the activity with UDP-GlcUA. The Km value for UDP-GlcUA was determined to be 1.1 ± 0.1 mm (Fig. 7 ; Table I ), whereas the Km value for UDP-Glc was 7.3 ± 0.9 mm (Fig. 8 ; Table I). The enzyme exhibited high activity using cyanidin 3-O-glucoside as acceptor. The Km value for cyanidin 3-O-glucoside was estimated to be 0.8 ± 0.2 mm. Substrate inhibition of BpUGT94B1 at acceptor concentrations above 1 mm prevented a more accurate assessment of this Km value (Fig. 9 ). Enzyme activity with the acceptor delphinidin-3-O-glucoside (Fig. 1) was of only 5% to 10% of the activity observed with cyanidin 3-O-glucoside.
Modulation of Sugar Donor Specificity
Residues in the PSPG motif of plant UGTs have previously been shown to be involved in sugar donor binding of UGTs (Kubo et al., 2004; Shao et al., 2005; Thorsoe et al., 2005; Offen et al., 2006). These residues are highly conserved and might not explain the various sugar specificities observed for plant UGTs. The model of BpUGT94B1 was analyzed to identify residues responsible for the high specificity of this enzyme for UDP-GlcUA. This identified an Arg residue (R25) far from the PSPG motif that interacted with the sugar part of the donor. The positively charged side chain of R25 is positioned pointing toward the negatively charged carboxylate group of the UDP-GlcUA of the sugar donor (Fig. 5B). This could serve to stabilize binding of the negatively charged sugar residue in the active site, explaining why BpUGT94B1 exhibits high specificity toward UDP-GlcUA. In other plant UGTs, the position corresponding to BpUGTT94B1 R25 is often a Ser or Pro. To investigate the role of the R25 residue experimentally, the mutations R25S, R25P, R25G, and R25K were introduced into BpUGT94B1. The mutant R25S mirrors S25 in MtUGT71G1 whereas R25P mirrors P23 in VvGT1. The R25G mutation would be expected to completely abolish a specific interaction with the charged carboxylate group of UDP-GlcUA, whereas the positively charged Lys side chain in the R25K mutation retains the positive charge but introduces a shorter side chain compared to Arg.
All mutated enzymes were tested for activity with the sugar donors UDP-GlcUA, UDP-GalUA, UDP-Glc, UDP-Xyl, and UDP-Gal, using cyanidin 3-O-glucoside as the acceptor. All the mutants exhibited significantly decreased activity (relative activity <2.5% of wild type) with UDP-GlcUA (Table I; Fig. 10 ). Despite the severe decrease in activity with UDP-GlcUA for the R25S mutant, the Km value for UDP-GlcUA only increased from 1.1 ± 0.1 mm to 1.4 ± 0.3 mm. In contrast, the Km value for the mutant R25K increased to 7.0 ± 0.5 mm (Fig. 7; Table I).
BpUGT94B1 and mutants R25S, R25G, R25P, and R25K were tested for their ability to use UDP-Glc as the sugar donor. Three of the four mutants (R25S, R25G, and R25K) showed 3-fold activity increases using UDP-Glc as donor compared to the wild type (Fig. 10). Km for UDP-Glc was determined for the mutant R25S and was 2.9 ± 0.5 mm (Fig. 8; Table I) as compared to 7.3 ± 0.9 mm for the wild-type enzyme (Fig. 8; Table I), and the relative Kcat value was more than twice as high as for the wild-type enzyme. No activity was detected with UDP-GalUA and UDP-Xyl. In contrast to the wild-type enzyme, the mutants R25S, R25G, and R25K showed marginal activity with UDP-Gal.
Modulation of Sugar Acceptor Specificity
In vivo, BpUGT94B1 glucuronosylates cyanidin 3-O-glucoside at the 2″-OH (Fig. 1; Toki et al., 1991; Sawada et al., 2005). When delphinidin-3-O-glucoside was used as substrate, the conversion efficiency dropped to 5% of that obtained with cyanidin 3-O-glucoside. Delphinidin 3-O-glucoside has a 3′,4′,5′-hydroxylation pattern at the B ring compared to the 3′,4′-hydroxylation pattern of cyanidin (Fig. 1). To identify amino acid residues in BpUGT94B1 involved in acceptor interactions and enabling the enzyme to discriminate so effectively between the di- and trihydroxylated acceptors, the model of BpUGT94B1 was analyzed. Residues I187, P174, L148, and D152 were found to form part of the acceptor pocket and accommodate the acceptor B ring (Fig. 5C). The amino acids I187, P174, and L148 provide a hydrophobic environment for the acceptor and the residue D152 is positioned to form a stabilizing H bond with the 3′-OH of the acceptor. Accommodation of a delphinidin acceptor with an additional OH group on the B ring would require more space around the B ring, but also propose an additional OH group to interact in H-bond formation compared to cyanidin. To test if more space or amino acids proposing H-bond interactions could increase activity with delphinidin 3-O-glucoside, the point mutations I187A, I187S, P174G, D152A, and L148A were independently introduced in the BpUGT94B1 sequence. The residues I187, P174, and L148 were changed into the amino acid Ala or Gly with very small or no side chains to provide more space in this area of the acceptor pocket. The mutant I187S was made as the Ser residue could offer a stabilizing interaction in the form of H-bond formation with the acceptor. The mutant D152A was made to test whether the predicted H-bond formation between this residue and the acceptor was of importance for substrate interaction and activity.
The mutated proteins were assayed for their activity toward the acceptors cyanidin 3-O-glucoside and delphinidin 3-O-glucoside. For both acceptors, the mutated proteins all showed less than 15% of the activity obtained with the wild-type protein. The most severe effect was observed with the D152A mutant, in which the activity toward cyanidine 3-O-glucoside dropped to less than 5% of wild type (Fig. 10). Km values of the mutant enzymes with respect to the two sugar acceptors were not determined because of the observed substrate inhibition (Fig. 9). To observe if the affinity for the sugar donor could account for the reduced relative activity of the mutants, the Km values for the sugar donor UDP-GlcUA were determined for the mutants L148A D152A and I187A (Fig. 7; Table I). The mutated proteins had Km values for the sugar donor UDP-GlcUA 1 to 3 times higher than wild type.
Analysis of the acceptor pocket in the model identified an additional residue predicted to be important for acceptor stabilization in the active site. This was the residue N123 that proposes H bonding to the sugar part of the acceptor. To verify a role of this residue for activity, the mutation N123A was introduced in the BpUGT94B1 sequence. Introduction of an Ala residue would abolish any interaction between this residue and the acceptor. The N123A mutation caused relative enzyme activity to decrease to less than 15% of wild type for both acceptors (Fig. 10).
Recent crystallization of four plant UGTs, MtUGT71G1 (Shao et al., 2005), VvGT1 (Offen et al., 2006), MtUGT85H2 (Li et al., 2007), and AtUGT72B1 (Brazier-Hicks et al., 2007), provides structural templates for homology modeling of plant UGTs. This increases the reliability of plant UGT modeling, formerly performed using crystal structure coordinates of more distant bacterial family 1 GTs as scaffolds (Hans et al., 2004; Thorsoe et al., 2005). UGT94B1 from red daisy exhibits high substrate specificity both with respect to the sugar donor and the sugar acceptor (Sawada et al., 2005). In this study a model of BpUGT94B1 was constructed to reveal structural features responsible for the tight specificity of this enzyme and to identify strategies to change the substrate specificity.
In the molecular model of BpUGT94B1, the substrates are tightly fitted in the cleft between the N- and C-terminal domains deeply buried within the enzyme (Fig. 4). This suggests cleft opening as a prerequisite for substrate binding. This is in accordance with studies of several GT crystal structures, suggesting accommodation of substrates within GT-B-fold GTs proceeds by slight movements of the linker region between the N- and C-terminal domains (Hu et al., 2003; Mulichak et al., 2003; Shao et al., 2005).
Sugar Donor Specificity of UGT94B1
In the model of BpUGT94B1, the positively charged side chain of R25 is positioned so that it points toward the carboxylate group of the UDP-GlcUA sugar donor. The crucial importance of the residue R25 was substantiated by biochemical analyses of BpUGT94B1 R25 mutants. Mutant proteins R25S, R25G, R25K, and R25P all possessed only 0.5% to 2.5% of wild-type activity with the sugar donor UDP-GlcUA. For the R25K mutant, this was paralleled by a 7-fold increase in Km. These results demonstrated the importance of R25 for enzyme activity with UDP-GlcUA as sugar donor.
In contrast to the observed loss of activity with UDP-GlcUA, the three point mutants R25S, R25G, and R25K showed a 3- to 4-fold increase in activity with UDP-Glc as sugar donor paralleled by a decrease of Km for UDP-Glc from 7.3 mm for the wild-type BpUGT94B1 to 2.9 mm for the R25S mutant. Even the R25K mutation that retains a positively charged side chain shows loss of activity with UDP-GlcUA and increased activity with UDP-Glc. Apparently even a minor displacement of the positive charge away from the GlcUA, as mediated by the shorter side chain of the Lys residue as compared to Arg, is enough to abolish activity with UDP-GlcUA as the sugar donor. The mutant R25P showed decreased activity with both sugar donors. The severity of this mutation probably reflects that the Pro residue causes a changed torsion of the backbone.
The crystal structures of UGTs show the majority of interactions with the UDP-sugar donor to be provided by amino acid residues within the highly conserved PSPG motif (Shao et al., 2005; Offen et al., 2006; Brazier-Hicks et al., 2007). This is in accordance with the fact that the major part of the donor is the invariant nucleotide unit. In the BpUGT94B1 model, nine of the 14 amino acid residues positioned close to the donor are from the PSPG motif. Of these nine residues, five are identical and four are conserved when compared to the PSPG motif in MtUGT71G1 (Fig. 2). Despite the high conservation of sugar donor interacting PSPG motif residues, most UGTs are highly specific with respect to their sugar donor preference. Several studies have searched for residues within the PSPG motif responsible for specificity toward specific sugars but with very limited success (Kubo et al., 2004; Shao et al., 2005; Offen et al., 2006). The R25 residue of BpUGT94B1 lies outside the PSPG motif and is neither conserved among plant UGTs nor in mammalian UGTs known to use UDP-GlcUA as sugar donor. This was demonstrated by Clustal alignments of a large number of UGT amino acid sequences (results not shown), including the plant UGT UBGAT from S. baicalensis that also uses UDP-GlcUA as the sugar donor (Nagashima et al., 2000). Alignment of the S. baicalensis UBGAT with BpUGT94B1 suggests an Ala to take the R25 position, and there are no charged residues nearby this position. The mechanism responsible for BpUGT94B1 activity with UDP-GlcUA thus seems to be specific for this particular UGT and is not a conserved feature of UDP-glucuronosyltransferases. In the Sorghum bicolor UGT85B1 sharing 24% amino acid identity with BpUGT94B1, a Pro residue is found at the position that corresponds to R25 in BpUGT94B1. Replacement of this Pro residue with an Arg residue did not confer activity with UDP-GlcUA (data not shown). Presence of an Arg residue at this position is therefore not sufficient for activity with UDP-GlcUA. The involvement of N-terminal residues as decisive for sugar donor specificity has also been observed for UGT93A2 (ZOG1) from Phaseoulus lunatus and UGT93A1 (ZOX1) from Phaseolus vulgaris, where domain swop identified a region of the N-terminal domain to be determinant for ZOG1 activity with UDP-Glc (Martin, 2000).
The observation that a single amino acid residue in BpUGT94B1 may be decisive with respect to UDP-sugar specificity is in accordance with other studies of the sugar specificity of UGTs. In the A. cordata UGT78A2 (GaT), a single residue (H374) was observed to dictate specificity for UDP-Gal (Kubo et al., 2004). This residue lies within the PSPG motif and is conserved in galactosyltransferases. Sugar specificity was shifted to UDP-Glc by the point mutation H374Q. However, the inverse mutation in S. baicalensis UGT UBGT did not confer galactosyltransferase activity, illustrating that several other structural features have to be in place to mediate such a shift in specificity (Hans et al., 2004; Kubo et al., 2004). Other studies add to the conclusion that UDP-sugar recognition is highly complex (Thorsoe et al., 2005; Kohara et al., 2007; Modolo et al., 2007; Yonekura-Sakakibara et al., 2007). The sugar residue Rha has the same C-4 configuration as Gal. In the Arabidopsis rhamnosyltransferase UGT89C1, a His residue is present at the position corresponding to H374 in the AcUGT78A2 galactosyltransferase. However, this is not the case for the rhamnosyltransferase AtUGT78D1 (Yonekura-Sakakibara et al., 2007), and in MtUGT78G1 that uses both UDP-Glc and UDP-Gal as sugar donor, this position is occupied by a Gln residue (Modolo et al., 2007). These observations on the specificity of UGTs with respect to the sugar donor lead to the conclusion that multiple amino acid residues contribute to the determination of sugar specificity. Thus, while single amino acid residues might be decisive for sugar recognition of a specific UGT, these are not general determinants of sugar specificity.
Studies of GTs from animals support the involvement of single residues as determinants of sugar specificity in specific GTs (Qasba et al., 2005). In the human GT-A-fold enzyme GlcAT-I involved in proteoglycan biosynthesis, the mutation H308R switches the sugar donor specificity of the enzyme from UDP-GlcUA to UDP-Glc. In addition, an Arg residue R277 was found to be of crucial importance for the glucuronosylation to proceed (Ouzzine et al., 2002). This Arg residue was suggested to stabilize the acidic group of the sugar donor in a similar fashion to that proposed for R25 of BpUGT94B1 in this study. Most of the plant UGTs so far studied and all of the four crystallized plant UGTs are glucosyltransferases. As more biochemical data, crystal structures, and modeling work on UGTs with other sugar preferences become available, a more comprehensive knowledge of key residues in sugar donor recognition is likely to emerge.
Sugar Acceptor Specificity of BpUGT94B1
BpUGT94B1 glucuronosylates cyanidine 3-O-glucoside at the 2″-OH position (Fig. 1). A very tight acceptor specificity of BpUGT94B1 is evidenced by the low activity with the acceptor delphinidin 3-O-glucoside, relative activity being only 5% of the activity with cyanidin 3-O-glucoside. Cyanidin 3-O-glucoside and delphinidine 3-O-glucoside offer the same structural epitope for glycosylation at the 2″-OH position, and only differ by the presence of an additional hydroxyl group on the B ring of the aglycone (Fig. 1). This shows that the entire structure of the substrates and not just the site of the accepting atom is being recognized by BpUGT94B1.
To investigate residues in BpUGT94B1 responsible for this very tight substrate specificity distinguishing between cyanidin and delphinidin, independent point mutations of residues forming the acceptor pocket were made. The mutations I187S, I187A, and L148A all severely compromised activity toward cyanidin as well as delphinidin 3-O-glucoside. This showed that even minor structural changes of the residues forming the substrate-binding pocket can have a large impact on enzyme activity. To further investigate residues important for substrate interaction, two additional residues were mutated. The mutation D152A was expected to result in reduced activity because of lost ability to form a stabilizing H bond with the 4′-OH on the B ring of the two acceptors. The mutant N123A was made as this residue is positioned favorably for interaction with the sugar part of the acceptor. These mutants showed loss of activity with the D152A mutation being the most severe (activity <5% of wild type). These results verify the predictive power of homology modeling in identifying residues directly involved in substrate binding and enzymatic activity.
The importance of several residues in the acceptor pocket for enzymatic activity has also been observed in a study of MtUGT71G1 (He et al., 2006). Based on the crystal structure available for this enzyme, a number of single point mutations of residues lining the acceptor pocket were produced. Regiospecific glucosylation of several acceptors by MtUGT71G1 was altered by introduction of the mutation F148V or Y202A. The changes were explained by changed size of the acceptor pocket resulting from introduction of smaller amino acid side chains, allowing for different positioning of the acceptor molecules (He et al., 2006). Change in the size of a single amino acid residue has also been observed to alter acceptor specificity of other plant UGTs (Masada et al., 2007; Cartwright et al., 2008). Eight other point mutations in the acceptor pocket of MtUGT71G1 resulted in activities of 20% to 90% of wild-type protein (He et al., 2006). The successful change of MtUGT71G1 glucosylation pattern by a single point mutation stands in contrast with our attempt to change the sugar acceptor specificity of BpUGT94B1. Broad acceptor specificity of MtUGT71G1 as compared to BpUGT94B1 might explain this difference. The very tight fit of BpUGT94B1 around the acceptor seems to be highly important for activity and at the same time responsible for the narrow acceptor specificity of this enzyme. MtUGT71G1 offers more plasticity with respect to acceptor positioning. At the same time the decreased activity of the eight independent MtUGT71G1 point mutants (He et al., 2006) is in accordance with the observations of the BpUGT94B1 point mutations and support the idea that acceptor specificity is determined by the overall shape and size of the acceptor pocket.
In Vivo Activity
In vivo the activity of BpUGT94B1 is a step in the synthesis pathway of the main anthocyanin in red daisy cyanidin 3-O-2″-O-glucuronosyl-6″-O-malonylglucoside (Fig. 1; Sawada et al., 2005). In vitro regiospecific activity at the 2″-OH position is reported for the two acceptors cyanidin 3-O-6″-O-malonylglucoside and cyanidin 3-O-glucoside (Fig. 1; Sawada et al., 2005), both intermediates in the synthesis pathway of cyanidin 3-O-2″-O-glucuronosyl-6″-O-malonylglucoside. This regiospecificity fits nicely with the model of BpUGT94B1, because suitable positioning of each of the other OH groups of the acceptor for glucuronosylation would require protein backbone rearrangement of the model. Additionally, the manual docking of either the acceptor sugar moiety 6″-OH or the B-ring 4′-OH showed that this demands the acceptor AC ring to be positioned pointing inward into the enzyme instead of outwards in the cleft region between the domains. This would require a larger movement of the domains for substrate binding and release.
The in vitro activity does not reveal whether the glucuronosylating activity of BpUGT94B1 in vivo takes place before or after introduction of malonic acid at the C 6″-OH of the cyanidin 3-O-glucoside. This cannot be deduced from the model either, because both substrates can be fitted into the acceptor pocket (Fig. 4). The site surrounding the acceptor hydroxyl group to be glucuronosylated is the same on both acceptors, but the malonic acid ester residue would take up additional space in the narrow acceptor pocket. Successful docking of the malonylated anthocyanin requires a twist of the malonic acid ester residue toward the aglycone, favored by intramolecular H-bond formation between the malonic acid and the cyanidine A ring (Dangles, 1997). In the absence of malonic acid, the residue N123 is positioned so that it can stabilize the sugar part of the donor. In the presence of the malonic acid ester, a more restricted and tight fit is envisioned to hold the acceptor in place. These observations may explain the activity with both acceptors of the otherwise very substrate-specific BpUGT94B1.
Cyanidin 3-O-2″-O-glucuronosyl-6″-O-malonylglucoside constitutes 85% of total anthocyanins in the petals of red daisy and very low amounts of the putative intermediates in the biosynthetic pathway are present (Sawada et al., 2005). Accordingly, the malonylation and glucuronosylation steps must proceed with high affinity. It has been suggested that the malonyltransferase and BpUGT94B1 are positioned in a metabolic grid where either activity may occur first (Sawada et al., 2005). This is in accordance with the observed favorable docking of both acceptor substrates into the model.
The very narrow in vitro substrate specificity of BpUGT94B1 is a general feature of several other UGTs (Fukuchi-Mizutani et al., 2003; Kramer et al., 2003; Yonekura-Sakakibara et al., 2007). The narrow substrate specificity stands in contrast to the fact that many UGTs show a very broad specificity in vitro (Hansen et al., 2003; Hefner and Stockigt, 2003; Caputi et al., 2008). This highlights the question of how narrow versus broad substrate specificity of UGTs is regulated in vivo. In vivo specificity of UGTs have been suggested to depend on their incorporation into metabolons where binding of the UGT per se could narrow its substrate specificity or serve to restrict access to its active site (Jorgensen et al., 2005; Kristensen et al., 2005; Nielsen et al., 2008).
The conclusion of this study is that a few specific amino acid residues as well as the overall size and shape of the acceptor pocket define substrate specificity. The residues in the BpUGT94B1 model forming the acceptor pocket are mostly situated in the N-terminal domain, the least well conserved domain between UGTs. The presence of several highly variable loops in the N-terminal domain adds to the possible diversity in overall shape and size of the substrate pocket of UGTs. This explains why substrate specificity cannot be assigned based on primary structure alone. It also implies that novel combinations of donor and acceptor specificity might be obtained by combining entire N- and C-terminal domains of different UGTs. In recent studies (Cartwright et al., 2008; Weis et al., 2008) it has been possible to construct such functionally active chimeric UGTs that harbor the N- and C-terminal domains from different plant UGTs.
The importance of glucuronosylation for in planta stability, transport, and storage of bioactive natural products including anthocyanins is not yet understood. Nevertheless, engineering of specific UDP-glucuronosyltransferase activities into plants offers a wide range of interesting applications. Glucuronosylation of anthocyanins may be used to alter the color tonation of flowers, fruits, and vegetables. Increased stability during exposure to high or low pH, light, elevated temperatures, oxygen, and ascorbic acid are desired properties, which may be gained by glucuronosylation and open up new uses for anthocyanins as food colorants. A chimeric UGT able to use delphinidin 3-O-glucoside as sugar acceptor and UDP-GlcUA as sugar donor would offer the possibility to produce a blue plant pigment with improved stability for use in fermented foods like yogurt. Likewise, introduction of glucuronosylated defense compounds into plants by genetic engineering may improve resistance to biotic attacks.
Homology Modeling of UGT94B1
Homology modeling was performed according to guidelines for molecular modeling of GTs (Imberty et al., 2006). The secondary structure of red daisy (Bellis perennis) UGT94B1 was predicted using the softwares Phyre (http://www.sbg.bio.ic.ac.uk/phyre/html/index.html) and JPred (http://www.compbio.dundee.ac.uk/approximately www-jpred/submit.html), and the predictions were compared to the distribution of α-helices and β-strands in the crystal structures of Medicago truncatula UGT73G1 (Shao et al., 2005) and Vitis vinifera VvGT1 (Offen et al., 2006). A structural alignment of UGT94B1 against these two UGT sequences was constructed manually, and SCRs defined (Fig. 2). A model of BpUGT94B1 including all SCRs was built from the structural alignment using the Composer function of the modeling software Sybyl from Tripos. Regions between two SCRs were defined as loops. Loops that could not be modeled from the templates were modeled by screening against nonredundant protein-fold database in Composer (Sybyl modeling software from Tripos). This was the case for the loops 1, 7, 9, 11, and 18 (Fig. 2). Loop 9 (residues 204–208) and the N-terminal 14 amino acids were left out as no satisfactory modeling of these loops could be made. The model was optimized by several cycles of energy minimization using Sybyl force field, and consultation of Ramachandran maps (PROCHECK) to verify backbone conformation and chirality of amino acids. Hydrogen atoms were added and atomic charges derived using the Pullman procedure. Model quality was verified by calculation of PROCHECK g scores (Laskowski et al., 1992) and ProSA Z score (Wiederstein and Sippl, 2007). The Figures 3, 4, and 5 showing the model of BpUGT94B1 were made using the software Pymol (DeLano, 2002).
Insertion of Sugar Donor and Acceptor into the Model
Docking of the sugar donor was performed by homology docking (Imberty et al., 2006), where the substrate is manually inserted guided by substrate position in the crystal structure of a homologous protein. For the sugar donor UDP-GlcUA, this was performed using the coordinates of MtUGT71G1 crystallized in the presence of UDP-Glc. For the sugar acceptor cyanidin 3-O-glucoside, this was performed guided by coordinates of the VvGT1 crystal structure with the sugar acceptor kaempferol (Offen et al., 2006) and the bacterial family 1 GT GtfD, crystallized with the acceptor l-vancosamine (Mulichak et al., 2004). The docking was adjusted manually to optimize the position of the reactive 2″-OH group of the Glc moiety of the acceptor. Energy optimization of the amino acid side chains surrounding donor and acceptor was performed.
Liquid Chromatography-Mass Spectrometry Analyses
Analytical liquid chromatography-mass spectrometry was carried out using an Agilent 1100 Series LC (Agilent Technologies) hyphenated to a HCTplus ion trap mass spectrometer (Bruker Daltonics) and fitted with a Zorbax SB-C18 RRHT column (2.1 × 50 mm, 1.8 μm [Agilent], protected by a Phenomenex Gemini C18 security guard cartridge, flow rate: 0.2 mL min−1). The mobile phases were: A, 2% (v/v) formic acid in water; B, 0.1% (v/v) formic acid in acetonitrile. The gradient program was: 0 to 8 min, linear gradient 5% to 25% (v/v) B; 8 to 9 min, linear gradient 25% to 100% B; 9 to 10.5 min 100% B, followed by equlibration at 5% B for 3.5 min.
Cloning of UGT94B1 and Generation of Mutated Proteins by Site-Directed Mutagenesis
For the cloning of UGT94B1, mRNA was prepared from petals of red daisy using MicropolyA purist kit (Ambion). cDNA was prepared from the isolated mRNA and used as template for PCR with the UGT94B1-specific primers 5′gcatggtaccttaattattcatttcac3′ and 5′gctactcgagatggattcaaaaatcg3′ introducing KpnI and XhoI sites in the 5′ and 3′ ends, respectively. The construct was cloned first into pCR-blunt II-TOPO vector (Invitrogen) and then transferred into pET30a+ vector (Novagen).
Single point mutations were introduced by PCR using UGT94B1 inserted into the PET30a+ vector (KpnI and XhoI sites) as template. PCR was performed using Phusion polymerase and thermocycling parameters were 95°C 1 min, 15 cycles of 95°C 30 s, 55°C 1 min, 68°C 12 min. PCR products were digested with DpnI (1–2 h, 37°C) and transformed into competent Escherichia coli cells. Plasmids were extracted from overnight cultures of transformed colonies and sequenced to verify the correctness of mutated sequences harboring the following changes: R25P, R25S, R25K, R25G, I187A, I187S, N123A, D152A, and P174G. The primers used to introduce these point mutations were: R25P fw: 5′GCATATAGTCACATATCTCCCTTCCTAGTTTTTGCC3′, R25P rew: 5′GGCAAAAACTAGGAAGGGAGATATGTGACTATATGC3′; R25 S fw: 5′GCATATAGTCACATATCTTCCTTCCTAGTTTTTGCC3′, R25 S rew 5′GGCAAAAACTAGGAAGGGAGATATGTGACTATATGC3′; R25G fw: 5′GCATATAGTCACATATCTGGCTTCCTAGTTTTTGCC3′, R25G rev: 5′GGCAAAAACTAGGAAGCCAGATATGTGACTATATGC3′; R25K fw: 5′GCATATAGTCACATATCTAAGTTCCTAGTTTTTGCC3′, R25K rev: 5′GGCAAAAACTAGGAACTTAGATATGTGACTATATGC3′; I187A fw: 5′GGTGGTTCGAAATATGCTGAACGCTTTGTTGATTGC3′, I187A rew: 5′GCAATCAACAAAGCGTTCAGCATATTTCGAACCACC3′; D152A fw: 5′GCGTTATATGCTTTGGCTGCTCATCTCTACAC3′, D152A rew: 5′GTGTAGAGATGAGCAGCCAAAGCATATAACGC3′; I187S fw: 5′GGTGGTTCGAAATATAGTGAACGCTTTGTTGATTGC3′, I187S rev: 5′GCAATCAACAAAGCGTTCACTATATTTCGAACCACC3′; P174G fw: 5′TTCCTGAAATATATGGTAAAAATCGCGATATT3′, P174G rew: 5′AATATCGCGATTTTTACCATATATTTCAGGAA3′; and N123A fw: 5′GGTTATATATGATTTTGCCCAATTGTGGGCCCCA3′, N123A rev: 5′TGGGGCCCACAATTGGGCAAAATCATATATAACC3′.
Expression and Purification of UGT94B1 and Derivatives
PET30a+ plasmids (Novagen) harboring BpUGT94B1 or mutated derivatives were transformed into the E. coli XJa DE30 autolysis strain (Zymo research). This strain has a chromosomally inserted λ lysosyme gene (encoding λ lysosyme/endolysin) inducible by l-Ara. Cultures were inoculated and grown in Luria-Bertani media with kanamycin (overnight, 28°C). Expression of recombinant enzymes was induced by adding isopropylthio-β-galactoside (0.1 mm) and cell lysis prepared by addition of l-Ara (3 mm) to express λ lysosyme. Cells were then grown overnight at 20°C, harvested, and resuspended in buffer (10 mm Tris, 5 mm CaCl2, 1 mm dithiothreitol, protease inhibitor [Roche complete tablets]). Samples were frozen at −80°C for cell membrane disruption to expose the peptidoglycan layer of the cell walls to degradation by λ lysosyme. Samples were thawed, supplemented with DNase (350 μg/mL), and incubated 5 min in a 37°C water bath. Samples were added 0.33 volumes of binding buffer (2 m NaCl, 80 mm Tris-HCl, pH 7.5) and the cleared cell lysate obtained after centrifugation (11,000 rpm, 15 min, 4°C) was incubated with HIS-Select (Sigma) column material (2 h, 4°C) with gentle rotation. Column material was washed three times with 1× binding buffer and His-tagged protein eluted with imidazole (250 mm). Purified enzymes were kept at −20°C in 50% glycerol stock. Quantification of purified enzymes was achieved by running different dilutions of the purified protein samples and standard protein of known concentration (bovine serum albumin) on SDS-PAGE gels. Intensity of the corresponding Coomassie-stained protein bands on the gels were quantified using LabWorks software (UVP BioImaging). All samples were then diluted to obtain 1 mg/mL of purified recombinant enzyme for enzymatic assays.
Enzymatic Activity and Determination of Kinetic Parameters
The activity of UGT94B1 and mutants was determined in assay mixtures (total volume: 20 μL) containing UDP-sugar donor and acceptor as specified for individual experiments, enzyme (2 μg protein), and 100 mm Tris-HCl pH 7.5. After incubation (30°C, 450 rpm, 2–60 min), the reactions were stopped by addition of 1 volume MeOH/6% formic acid. After centrifugation (10 min, 4°C, 10,000g), product formation was analyzed by liquid chromatography-mass spectrometry analysis of supernatants. Sugar acceptor concentration (cyanidin 3-O-glucoside) was 650 μm for all experiments (except determination of cyanidine 3-O-glucoside kinetic parameters). Sugar donor concentrations were as stated in Figures 7, 8, and 9 for determining kinetic parameters and were 2.5 mm for determining relative activity. For all enzymatic assays both donor and acceptor were added to obtain a 5 to 10 times substrate excess during the entire time course of the enzymatic reactions. Calculation of kinetic parameters Km ± sd (mm) and relative Vmax ± sd (calculated as relative product formation min−1 mol−1 also called kcat) were performed using the software R (version 2.6.2), fitting initial velocity data (experimental points) to the Michaelis Menten (V0 = Vmax[S]/Km + [S]) equation by means of nonlinear regression analysis.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Birger Lindberg Møller (blm{at}life.ku.dk).
↵1 This work was supported by the Ministry of Science and Innovation, Program Committee for Foods and Health, the EU–FP6–2005 Life Science Health Programme to the "CAPELLA" project, the Center of Molecular Plant Physiology, the VKR Research Centre Pro-Active Plants, and a Ph.D. stipend from the University of Copenhagen (to S.A.O.).
↵[C] Some figures in this article are displayed in color online but in black and white in the print edition.
↵[OA] Open Access articles can be viewed online without a subscription.
- Received August 20, 2008.
- Accepted September 19, 2008.
- Published October 1, 2008.