cytochrome p450 enzymes 
 ( p450s ) are heme - containing monooxygenases 
 that catalyze a variety of oxidative transformations in primary and 
 secondary metabolism . p450 enzymes are 
 widely distributed in natural product biosynthetic pathways from bacteria , 
 fungi , and plants . these enzymes play a key role in the generation 
 of structural complexity , including hydroxylation by activation of 
 c  h bonds , epoxidation , c  c bond cleavage , and functional group rearrangement / migration . in addition , p450s from secondary metabolism 
 have also been shown to catalyze less common reactions , including 
 recent examples of heterocycle formation and cationic terpene cyclization . an 
 increasing number of oxidative enzymes have been shown to catalyze 
 multistep oxidation at distinct carbon atoms of substrates , resulting 
 in drastic structural transformations ( figure 1 ) . for example , during lovastatin biosynthesis , lova 
 catalyzes consecutive oxidations at opposite sides of the decalin 
 ring to yield the precursor monacolin j , while tami catalyzes successive hydroxylation and epoxidation reactions 
 to afford the bicyclic ketal system in tirandamycin . considering the central roles of such p450s in biosynthetic 
 pathways , as well as the potential applications of these enzymes as 
 c h activation biocatalysts , discovery of novel multifunctional 
 p450s from nature is of both fundamental mechanistic significance 
 and practical importance . examples of natural products of which multifunctional 
 p450s play 
 important roles in the biosynthesis . fumagillin ( 1 ) from aspergillus fumigatus is a meroterpenoid that has numerous biological activities . compound 1 and its derivatives have been intensely studied for their 
 potential use in the treatment of amebiasis , microsporidiosis , rheumatoid arthritis , and for their antiangiogenic properties by the 
 irreversible inhibition of human type 2 methionine aminopeptidase 
 ( metap2 ) . structurally , 1 consists of 
 a highly oxygenated , rearranged sesquiterpene esterified to a polyketide - derived 
 tetraenoic diacid . the terpenoid portion , fumagillol ( 2 ) is derived by cleavage and multiple oxidation of the bicyclic sesquiterpene 
 hydrocarbon intermediate -trans - bergamotene 
 ( 3 ) ( figure 2 ) . the transformation of 3 to 2 involves 
 a dramatic skeletal rearrangement in which the strained cyclobutane 
 bridge is opened to yield the 1,8-bisepoxide - containing cyclohexanediol 
 with six contiguous stereocenters . therefore , given the unusual c c 
 cleavage and the requisite pair of c  h activation reactions 
 starting from 3 , it appeared likely that a p450 might 
 be centrally involved in the biosynthesis of 2 and 1 . this fma cluster 
 contains the first reported membrane 
 bound class i terpene cyclase ( fma - tc ) that catalyzes 
 the formation of 3 from farnesyl pyrophosphate ( fpp ) . the fma cluster also encodes a polyketide synthase 
 ( fma - pks ) that synthesizes a dodecapentaenoate precursor 
 that is trans - esterified to 2 by the acyltransferase 
 fma - at to yield the intermediate prefumagillin 4 . it was proposed that the polyene portion of 4 would be oxidatively cleaved to yield 1 . following 
 our discovery , wiemann et al . reported that the fma gene cluster is embedded within a supercluster on chromosome 8 of a. fumigatus that also encodes the biosynthetic pathways 
 for fumitremorgin and pseurotin . the fma gene cluster ( figure 3a ) contains 
 four oxygenases that are most likely responsible for the oxidative 
 tailoring steps that transform 3 to 1 , including af470 ( antibiotic biosynthesis monoxygenase superfamily 
 monooxygenase ) , af480 ( nonheme iron - dependent dioxygenase ) , af510 ( cytochrome p450 monooxygenase ) and af440 ( flavin - binding monooxygenase / methyltransferase ) . an additional 
 redox enzyme in the fma gene cluster is the partial 
 pks ( af490 ) , in which only the dehydratase ( dh ) and 
 ketoreductase ( kr ) domains are present . the mechanistic role of each 
 of these enzymes , especially that of the p450 ( encoded by af510 ) , which is hypothesized to play a central role in 
 the conversion of 3 to 2 , has remained unknown . here we describe the complete characterization of the biosynthetic 
 pathway of fumagillin ( 1 ) . using a combination of genetic 
 knockout , chemical complementation , heterologous reconstitution in saccharomyces cerevisiae , and in vitro biochemical assays , 
 we have identified the role of each of the required pathway enzymes 
 and biosynthetic intermediates . we show that af510 
 indeed encodes a multifunctional p450 with a spectacular range of 
 catalytic prowess , including hydroxylation of 3 , oxidative 
 cleavage of the bridging cyclobutane ring , and two epoxidation reactions . in addition to the on - pathway intermediates , we also demonstrate the 
 range of off - pathway compounds that can be obtained using p450 starting 
 from 3 . another notable finding includes the functional 
 assignment of af490 as a stereoselective ketoreductase 
 in the biosynthesis of 2 . we first set 
 out to determine the roles and timing of the several 
 redox enzymes in the fma gene cluster . we individually 
 deleted five genes , af510 , af490 , af480 , af470 , and af440 
 in a. fumigatus cea17 akub strain ( pyrg89 , akub ) , which is deficient in nonhomologous 
 end joining ( supporting information ) . in comparison to the isogenic control strain , 
 the metabolic profile of af510 , af480 and af470 showed complete 
 abolishment of the formation of 1 ( figure 3b ) , while af440 retained production 
 of 1 . the af490 strain showed 
 decreased titers of 1 ( 86% decreasing in the culture 
 of 4 days using cya medium ) . the nonessential role of af440 is in accordance with that reported by wiemann et al . , in which af440 ( which corresponds to psof ) however in contrast to the wiemann report , in the analysis of the 
 af470 profile , we found the accumulation of 4 with m / z 471 [ m+na ] ( figure 3b ) , therefore suggesting 
 a role for af470 in the oxidative cleavage of the 
 terminal alkene of the dodecapentaenoate side chain into the carboxylic 
 acid present in 1 . on the other hand , deletion of af480 led to the accumulation of a polyene - containing compound 5 , with maximum uv absorbance at 333 nm and m / z 451 [ m+na ] ( figure 3b ) . compound 5 was isolated from a 6-day liquid 
 culture of a. fumigatus af480 mutant strain and structurally characterized . on the basis of 
 the h , c and 2d nmr analyses , 5 was elucidated as 6-demethoxyfumagillin , in contrast to an aldehyde 
 shunt product reported by wiemann et al . the structure of 5 revealed that the nonheme iron - dependent 
 oxygenase encoded by af480 catalyzes hydroxylation 
 of c6 in the biosynthesis of 1 . ( b ) hplc analysis of metabolites extracted 
 from isogenic control and af510 , af480 , and af470 strain showing loss of 1 and accumulation of 5 from af480 and 4 from af470 strain , 
 respectively . ( c ) gc - fid analysis of the af510 strain showing the accumulation of 3 . the only remaining unassigned oxygenase in the 
 gene cluster was 
 therefore the p450 gene , af510 , which shows the strongest 
 sequence homology to the multifunctional p450 orda in aflatoxin biosynthesis ( 47% protein identity ) . wiemann 
 et al . reported that deletion of af510 in a. fumigatus led to no detectable accumulation of any intermediates . 
 while the same phenotype was observed here using lc ms assay 
 conditions , a more careful extraction with hexane and analysis of 
 the hydrocarbon extract by gc - fid revealed the presence of -trans - bergamotene ( 3 ) ( figure 3c ) . in the isogenic control , this result suggested that the initial oxidation of 3 is catalyzed by the enzyme encoded by af510 . the enzyme encoded by af470 ( fma - abm ) was initially revealed to harbor a duf4188 
 conserved domain of unknown function by ncbi conserved domain search . 
 a further homology modeling search with phyre2 showed , however , that fma - abm appears to be related to 
 the cofactor - independent abm superfamily monoxygenases with a ferrodoxin - like 
 fold . to further test the role of fma - abm , 
 we cloned the intron - less af470 ( for reconstruction 
 of the gene from mrna , see supporting information ) into a yeast 2 m expression vector which was transformed 
 into s. cerevisiae strain bj5464-npga . upon supplementing 4 to the yeast culture expressing af470 , on the basis of protein structure prediction 
 ( supporting information , figure s7a ) , fma - abm was predicted to be a membrane - bound protein . upon 
 purification of the microsomal fractions of the yeast strain expressing 
 fma - abm and incubation with 4 , the same 
 conversion to 1 these results confirmed the role of fma - abm in the oxidative 
 cleavage of 4 to 1 . verification of the function 
 of fma - abm , fma - c6h , and fma - mt . ( a ) 
 lc  ms detection of 1 upon expression of fma - abm 
 in s. cerevisiae bj5464-npga with supplement of 4 ( b ) lc  ms analysis of in vitro assays of yeast 
 microsomes containing fma - abm toward 4 . ( c ) lc  ms 
 analysis of in vitro assays of fma - c6h with 6 ; and combined 
 fma - c6h and fma - mt with 6 , respectively . to confirm the role of the enzyme 
 encoded by af480 ( fma - c6h ) , the 
 6xhis - tagged enzyme was solubly 
 expressed from e. coli bl21 ( de3 ) and purified to 
 homogeneity ( supporting information , figure 
 s5 ) . bioinformatics analysis showed that fma - c6h shares a similar 
 sequence with the members of the phytanoyl - coa dioxygenase ( phyh ) 
 superfamily , thereby requiring fe ( ii ) and -ketoglutarate ( -kg ) 
 for its activity . incubation of recombinant fma - c6h 
 with -ketoglutarate , sodium ascorbate , and substrate 5 , however , did not lead to the formation of any hydroxylated 
 product . we therefore reasoned that fma - c6h may function 
 upstream in the pathway prior to attachment of the polyene portion . to obtain such a substrate , the hydrolysis of 5 under 
 basic condition was performed to afford 6-demethoxyfumagillol ( 6 ) ( supporting information ) . when 
 recombinant fma - c6h and 6 were incubated 
 under the same condition as above , complete conversion of 6 to 6-hydroxylfumagillol ( 7 ) was obtained ( figure 4c ) . furthermore , coincubation of fma - c6h with the recombinant methyltransferase ( fma - mt ) encoded by af390400 in the presence 
 of the necessary cofactors ( -ketoglutarate , sodium ascorbate , 
 and sam ) and 6 resulted in the formation of the expected 2 ( figure 4c ) . collectively , these 
 studies confirmed the function of af480 and revealed 
 that 6 ( or a related compound ) may be a key intermediate 
 in the biosynthetic pathway of 1 . to test this hypothesis , 
 feeding of 6 to a. fumigatus blocked 
 in bergamotene formation ( deletion of fma - tc , af520 ) restored biosynthesis of 1 as well as 
 the production of 5 ( see compilation of chemical complementation 
 traces in figure 7 ) . with this information 
 in hand , we then turned to investigation of the possible routes of 
 oxidative modification of 3 into 6 . the transformation of 3 into 6 requires at least three oxidation steps 
 to introduce the one c5 hydroxyl group and the two epoxide functionalities . in addition , the cleavage of the c  c bond between c5 and c8 
 must also take place . several proposals for this mechanistically unresolved 
 transformation have been advanced , including desaturation of the c5c6 
 bond followed by concomitant cleavage of the c5c8 bond . in light of the functional assignment of af440 , af470 , and af480 
 described above , it appeared likely that the p450 encoded by af510 ( fma - p450 ) is a multifunctional p450 that is responsible 
 for most , if not all , of the oxidative steps between 3 and an advanced intermediate such as 6 . we previously 
 showed that expression of fma - tc alone in s. cerevisiae can lead to accumulation of -trans - bergamotene 
 ( 3 ) . to investigate the 
 activity of fma - p450 toward 3 , we cloned 
 the intron - less af510 into a yeast 2 m expression 
 vector for microsomal expression . to equip fma - p450 
 with the optimal redox partner , we also cloned the a. fumigatus cytochrome p450 oxidoreductase ( afcpr ) for coexpression . all three genes ( encoding fma - tc , fma - p450 , and afcpr ) were placed under the adh2 promoter and transformed into bj5464-npga . after four 
 days of culturing followed by extraction with hexanes / etoac ( 1:1 ) , 
 we observed a series of sesquiterpene compounds ( 6 , 819 ) that are derived from 3 ( figure5a ) . of these compounds , 10 , 11 , 14 , and 17 were the 
 major products ( 0.61.1 mg / l ) ; 8 and 12 were minor products ( 0.30.5 mg / l ) ; and the remaining ( 6 , 9 , 13 , 15 , 16 , 18 , and 19 ) were present at 
 less amounts ( < 0.3 mg / l ) . the same set of metabolites was also 
 observed when 3 was directly supplied to the yeast culture 
 expressing only fma - p450 and afcpr ( figure 5b ) . in the absence of 3 or fma - p450 , to elucidate the structures of these products , large scale 
 ( 12 l ) fermentation of the triply transformed yeast strain was performed , 
 followed by isolation and characterization of each compound ( figure 5d and supporting information ) . compounds 6 and 9 were identified as 6-demethoxyfumagillol and cordycol , respectively , 
 based on ms and nmr comparison to standards . 9 is the 
 monoepoxide relative of 6 and may therefore be an immediate 
 precursor of 6 . formation of these compounds does indicate 
 that fma - p450 alone is sufficient to transform 3 into 6 . however , both compounds are found in 
 very minute quantities ( figure 5a ) . surprisingly , 
 the 5-epimeric forms of 6 and 9 , which are 
 5-epi - demethoxyfumagillol ( 11 ) and 5-epi - cordycol ( 10 ) , respectively , were present 
 as major products . the opposite stereochemistry of the c5 hydroxyl 
 groups was readily established through the coupling patterns of h-5 
 in h nmr ( 6 and 9 : br.s ; 10 and 11 : br.t , j = 9.4 and 
 10.5 hz , respectively ) . to identify which of these compounds are on - pathway 
 intermediates in the biosynthesis of 1 , each compound 
 was supplied to the af520 blocked mutant at 
 40 g / ml . as expected , only 6 and 9 restored production of 1 , even though each 
 strain also produced the fumagillin analogues 5 and 20 , respectively ( figure 7a and supporting information , figure s11 ) , through 
 the direct esterification with the polyketide side chain ( more on 
 this below ) . in contrast , neither 10 nor 11 restored biosynthesis of 1 ( figure 7a and supporting information , figure 
 s11 ) , thereby confirming that they are shunt products in s. 
 cerevisiae , and no further epimerization of the c5 hydroxyl 
 can take place in a. fumigatus . this result also 
 provides insight into the intrinsic stereoselectivity of the fma - at which prefers the natural r - oh group 
 at c5 . ( a ) lc  ms metabolic profiles of coexpression 
 of fma - tc ( encoded by af520 ) , fma - p450 ( encoded by af510 ) , and afcpr . compounds 13 , 15 , 16 , 18 , and 19 are present at trace quantities and are not shown . ( b ) 
 lc  ms analysis of coexpression of fma - p450 
 and afcpr in s. cerevisiae bj5464-npga with supplement 
 of 3 ; ( c ) same as trace b but with supplement of 8 . compound 8 contains 
 one additional oxygen atom compared to 3 , and was found 
 to be 5r - hydroxyl--trans - bergamotene 
 ( supporting information , table s5/figures 
 s21s26 ) . 8 is therefore the product of direct 
 oxidation of c5 of 3 by fma - p450 . nmr characterization revealed the structures of 14 and 15 to be 5,9-dihydroxyl--trans - bergamotene and 5,10-dihydroxyl - trans - bergamotene , 
 respectively ( supporting information , tables 
 s11s12/figures s48s57 ) . all three compounds retained 
 the trans - bergamotene scaffold found in 3 , and therefore represent possible early intermediates in the pathway 
 prior to the c5c8 bond cleavage step . interestingly , only 
 the feeding of 8 to the af520 
 blocked mutant restored production of 1 , while addition 
 of either 14 or 15 failed to do so ( figure 7a and supporting information , figure s11 ) . therefore 14 and 15 are 
 shunt products of fma - p450 activity in yeast . in 
 particular , the high levels of 14 in the yeast culture 
 extract hints that c9 is the site of the second p450 oxidation and 
 may be subject to rapid hydroxylation ( see discussion below ) . to prove the multifunctional nature of fma - p450 no restoration of 1 was observed , as would be expected for any additional role 
 of fma - p450 in the pathway . finally , addition of 
 purified 8 to the yeast culture expressing fma - p450 and 
 afcpr similarly led to the formation of more oxidized products such 
 as 11 and 14 ( figure 5c ) . among the 
 products isolated , 5-keto - isocordycol ( 12 ) contains a ketone functionality at c5 and an allylic alcohol resulting 
 from prototopic ring - opening / rearrangement of the c8c9 epoxide 
 ( supporting information , table s9/figure 
 s40s41 ) . although this compound is only present in minor quantities , 
 the unexpected cyclohexanone shunt product suggests that the c5 hydroxyl 
 ( in 8 , for example ) may serve as the source of electrons 
 in the oxidative cleavage of the cyclobutane ring , thereby transforming 
 the bicyclic trans - bergamotene scaffold into an 2,5,7-trisubstituted 
 cyclohexanone . the corresponding c5-reduced derivative , epi - isocordycol ( 13 ) , was also found in the extract with 
 the 5s - hydroxyl ( supporting information , table s10/figures s42s47 ) . the other four compounds 1619 were all found to be structurally 
 related , but clearly off - pathway products . compound 16 is particularly interesting with its trisubstituted sesquifenchyl 
 core ( supporting information , table s13/figures 
 s58s62 ) . the 5-hydroxy--cis - bergamotene 
 ( 17 ) , 5,10-dihydroxyl--cis - bergamotene 
 ( 18 ) , and 19 all have the unexpected -cis - bergamotene scaffold and are presumably formed by isomerization 
 of reactive intermediates derived from 8 ( supporting information , tables s14s16/figures 
 s63s80 ) . 17 is a major product from the yeast 
 expression of fma - p450 , while 18 and 19 are further oxidized products of 17 . the feeding 
 of 17 to the af520 blocked mutant 
 did not restore production of 1 ( figure 7a ) . possible mechanisms of formation of 1619 are shown in figure 10 and will 
 be discussed below . the array of products recovered 
 from s. cerevisiae clearly showcased the multifunctional 
 capability of fma - p450 ( hydroxylation , epoxidation , 
 rearrangement ) in the oxidation of -trans - bergamotene 
 ( 3 ) . the large number of shunt products , however , may 
 be a result of the weak expression of fungal membrane - bound p450 in 
 yeast . as a result of the low concentration of the p450 , reactive 
 intermediates may be readily intercepted or hydrolyzed to yield off 
 pathway products such as 14 and 17 . therefore , 
 to analyze the function of fma - p450 under more controlled conditions , 
 we prepared microsomal fractions from the yeast strain that overexpressed 
 fma - p450 and afcpr for in vitro assay . ms 
 analysis of in vitro assays of yeast microsomes containing af510 and afcpr toward ( a ) 3 and ( b ) 9 . when 3 was incubated with 10 mg / ml of microsomal 
 fractions 
 and nadph overnight , we detected 11 as essentially the 
 single product in the extract ( figure 6a ) . other major compounds observed in vivo , such as 10 , 14 , and 17 , etc . are only present at very low 
 levels when searched for using selective ion monitoring . similarly , 
 when the assay is repeated using 8 as the substrate , 
 near complete conversion of 8 to 11 was 
 observed with nearly no side products ( supporting 
 information , figure s8 ) . to analyze one of the individual steps 
 catalyzed by the multifunctional p450 , we assayed the epoxidation 
 of 9 to 6 using the same microsomal extract . as shown in figure 6b , we could observe incomplete 
 conversion of 9 to 6 only in the presence 
 of fma - p450 , thereby confirming the ability of this enzyme to catalyze 
 the specific formation of epoxide . collectively , these in vivo 
 and vitro results confirmed the multifunctional 
 role of fma - p450 , including c5 hydroxylation , coupled 
 or sequential 4e - oxidative cleavage , and rearrangement 
 to yield epoxycyclohexanone , and the tandem epoxidation . at the same 
 time , it is also evident that fma - p450 alone is insufficient 
 to generate the correct stereoisomer of 5r - demethoxyfumagillol 6 , instead the 5s diastereomer 11 being formed in yeast or using yeast microsomes . therefore , given 
 that a ketone intermediate 12 could be isolated , the 
 transformation of 3 most likely involves a c5 ketone 
 intermediate that is stereoselectively reduced into the 5r isomer . several ketoreductases ( krs ) such as the 3-kr in sterol 
 biosynthesis 
 and 3-kr for long - chain fatty acid synthase have been reported in s. cerevisiae , hence it is 
 possible that an endogenous yeast kr could catalyze the reduction 
 of the c-5 ketone to form the 5s - hydroxy moiety observed 
 in compounds 10 , 11 , and 13 . to test this hypothesis , we prepared both soluble and microsomal 
 extracts of the untransformed host bj5464-npga . we first tested if 
 the isolated ketone 12 could be selectively reduced to 13 . as shown in supporting information , figure s9 , complete reduction of 12 to 13 was observed . the r - isomer of 13 may 
 be present at a very low concentrations as suggested by ion - monitoring , 
 but the amount was insufficient for purification and characterization . 
 to further test whether this endogenous yeast kr activity is responsible 
 for formation of the other 5s - containing products 
 shown in figure 5d , we chemically prepared 
 the three ketone substrates , 5-keto - cordycol ( 21 ) , 5-keto - demethoxyfumagillol ( 22 ) , and 5-keto - fumagillol ( 23 ) ( figure 7d , spectroscopic data in supporting 
 information , tables s17s19 and figures s81s86 ) . these compounds were synthesized from 10 , 11 , and 2 by pcc oxidation , respectively . when either 21 or 22 was added to the control yeast extract 
 ( soluble or microsomal ) , we observed the corresponding ketoreduction 
 to 10 or 11 ( supporting 
 information , figure s10 ) , respectively , thereby validating 
 the involvement of endogenous yeast krs in the formation of compounds 
 with the 5s hydroxyl functional group . compounds are supplemented at 40 
 g / ml to ( a ) af520 ; ( b ) af510 ; ( c ) af480 . ( d ) structures 
 of 2123 . with the ketones 2123 in 
 hand , 
 we performed chemical complementation experiments by feeding each 
 of these compounds individually to the af520-blocked 
 mutant . in each case , complete restoration of biosynthesis of 1 was observed ( figure 7a ) . therefore , the c5-ketone moiety in each of the 
 compounds can be processed correctly . furthermore , chemical complementation 
 of af510 mutant by 22 restored 
 fumagillin production , indicating that fma - p450 is 
 not involved in the biosynthetic pathway beyond 22 and 
 is not essential for reduction of the c5 ketone . lastly , complementation 
 of 23 to the af480 mutant efficiently 
 restored fumagillin production , hinting that reduction of c5 may occur 
 as the last step in the formation of 2 . we hypothesized 
 that a kr encoded in the fma gene cluster might 
 be responsible for the stereospecific 5r reduction 
 of the ketone during the biosynthesis of 1 . although 
 no standalone kr is found in the fma cluster shown in figure 3a , af490 encodes a partial pks 
 in which only the dh - kr domains are present . upon initial discovery 
 of the fma cluster , the pseudo / partial pks was assumed 
 to be an inactive enzyme that had no likely role in the biosynthetic 
 pathway . although deletion of af490 did not completely 
 abolish the production of 1 , we did notice an 86% decrease 
 in the titer of fumagillin ( 1 ) ( figure 3b ) . the incomplete abolishment of the biosynthesis of 1 may be due to the presence of endogenous krs in a. fumigatus that have overlapping functions to that encoded 
 by the pathway enzyme af490 ( fma - kr ) . to investigate the role of fma - kr , the 110 
 kda protein was therefore expressed as a 6xhis tag fusion and purified 
 from s. cerevisiae ( supporting 
 information , figure s6 ) . upon individual incubation of each 
 of the ketone derivatives 21 , 22 , or 23 with recombinant fma - kr , we observed complete 
 conversion to the corresponding 5r - hydroxy - containing 
 compounds , 9 , 6 , or 2 , respectively 
 ( figure 8a  c ) . lastly , to examine 
 the product profile of fma - p450 in the presence of 
 fma - kr , we repeated the biotransformation of 3 in s. cerevisiae by coexpression of fma - p450 , fma - kr , and afcpr . as shown in figure 8d , the product profile is altered considerably to 
 reflect the presence of the dedicated c5 reductase . the 5r - hydroxy isomers 6 and 9 are now clearly 
 major products of the assay , while the 5s - hydroxy 
 diastereomers 10 and 11 have essentially 
 disappeared . the same results were obtained in the in vitro assay 
 when fma - p450-containing yeast microsomes were coincubated 
 with purified fma - kr . whereas 11 was 
 formed as the single product in the previous assay shown in figure 6a , inclusion fma - kr led to the 
 production of the natural diastereomer 6 ( figure 8e ) . ( a  c ) lc  ms analysis of in vitro assays of fma - kr with 2123 . ( d ) lc  ms 
 analysis of coexpression of fma - kr , fma - p450 , and afcpr in s. cerevisiae bj5464-npga with 
 supplement of 3 . ( e ) lc  ms analysis of in vitro 
 assay of fma - kr , and yeast microsomes containing af510 and afcpr plus 8 . on the 
 basis of the new enzymes characterized and the new compounds isolated 
 and identified in this work , we can now deduce a complete biosynthetic 
 pathway for fumagillin ( 1 ) , as shown in figure 9 . the pathway begins with the conversion of fpp 
 to -trans - bergamotene ( 3 ) by 
 a membrane - bound fma - tc . the initial 
 oxidation of 3 by fma - p450 involves c  h hydroxylation 
 at the bridgehead c5 position to yield 8 . subsequently , 
 a four electron oxidation initiated at c-9 coupled to cleavage of 
 the cyclobutane c5c8 bond of the bicyclo[3.1.1 ] core yields 
 the on - pathway intermediate epoxyketone 21 . an additional 
 epoxidation reaction also catalyzed by fma - p450 then furnishes the 
 characteristic bisepoxide ketone 22 . a possible mechanism 
 for the fma - p450-catalyzed conversion of 3 to 22 the diepoxyketone 22 is then subjected 
 to successive c-6 hydroxylation and o - methylation 
 by fma - c6h and fma - mt , respectively , to yield 23 , which is then stereoselectively reduced by fma - kr to 5r - hydroxy - seco - sesquiterpene 2 . acylation catalyzed by the fma - at with the dodecapentaenoyl group on fma - pks to yield 4 has been previously demonstrated . finally , oxidative cleavage of 4 by the oxygenase ( fma - abm ) 
 encoded by af470 arrives at 1 . we have also shown that fma - kr can stereospecifically 
 reduce each of the three potential ketone intermediates 2123 into the corresponding 5r - hydroxyl compounds , thereby implying that this ketoreduction step 
 may occur at different stages during the biosynthesis of 2 . in the proposed pathway , however , we have assigned the enzyme to 
 catalyze reduction of the most advanced intermediate 23 . we have shown that compounds 6 and 9 , 
 which can be produced by reduction of 22 and 21 , respectively , can each be acylated efficiently by fma - at to yield the analogues 5 and 20 . for 
 example , feeding 6 to the af520 
 strain resulted in the production of both 5 and 1 ( figure 7a ) , with a relative ratio 
 around 4:1 . this indicated that c-6 hydroxylation by fma - c6h may be slower than the c-5 acylation catalyzed by fma - at . the 
 formation of 5 and 20 also demonstrates 
 that fma - at has significant tolerance toward the 
 substitution pattern of the terpene substrate , and can intercept these 5r - reduced products in vivo . in contrast , we have established 
 that fma - c6h is highly specific toward the unacylated 
 intermediate 6 . therefore , 5 and 20 can not be oxidized to 1 . however , no trace of 5 or 20 is recovered from a. fumigatus , thereby suggesting that neither 6 nor 9 is an intermediate in vivo . hence , we proposed that reduction of 
 the c5 ketone takes place after the 6-methoxyl function has been installed 
 in 23 , thereby preventing premature c5 acylation of the 
 terpenoid by fma - at . in 
 this study , we have identified all the enzymes in the fumagillin 
 pathway that transform the bicyclo[3.1.1 ] sesquiterpene 3 into the richly decorated pharmacophore 2 , which acts 
 by binding to type 1 and 2 metap . the 
 highly oxidized cyclohexane portion of 2 , which is also 
 present in the metabolite ovalicin from pseudeurotium 
 ovalis , has been the 
 subject of intensive synthetic efforts ( see review in yamagushi et 
 al . and references therein ) . considering 
 the number of oxidative steps required to modify 3 into 2 , it is remarkable that only two oxygenases are responsible . 
 most impressively , fma - p450 encoded by af510 alone 
 catalyzes the eight electron oxidation of 3 into the 
 intermediate 22 ( figure 9 ) . the 
 same set of reactions must therefore also be involved in the conversion 
 of 3 into ovalicin , which is structurally identical to 23 except for the additional hydroxylation at c7 ( figure 8) . the biosynthesis of ovalicin has previously been 
 studied using labeled samples of mevalonate and of 3 . from these investigations , several mechanisms and intermediates were ruled out , including the involvement 8,9-didehydrobergamotene 
 that had been suggested by birch ( figure 9 ) . the pathway deduced from our genetic and biochemical 
 experiments is fully consistent with these earlier findings . the multifunctional fma - p450 is the central player 
 in the consecutive series of chemically and mechanistically distinct 
 catalytic processes . this enzyme initiates the oxidative transformation 
 on 3 by performing c-5 hydroxylation , followed by oxidative 
 ring - opening coupled to epoxidation and a second tandem epoxidation 
 to generate the key diepoxyketone intermediate 22 that 
 serves as the substrate for later hydroxylation , methylation , ketoreduction , 
 and acylation catalyzed by other pathway enzymes from the fma cluster . when fma - p450 was reconstituted in s. cerevesaie , we observed numerous oxidized products derived 
 from the bioconversion of 3 . on the basis of the structures 
 of these compounds , we can propose a detailed mechanism for fma - p450 . either radical or cationic rearrangement mechanisms 
 can be proposed for this enzyme and are shown in figure 10 and supporting information , figure s12 . although most of the p450-catalyzed c  h bond 
 activation reaction has been shown to involve caged radical intermediates 
 rather than carbocations , several recently discovered p450-catalyzed reactions almost certainly 
 proceed through carbocationic mechanisms . it 
 is possible that the various products produced by af510 result from a partitioning between radical pair and cationic 
 intermediates . the first c  o bond installation occurs 
 by c  h bond 
 cleavage and hydroxylation at the c-5 of hydrocarbon 3 to form 5-hydroxybergamotene 8 . next , the hydrogen 
 atom at c-9 is abstracted by the key ferryl - oxo intermediate ( fe = o , porphyrin  cation radical ) to generate 
 the hydroxycyclobutylcarbinyl radical , a. this reactive 
 intermediate can either undergo an oxygen rebound to generate 14 or be converted to a hydroxycyclobutylcarbinyl cation ( b ) after one - electron oxidation by the oxidative ion ( iv)-hydroxo 
 intermediate . both reactions result in 
 a ferric heme center and resume the catalytic cycle of fma - p450 in 
 the presence of afcpr . carbocation b can be subjected 
 to attack by the  supernucleophile  iron(iii)-peroxo 
 intermediate generated from the following p450 cycle . the later reaction produces intermediate c and induces the subsequent ring - opening rearrangement through c5c8 
 bond cleavage to form the 8,9-epoxide 21 ( figure 10 ) , followed by a 1,2-epoxidation to generate 22 . nucleophilic addition from water or ferric hydroxo ( fe  oh ) complex to b also generates 14 . this shunt pathway can readily take place , as indicated 
 by the high level of 14 observed in yeast ( figure 5a ) . formation of the shunt -cis - bergamotene derivatives 1719 can 
 result from ring - opening / closure of the c-9 radical intermediate ( d ) to generate the isomeric radical ( or cation ) cis-5-hydroxybergamotene intermediate . compounds 1719 can then readily be obtained through direct nucleophilic 
 attack of water or intervening 1,2-hydride shift . lastly , if fma - p450 
 epoxidizes the 1,2-exomethylene double bond of 3 prior 
 to c-5 hydroxylation , the resulting epoxide intermediate could undergo 
 well precedented cationic rearrangement of its bicyclo[3.1.1 ] skeleton 
 to form the tetra - substituted sesquifenchol derivative 16 . overall , the oxidative cleavage of 8 to 21 is particularly intriguing , and would 
 parallel the proposed conversion of loganin to secologanin , as well as that involved in the formation of 
 furanocoumarin . proposed mechanism of 
 fma - p450 . radical mechanism from a to 21 was shown in supporting information , 
 figure s12 . the af490 ( fma - kr ) gene has previously been annotated 
 as a pks - like enzyme that harbors conserved motifs characteristic 
 of the pks - dh ( smart00826 ) superfamily and pks - kr ( smart00822 ) domains 
 as revealed by a ncbi conserved domain - search . the discovery that 
 the truncated pks gene af490 with only dh - kr domains 
 encodes a functional enzyme that catalyzes stereospecific 5-ketoreduction 
 of cyclohexanone 23 to 2 is intriguing given 
 that pks kr domain typically acts on -ketoacyl thioester substrates 
 attached on the acyl carrier proteins . interestingly , a blast search 
 for similar fma - kr - like enzymes indicates that in a. fumigatus the hybrid pks / nrps ( psoa ) in pseurotin biosynthesis ( and the corresponding orthologues 
 in a. clavatus and m. anisopliae ) are the closest match with up to 56% 
 protein identity ( 65% nucleotide identity ) within the kr domain region . hence , it appears that the fma - kr may be an evolutionary 
 product originating from classic gene duplication , divergence , and 
 differential loss , but with the mechanisms acting on the functional 
 domain level rather than on the entire gene . although both a. clavatus and m. anisopliae have a close 
 orthologue of psoa , the fma - kr gene 
 appears to be absent , suggesting the duplication has only occurred 
 in a. fumigatus . curiously , the pseurotin biosynthetic 
 gene cluster is intertwined with the fma gene cluster and was shown recently to be coregulated 
 by a single transcriptional factor ( fapr ) embedded in this fma - pso supercluster . the possibility 
 that af490 originated from a duplicated partial psoa gene but neofunctionalized to participate in the fma pathway further adds to the complex evolutionary history 
 of this supercluster . even more interesting , although we verified 
 the role of fma - kr in vitro and in s. cerevisiae , deletion of this gene in a. fumigatus did not lead 
 to complete abolition of fumagillin ( 1 ) biosynthesis . it remains highly possible that the activity of the kr domain of psoa 
 can partially complement the deletion of af490 , therefore 
 maintaining the ketoreductase activity , albeit at lower efficiency 
 as indicated by the lowered titer of 1 in the af490 strain . alternatively , other endogenous krs in a. fumigatus may also possess this activity and catalyze 
 the requisite reduction . fma - abm encoded by af470 has been shown to be 
 the oxygenase responsible for the c10-c11 cleavage of the dodecapentaenoate of 4 and further 
 oxidation of the aldehyde intermediate to the decatetraenedioic ester 1 . oxidative cleavage of olefins by a single oxygenase has 
 been implicated in the biosynthesis of carotenoids , as well as in the formation of microbial secondary metabolites 
 such as rifamycin . the carotenoid oxygenases 
 are nonheme iron - dependent dioxygenases , while the cleavaging enzyme 
 in the rifamycin pathway is a p450 monooxygenase . interestingly , fma - abm 
 belongs to a potentially new class of oxygenases that catalyze such 
 reactions . it contains a duf4188 conserved domain of still unknown 
 function but homology modeling using phyre2 suggests that the main 
 portion of fma - abm is structurally related to aldoxime dehydratase 
 ( supporting information , figure s7b ) which 
 is a heme - containing enzyme from bacillus sp . , as well as the cofactor - free abm superfamily 
 of monooxygenases , which is typified by actva - orf6 in actinorhodin 
 biosynthesis and snoab in nogalonic acid biosynthesis . it is uncertain whether fma - abm requires a heme 
 as a prosthetic group as it is much smaller than aldoxime hydratase 
 ( 275 versus 373 amino acids ) . further biochemical investigation of 
 fma - abm enzyme is warranted as a blast search reveals that fma - abm 
 homologues are ubiquitous in fungi . we 
 have uncovered the roles of three oxygenases , fma - p450 , fma - c6h , and fma - abm , as well as an o - methyltransferase , fma - mt , involved in the biosynthesis 
 of fumagillin ( 1 ) . among these enzymes , fma - p450 , a p450 monooxygenase , catalyzes a multistep transformation 
 that includes a simple hydroxylation followed by ring - opening of the 
 bicyclic substrate coupled to generation of an epoxide , followed by 
 a second epoxidation that results in the conversion of -trans - bergamotene ( 3 ) to a highly oxygenated 
 structure , 5-keto - demethoxyfumagillol ( 22 ) . in the course of these studies we identified nine compounds as 
 off - pathway products that shed light on the mechanism of action and 
 catalytic potential of fma - p450 . the catalytic versatility 
 of fma - p450 therefore significantly augments the already impressive 
 chemical virtuosity of this important class of enzymes . we have 
 also characterized the function of fma - kr as the 
 ketoreductase that controls the configuration at c-5 of 
 hydroxylated intermediates that then undergo further acylation catalyzed 
 by fma - at . this study has elucidated all the tailoring reactions in 
 the biosynthesis of 1 and will provide opportunities 
 for derivatization of 1 using enzymatic approaches . a. fumigatus strain used in this study was a. fumigatus cea17 akub strain 
 ( pyrg89 , akub ) , which is deficient 
 in nonhomologous end joining and was 
 maintained on czapek - dox agar or glucose minimal agar ( gmm ) . e. coli top10 ( invitrogen ) and xl1-blue ( stratagene ) 
 were used for dna manipulation , and bl21 ( de3 ) was used for protein 
 expression . saccharomyces cerevisiae strain bj5464-npga 
 ( mat ura352 his3- 200 leu2- 1 
 trp1 pep4::his3prb1 1.6r can1 gal ) was used as the 
 yeast expression host . polymerase chain 
 reactions were performed using phusion dna polymerase ( new england 
 biolabs ) or platinum pfx dna polymerase ( invitrogen ) . dna restriction 
 enzymes were used as recommended by the manufacturer ( new england 
 biolabs ) . rna extraction was performed using a ribopure yeast kit 
 ( ambion ) , and improm - ii reverse transcription system for rt - pcr ( invitrogen ) 
 was used to synthesize complementary dna ( cdna ) from total rna . pcr 
 products were subcloned to a pcr - blunt vector ( invitrogen ) and confirmed 
 by dna sequencing . primers used to amplify the genes were synthesized 
 by integrated dna technologies and are listed in supporting information , table s2 . in vivo yeast recombination 
 cloning was performed by transforming the s. cerevisiae bj5464-npga with dna fragments with > 35 bp overlaps and includes 
 a 2 m plasmid backbone ( derived from yeplac195 or yeplac112 ) 
 using an s.c . easycomp transformation kit ( invitrogen ) . for production of 1 and 
 other metabolites , a. fumigatus and mutant strains 
 were cultured in cya medium ( total rna for rt - pcr was extracted from a. 
 fumigatus grown on czapek - dox liquid medium with 5 g / l yeast 
 extract ( cyb ) after 4 days of cultivation . for chemical complementation 
 of af520 , af510 , and 
 af480 mutants , purified compounds are supplemented 
 to the cya medium at 0.2 mg / ml individually . lc  ms analyses 
 of conversion of 7 to 8 by af480 and 7 to 2 by af480 
 and af390400 were performed on prevail 3 
 m , 2.1  100 mm c18 reversed - phase column 
 ( alltech ) and separated on a 595% ( v / v ) ch3cn linear 
 gradient in h2o supplemented with 0.05% ( v / v ) formic acid 
 at a flow rate of 125 l / min . all lc  ms analyses except 
 the aforementioned were performed on a shimadzu 2010 ev lc ms 
 ( phenomenex luna , 5 m , 2.0  100 mm , c18 column ) 
 using positive and negative mode electrospray ionization with a linear 
 gradient of 595% mecn - h2o in 30 min followed by 
 95% mecn for 15 min with a flow rate of 0.1 ml / min . h , c , and 2d nmr spectra were obtained on bruker av500 spectrometer 
 with a 5 mm dual cryoprobe at the ucla molecular instrumentation center . af390 and af400 were obtained as one transcript by rt - pcr verifying that af400 
 was misannotated ( for revised annotation , see supporting information ) . the dna fragemnt of af390400 and af480 from cdna were inserted 
 into pet21 ( novagen ) digested with ndei and xhoi to yield pkw20174 and pkw20172 , respectively . primers 
 used for the amplification and cloning are listed in supporting information , table s2 . recombinant enzymes were 
 expressed with c - terminal his6-tagged in e. coli bl21 ( de3 ) and purified by nickel affinity chromatography . the cells 
 were cultured at 37 c , 250 rpm in 500 ml of lb medium with 35 
 g / ml carbenicillin . isopropylthio--d - galactoside 
 ( iptg , 0.1 mm ) to induce protein expression was added at od600 between 0.4 to 0.6 and the cells were further cultured for 1216 
 h at 16 c . the cells were then harvested by centrifugation ( 3500 
 rpm , 15 min , 4 c ) , resuspended in 25 ml of lysis buffer 
 ( 100 mm tris - hcl , ph 7.4 , 0.1 m nacl , 20 mm imidazole ) , followed by 
 lysed by sonication on ice . cell debris was removed by centrifugation 
 ( 15  000 rpm , 30 min , 4 c ) . the his6-tagged proteins were 
 purified by using ni - nta agarose ( qiagen ) according to manufacturer s 
 instructions . purified enzyme was concentrated and exchanged into 
 buffer a ( 50 mm tris - hcl , ph 7.9 , 2 mm edta , 2 mm dtt ) + 10% glycerol 
 with the centriprep filters ( amicon ) and stored at 80 c 
 for enzyme assays . for in vitro synthesis of 2 and 7 , 10 m 
 fma - c6h480 was incubated with 20 m substrate 6 , 1 mm sodium ascorbate , 1 mm -ketoglutarate , and 100 mm nacl 
 in 100 mm tri - hcl ( ph 7.4 ) . for in vitro synthesis of 2 , the assay was performed using the same condition as above with 
 additional 1 mm sam and 10 m fma - mt . the organic phases 
 were dried and dissolved in 20 l of meoh and subjected for 
 analysis by lc ms as described in chemical 
 analysis . for biotransformation 
 in s.c . of fma - p450 and afcpr , s. cerevisiae strain bj5464-npga harboring both fma - p450 and afcpr plasmid were 
 inoculated to 4 ml of yeast synthetic drop - out medium without uracil 
 and leucine . a 15 l aliquot of the seed culture was inoculated 
 with 2 ml of ypd ( 10 g yeast extract , 20 g peptone , and 950 ml of 
 milli - q water ) supplemented with 1% dextrose . 3 , 8 , and 9 ( 0.5 mg in 10 l of dmso ) was 
 added to the culture after 48 h at 28 c with shaking and the 
 cells were cultivated for another 24 h. the cultures were extracted 
 by hexanes - ethyl acetate ( 1:1 ) twice , the organic layers were concentrated in vacuo and redissolved in 100 l of meoh . ms with the method 
 described in chemical analysis . for biotransformation 
 in s.c . of af470 , the culture of s. cerevisiae strain bj5464-npga harboring af470 was prepared by a similar method above and 4 ( 0.1 mg in 10 l of dmso ) was added to the culture after 48 
 h. details 
 in preparation of fma - p450 and afcpr - containing microsomes 
 for in vitro assay are shown in the supporting 
 information . for in vitro microsomes assay , 10 mg / ml ( wet weight ) 
 microsomal fractions containing af510 and afcpr , 1 mm substrates , 2 mm nadph , and nadph regeneration 
 system ( bd ) solution a ( 5 l ) and b ( 1 l ) , and 100 mm 
 pbs , ph 7.4 were incubated in a 100 l reaction . the reaction 
 was incubated at room temperature for overnight and extracted with 
 100 l of hexanes - ethyl acetate ( 1:1 ) twice . the organic phase 
 was dried and redissolved in 20 l of meoh for analysis by lc  ms . the amount of protein in 10 mg / ml microsomes was calculated to be 
 180 g / ml based on a modified bradford assay against a bsa standard 
 curve ( protein samples were predenatured in 0.1 m naoh ) . g / l 
 yeast extract , 20 g / l peptone ) supplemented with 1% dextrose and incubated 
 at 28 c with shaking for 72 h. the cells were harvested by centrifugation 
 ( 3750 rpm at 4 c for 10 min ) , and the cell pellet was resuspended 
 in 20 ml of lysis buffer ( 50 mm nah2po4 , 150 
 mm nacl , 10 mm imidazole , ph 8.0 ) and lysed by sonication on ice in 
 one minute intervals until homogeneous . to remove cellular debris , 
 the homogeneous mixture was centrifuged at 17000 rpm for 1 h at 4 
 c . ni - nta agarose resin was added to the supernatant ( 2 ml ) 
 and the solution was stirred at 4 c overnight . soluble fma - kr 
 was purified by gravity - flow column chromatography with increasing 
 concentrations of imidazole in buffer a ( 50 mm tris - hcl , 500 mm nacl , 
 20 mm250 mm imidazole , ph 7.9 ) . purified protein was concentrated 
 and buffer was exchanged into buffer b ( 50 mm tris - hcl , 2 mm edta , 
 100 mm nacl , ph 8.0 ) using an amicon ultra-15 centrifugal filter unit 
 and stored in 10% glycerol . the purified fma - kr was analyzed by sds - page 
 ( supporting information , figure s6 ) and 
 their concentration was calculated to be 8.7 mg / l , using the bradford 
 assay with bsa as a standard . for in vitro synthesis of 6 , 9 , and 2 , 10 m fma - kr 
 was incubated with 1 mm substrates 2123 , respectively , 2 mm nadph in 100 mm pbs , ph 7.4 in a total 100 l 
 reaction . the reaction was incubated at room temperature overnight 
 and extracted with 100 l of hexanes  ethyl acetate ( 1:1 ) 
 twice . the organic phase was dried and dissolved in 20 l of 
 meoh for analysis on lc  ms . the method of oxidation by pcc followed 
 that of asami et al . a suspension of 
 pcc ( 1 mg , 0.0046 mmol ) and 
 powdered molecular sieves 4a ( 3.0 mg ) was added to a solution of 2 ( 2.3 mg ) in 0.5 ml of ch2cl2 . the 
 mixture was stirred in an ice  water bath and for 2.5 h at room 
 temperature . florisil and ch2cl2 were added 
 to the mixture , and the suspension was filtered through a combination 
 of celite and florisil . the residue was purified by silica gel plate ( merck , tlc silica 
 gel 60 f254 , glass plates ) with 25% acetone - hexanes developed 
 by two times to give 23 ( 1.1 mg ) . a similar method was 
 used as above to obtain 21 ( 0.8 mg ) and 22 ( 1.0 mg ) from 10 ( 2.2 mg ) and 11 ( 3.0 
 mg ) , respectively .