The natural products (NPs) biosynthetic gene clusters (BGCs) represent the adapting biochemical toolkit for microorganisms to thrive different microenvironments. Despite their high diversity, particularly at the genomic level, detecting them in a shake-flask is challenging and remains the primary obstacle limiting our access to valuable chemicals. Studying the molecular mechanisms that regulate BGC expression is crucial to design of artificial conditions that derive on their expression. Here, we propose a phylogenetic analysis of regulatory elements linked to biosynthesis gene clusters, to classify BGCs to regulatory mechanisms based on protein domain information. We utilized Hidden Markov Models from the Pfam database to retrieve regulatory elements, such as histidine kinases and transcription factors, from BGCs in the MIBiG database, focusing on actinobacterial strains from three distinct environments: oligotrophic basins, rainforests, and marine environments. Despite the environmental variations, our isolated microorganisms share similar regulatory mechanisms, suggesting the potential to activate new BGCs using activators known to affect previously characterized BGCs. Our study represents the first approach that classifies biosynthetic gene clusters according to a regulatory structure. Through the study of known activators of well-characterized BGCs, we have identified common patterns in regulatory mechanisms, offering potential activators for previously unexplored BGCs. This research contributes to NP discovery by presenting a framework for identifying potential activators for novel BGCs, opening the door to the discovery of new Natural Products.The exploration of natural products (NPs) from microbial sources has played a key role in the search of pharmaceuticals, insecticides, and herbicides . HoweverThe goal of harnessing the full potential of NPs often centers around understanding the molecular mechanisms governing their biosynthesis. Notably, NP production is subject to regulation influenced by several factors, including pleiotropic and pathway-specific factors. This regulation often involves a complex interplay of regulatory genes (transcription factors and sigma factors), within the BGCs . While bStreptomyces and other relevant NP producers. For example, S. venezuelae and some of them show a regulatory function on antibiotics. Also, a pangenome analysis recently reported that Streptomyces Antibiotic Regulatory Proteins (SARPs) TFs are less conserved on Streptomyces species (strain-specific) and control the BGCs in which they are encoded  BGCs . While, In this study, we propose a framework to identify closely related regulatory elements from environmental strains from the Cuatro Cienegas Basin (CCB), the Calakmul rainforest, in Mexico; and the Comau fjord, Penas Gulf, and Valparaíso beach in Chile. By utilizing Hidden Markov Models from the Pfam database to detect key protein domains and a well curated BGC database (MIBiG), we generated a database of 2,694 BGCs including 784 BGCs from our strain collection. We conducted phylogenetic analyses of regulatory mechanisms (TFs and TCS) on both known and predicted BGCs to study activation mechanisms. This research contributes to our understanding of the diversity of regulatory mechanisms within BGCs and enhances our ability to predict activators in environmental isolates, thereby advancing the field of NP discovery.High quality genomic information from a total of 40 environmental strains are included in this study . 23 EnviBiosynthetic gene cluster was predicted in all genomes using antiSMASH version 6.0 . BGC com1 using Hidden Markov models (HMM) of CA domains .Histidine kinase class I and II have a different structure, class I has a dimerization and histidine phosphoacceptor (DHp) domain and class II has a histidine-containing phosphotransfer (HPt) domain, but both share a similar CAtalytic domain (CA domain). To detect all HK first all CA-containing were recovered and classified as class I (DHp-containing) and class II (HPt-containing). Proteins with Histidine Kinase CA domain, a domain present in HK class I and II, were used to recover all potential HK in MIBiG and genome sequences. Protein sequences with CA domain were detected with HMMER version 3.3.2 (Nov 2020) domains  from PfaHistidine phospho-acceptor domain (DHp) was detected in CA domain proteins. DHp was detected with HMMER using HMM from the Pfam database  at defauTFs from BGC were detected using selected TF domains using HMM from the Pfam database . TF domaProtein sequences with the same phosphoacceptor domain or transcription factor domain were used to calculate the phylogenetic tree. All sequences were aligned using MUSCLE  using deOur database comprises a total of 2,694 complete BGCs, with 1,910 originating from MIBiG and 784 newly reported from environmental samples in our strain collection. Of these BGCs, 204 contain at least one histidine kinase, while 1,291 encode at least one transcription factor. Notably, 2,213 BGCs do not contain either an HK or TF. We identified a total of 225 HKs, with 137 previously identified in MIBiG BGCs and 88 from environmental samples. Additionally, we identified a diverse array of 2,777 distinct TFs, encompassing 1,952 from MIBiG BGCs and 825 from environmental samples. The remaining 2,213 BGCs do not encode any HKs or TFs within the cluster.To analyze the preference for NP classes among actinobacterial strains from different environments, we examined the distribution of BGC classes. We calculated the percentage of BGC classes that contain at least one HK, as shown in Histidine kinases were predominantly associated with BGCs from Polyketides in the MIBiG database and rainforest samples, while BGCs from NRP were primarily linked to the Cuatro Cienegas Basin and marine strains. Alkaloid-containing BGCs were exclusively found in the MIBiG dataset, and saccharide-containing BGCs were identified in both Cuatro Cienegas Basin and MIBiG samples.On the other hand, Similar to the distribution of HKs, BGCs from the polyketide class were more prevalent in strains from the rainforest and the MIBiG dataset, with polyketides accounting for over half of the BGCs containing TFs in the MIBiG dataset. Similarly, NRPs with TFs were more common in both the Cuatro Cienegas Basin and marine strains. Notably, BGCs containing TFs from the alkaloid class were absent in rainforest and marine strains.We identified the presence of Two-Component system regulatory mechanisms when both a HK and a TF were encoded in tandem, transcribed within the same strand, and the TF encoded a response regulator protein domain (PF00072). Additionally, we classified HKs without a corresponding TF as “orphan HKs,” and TFs without an associated HK as “orphan TFs.” All regulatory proteins, including HKs and TFs, were categorized based on their regulatory mechanisms into TCS, orphan HKs, or orphan TFs, as depicted in Within the TCS, HisKA and HisKA3 were the predominant contributors among HKs, accounting for 49.3 and 49.7%, respectively . In contIn a similar pattern to HKs within the TCS, we observed that HisKA and HisKA3 domains were present in almost all orphan HKs, accounting for 58.7 and 40%, respectively, while HWE contributed 1.3% . Among tMicromonospora and Streptomyces genera are found in all environments, while most orders in the database were isolated from only one or two environments, with Bacillus being an exception, isolated from three different environments.To examine the impact of environmental factors on BGC diversity across different genera, we analyzed the diversity of BGC classes and compared them to the isolation environments of each genus. Our analysis revealed a clear relationship between the occurrence and diversity of BGCs and the variety of environments where microorganisms were isolated. Lentzea sp. in the Cuatro Cienegas Basin and another from Streptomyces sp. in the rainforest.Histidine kinase A (PF00512) and the catalytic ATP-binding domain (PF02518) were identified in a total of 111 protein sequences, with 75 originating from the MIBiG database and 36 from environmental samples. Specifically, HKs associated with lanthipeptide-producing BGCs formed a distinct clade within the phylogenetic tree  and the catalytic ATP-binding domain (PF02518) were identified in a total of 112 sequences, including 60 from MIBiG, 40 from the Cuatro Cienegas Basin, 7 from marine environments, and 5 from the rainforest. A distinct clade associated with calcium-dependent antibiotics BGCs was identified (Streptomyces sp. strains from MIBiG and was a subclade of a larger group containing the sensor domain (PF13796) in the sensor region. This sensor domain is associated with proteins containing histidine kinase 3 and the catalytic ATP-binding domain. Notably, the majority of these histidine kinases were associated with BGCs responsible for producing NRPs products.Another clade with BGCs responsible for producing macrolactams was identified  was detected in 191 sequences featuring a LuxR domain. Most of these sequences clustered within a distinct clade  and terpene (tiancilactone) products.Four closely situated clades primarily corresponded to LuxR proteins with response regulator activity. Approximately half of the response regulators were associated with NRPs metabolites, followed by polyketides (32.5%). Interestingly, response regulators were evenly distributed across these clades. Another small clade featuring the sigma 70 region was detected, with members predominantly originating from the Cuatro Ciénegas Basin and all corresponding to Pseudomonas, four to Burkholderia, and one to Methylobacter. Finally, the PAS domain was identified in 27 sequences clustered within a single clade, with six originating from environmental samples: five from the Cuatro Ciénegas Basin and one from the marine environment. Most of the transcription factors within this clade were associated with polyketide BGCs.Additionally, a clade contained LuxR proteins with the autoinducer binding domain, with all sequences originating from the MIBiG database and associated with polyketide and NR-like BGCs. Among these BGCs, four belonged to The TetR domain emerged as the most prevalent domain among transcription factor proteins associated with BGCs, with 22% of all transcription factors featuring this domain. Following closely was the LuxR domain, found in 21% of all transcription factors. Notably, 40% of these transcription factors were linked to polyketide BGCs, while 33% were associated with NRPs. Furthermore, 46% of all transcription factors were sourced from the MIBiG database.One clade comprising 42 proteins featured the TetR C-terminal 14  . The majA separate clade containing 40 transcription factors featured the TetR C-terminal 6 domain. This clade included a higher proportion of environmental samples (23 proteins). Similar to the TetR C-terminal 11 clade, sequences from the MIBiG database were predominantly associated with polyketides, while environmental samples showed a preference for NRPs. Additionally, a clade encompassing 66 proteins contained the TetR C-terminal 1 domain. Among all TetR C-terminal proteins, this domain was the most prevalent. Within this clade, polyketide and NRP BGCs were more common, with 22 and 20 BGCs, respectively. Lastly, from a review of the literature, a clade of transcription factors activated by γ-butyrolactone was identified. This clade comprised five proteins  clustered together.Streptomyces, accounting for 76% in our database . All product classes are evenly distributed along the tree  compared to SARP (25.3%).A total of 450 TFs encode an HTH domain (PF08279), with the majority originating from NRPs and polyketide-producing BGCs . The distribution by isolation site is similar to that of TetR, where 46% of the proteins come from MIBiG database, 30% from the Cuatro Cienegas Basin, 16% from rainforests, and 8% from marine environments. A significant portion of these proteins also contains a LuxR domain, as shown in the green area in TetR domains were identified in 40 TFs, and many of them are scattered throughout the phylogenetic tree. Among these 40 sequences, 7 form a distinct clade of TFs with the TetR domain. Most of them are associated with polyketide BGCs, followed by NRPs BGCs. The Iron repressor domain (PF01325) was detected in 125 TFs, forming two closely related clades. These two clades are separated by the TetR clade. Within all TFs containing an Iron repressor domain, 42% are associated with polyketide BGCs, and 38% are linked to NRPs BGCs. Additionally, potential siderophore-producing BGCs from environmental samples were identified, including heterobactin and coelichelin.The relationships between organisms and their environments can influence genetic variation among species, and this variation is correlated to the environment independently of physical locations . To inveOur analysis revealed that one-component systems are the primary regulatory system for BGCs. However, a substantial number of BGCs lack a regulatory mechanism within the cluster, suggesting external regulatory mechanisms. Regulatory genes located outside the BGCs, with multiple regulation targets or pleiotropic effects, control many secondary metabolites or morphological development . Most BGLentzea sp. CC55 from CCB and Streptomyces sp. KL110B from the rainforest), were also grouped in this clade. The aminoglycoside clade included gentamicin, sisomicin, and catenulisporolides (a glycosylated macrolide). Additionally, this clade included an environmental sample, Streptomyces sp. KL110B from the rainforest, which produces a polyketide.In the HisKA phylogenetic analysis, a clade of lanthipeptide BGCs was observed, with all HKs within this clade being part of a two-component system. For instance, nisin and Bovicin HJ50, two lanthipeptides, are well-known natural products with an autoinducible mechanism . Some deLentzea sp. CC55 and Streptomyces sp. SS52, both from CCB, with one of them having the potential to produce CDA1. Another different clade within the same analysis contained proteins with the putative sensor domain (PF13796), which was identified in proteins with the HisK 3 domain. This clade encompassed two subclades of cyclic proteins: macrolactams and cyclohexapeptides. The macrolactams subclade included BGCs from salinilactam, lobosamide, and sceliphrolactam, and one of the nodes corresponded to an environmental sample, Streptomyces sp. Vc67-4 from the marine environment, which has the potential to produce sceliphrolatam. The cyclohexapeptides subclade included BGCs similar to desotamide, curacomycin, dechlorocuracomycin, and ulleungmycin, along with two environmental samples from CCB, Lentzea sp. CC55, and Streptomyces sp. CC208A. Most of the members of the sensor clade produced cyclic NRPs.The HisK 3 domain phylogenetic tree has a marked clade with BGCs for calcium-dependent antibiotics from the NRPs class, which included two of our environmental samples, Nocardia species from CCB, were included, and they were predicted to produce NRPs.The LuxR domain was the predominant contributor to the response regulator. The LuxR family is associated with quorum sensing and molecular sensing . Within Streptomyces strains within the PAS clade, four of which were isolated from CCB  and one from rainforest (with a 2-methylisoborneol like BGC).In addition, a clade containing N-Acyl homoserine lactone (AHL) binding domains was identified from previously characterized Gram negative bacteria. AHL is a known molecule involved in quorum sensing in Gram negative bacteria while GBL molecules are used for Gram positives microorganisms . We alsoThe TetR domain emerged as the most prevalent protein family among TF BGCs, accounting for 21.9% of the cases. The TetR family typically acts as a repressor in most instances . SeveralStreptomyces sp. CC219B from CCB.The TetR phylogenetic tree revealed four marked clades. One of these branches includes the MftR C-terminal domain (PF17754) from environmental samples . Another branch included TFs with the TetR C-terminal domain 1 (PF02909) and BetI-type transcriptional repressor, C-terminal (PF13977), both of which are associated with antibiotic expression and osmoregulation via pumps . The TetOmpR domain proteins are typically part of SARP or TCS. Nearly all reported genes containing an OmpR domain positively regulate their BGCs , and manStreptomyces strains potentially producing venemycin, chloramphenicol, and 7-prenylisatin from CCB and the rainforest. The Iron-dependent repressor was commonly observed in multiple clades of the HTH phylogenetic tree, with four such clades and 27.8% of TFs featuring this domain. When considering a possible relationship between BGCs producing siderophores and the presence of the Iron-dependent repressor, we detected three siderophore BGCs (two for pyoverdine and one for yersiniabactin) from MIBiG, as well as one previously reported potential siderophore BGC (heterobactin) Rhodococcus sp. H-CA8f. However, none of these BGCs contained the Iron-dependent repressor. Some Nocardia strains from CCB possessed the Iron-dependent repressor and had the potential to produce a siderophore, although with similarities of 72% and 63% for heterobactin, and 27% for coelibactin.Most DNA-binding proteins possess a helix-turn-helix motif, which is responsible for DNA binding in almost 95% of transcription factors . In our In this study, we propose a phylogenetic analysis of regulatory elements linked to biosynthesis gene clusters. Our analysis allowed us to explore the diversity (based on protein domain information) of regulatory mechanisms present in curated BGCs from database. More importantly, our analysis permits to cross-correlate regulatory mechanisms of newly sequenced environmental samples in order to formulate informed hypothesis on activation of BGCs. As a proof of principle, we conducted a comprehensive genome analysis of Actinobacteria isolated from three distinct environments: the low-nutrient Cuatro Ciénegas Basin, the Calakmul Rainforest in Mexico, and the marine environments of Chile. This approach provided valuable insights into the regulatory mechanisms governing biosynthetic gene clusters (BGCs) across diverse ecological niches. Despite variations in isolation sites, our findings revealed striking similarities in the regulatory elements of similar natural products (NPs) compared to those previously documented in the MIBiG database. This innovative approach holds promise for selectively identifying and activating known NPs with analogous activity or structure, albeit with unique characteristics in environmental strains. Furthermore, it offers the potential to uncover potential elicitors for BGCs in strains suitable for NP discovery. However, it’s important to note that this method is most effective for BGCs with internal regulatory elements and may be less efficient for those relying on external activation mechanisms. This research significantly contributes to our understanding of BGC regulatory mechanisms by exploring the roles of transcription factors (TFs) and histidine kinases (HKs) in relation to NP structures and environmental contexts, offering valuable insights for NP mining endeavors.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/AR-S: Data curation, Formal analysis, Methodology, Writing – original draft, Writing – review and editing. LG-S: Data curation, Formal analysis, Writing – review and editing. LR-O: Data curation, Formal analysis, Writing – review and editing. ÁC: Formal analysis, Writing – review and editing. AU: Data curation, Writing – review and editing. BC: Data curation, Writing – review and editing. NS-M: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Writing – review and editing. CL-C: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing.


Brosimum alicastrum) is a novel food with high nutritional value. This research aimed to evaluate the nutritional and antioxidant properties of Maya nut flour (MNF) made from seeds dried by different methods (sun-dried and using hot air at 45 °C and 60 °C) to explore its incorporation into cookies and evaluate its nutritional and functional properties. The naturally sun-dried flour (NF) had the highest content of ash (3.64 ± 0.11 g/100 g), protein (6.35 ± 0.44 g/100 g), crude fiber (6.75 ± 0.29 g/100 g), and functional properties (water and oil absorption). The color of the flour was affected by the different drying methods. While the drying methods influenced the total polyphenolic content (TPC) and antioxidant activity (AA) of MNF, they did not affect the morphology of the native starch or generated important molecular-structural changes. The substitution of 60% of wheat flour with NF in the cookie’s formula increased the protein and fiber content, whereas 20% substitution increased its AA. MNF is a source of protein, dietary fiber, micronutrients, and functional compounds that can enrich cookie formulations.Maya nut ( Brosimun alicastrum). According to historical data, the Mayas cultivated the Ramon tree to use its seeds as food in their diet. Currently, the Ramon tree is an underexploited resource with high economic potential. In Mexico, conserving and restoring the Ramon tree is sought in forest systems. Its wood, leaves, fruits, and latex are used as a source of food and in traditional medicine  or greenness [0 to 60]); and b* values (yellowness [0 to 60] or blueness [0 to 60]) were measured for all the samples. Three readings were taken at different locations on the surface of the samples to obtain the mean values of L*, a*, and b*.We poured the MNF into Petri dishes until a 10 mm thick layer was formed. A colorimeter (3nh NR110 Precision Colorimeter) measures the We used a tabletop scanning electron microscope to observe the microstructure of MNF . The images were taken at an acceleration voltage of 15 kV and a magnification of 2000.The X-ray diffractometer  generated a diffractogram with an interval of 2θ angles that ranged from 7 °C to 50 °C at a rate of 4.3°/min and operating at a power of 40 kV/30 mA.−1 in the region from 4000 cm−1 to 600 cm−1 at room temperature.A PerkinElmer Frontier spectrometer with a universal attenuated total reflection (ATR) determined the main functional groups in the flour and worked in transmittance mode with a resolution of 4 cmv/v) solution, following Moo-Huchin et al. [g at 25 °C for 10 min, and the supernatant was recovered and stored in the refrigerator, protected from light, until analysis [The extraction of total polyphenols and antioxidant components was performed in the dark at room temperature. We weighed 100 mg of flour and mixed it with 5 mL of ethanol/water  was added. After a 90 min incubation at room temperature, we measured the absorbance at 750 nm using a spectrometer . A standard curve of gallic acid was prepared from 0 ppm to 400 ppm in 50% methanol. Phenolic content was expressed in gallic acid equivalents per g of sample (mg GAE/g).The TPC was estimated using the Folin-Ciocalteu procedure according to the method reported by Goiris et al. , with soThe antioxidant activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS+), and ferric reducing antioxidant power (FRAP) methods, described by González et al.  with sliFor the DPPH assay, we prepared a 60 μM solution of DPPH reagent with methanol. Then, 7 μL of each extract was mixed with 193 μL of DPPH solution at the desired absorbance (0.7 ± 0.02) and kept in the dark for 30 min. The absorbance was determined at 517 nm.2S2O8 (1:1 v/v). This ABTS solution was allowed to stand for 16 h in the dark at room temperature (25 °C ± 2), then it was diluted with ethanol to an absorbance of 0.7 ± 0.02 at 734 nm. Next, 5 μL of each sample and 95 μL of ABTS ± solution were placed in a microplate. Solutions were rested for 1 min before reading at 734 nm.To determine antioxidant activity with the ABTS assay, we prepared the ABTS solution at 7 mM with ethanol and mixed it with 2.45 mM K3 (20 mM), and acetate buffer  in the proportions of 1:1:10 v/v/v. Then, 10 µL of each of the sample solutions was added to the microplate followed by 290 µL of the FRAP solution. Subsequently, we incubated the microplate in the dark at 37 °C for 15 min. The absorbance was measured at 593 nm.In the case of the FRAP assay, we prepared the FRAP solution by mixing TPTZ  at 10 mM, FeClThe incorporation of MNF in the formulation of cookies was explored. We chose to develop the cookies with NF flour because it showed better techno-functional, nutritional, and functional properties than the F45 and F60. We compared the NF flour to the commercial flour (CF). We replaced 0% (F0), 20% (F20), 40% (F40) or 60% (F60) of wheat flour with NF or CF flour, and we obtained four formulations with substitutions and one control formulation. The ingredients  were incThe cookies were made by substituting 20%, 40%, and 60% wheat flour with MNF and compared to a control (100% wheat flour). Sensory acceptance tests of formulations were conducted with 50 semi-trained tasters (aged from 18 to 30 years old) recruited randomly from the staff of the Autonomous University of Coahuila and the Autonomous Agrarian University Antonio Narro. Samples were served in small cups at room temperature. Panelists were asked to show their level of liking for the taste, aroma, texture, color, and appearance attributes for each of the Maya nut cookie formulations. Panelists used a 5-point hedonic scale ranging from 1 (dislike extremely) to 5 (like extremely) with a medium value 3 (neither like nor dislike) to evaluate each formulation, following previously reported methods .All experiments were carried out at least in triplicate and results were reported as the average ± standard deviation (SD). The statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s test with a significance level of 5%, using INFOSTAT software version 2018 for Windows .The seeds correspond to dicotyledonous seeds, as seen in Brosimum alicastrum grows in areas with average annual temperatures of 18 °C to 27 °C, with annual rainfall of 600 mm (in the Mexican state of Tamaulipas) and up to 4000 mm (in the Mexican states of Chiapas and Tabasco) [Tabasco)  making iThe water content of the fresh Maya nut was 58.28 ± 4.24%. We obtained all MNF after drying and grinding. The moisture content and chemical composition of the flours are shown in NF’s moisture was higher (9.32%) than F45′s (6.05%) and F60′s (5.92%) moisture. This means that the drying method directly affects this parameter. Low moisture values could result in improved stability for long-term storage with an impact on improving the quality and stability of the product .The ash content of the flours was highest for NF (3.64 ± 0.11 g/100 g), while the lowest was found in F45 (3.08 ± 0.01 g/100 g). The highest protein value was also recorded for NF (6.35 ± 0.44 g/100 g). The lowest protein content was for flour F45 (4.48 ± 0.07 g/100 g). There were no significant differences between crude fat values, which ranged from 1.24 to 1.62 g/100 g across all flours. The highest carbohydrates value was for F45 (84.87 ± 0.11 g/100 g), even higher than CF.These results differ from those obtained by Subiria-Cueto et al. , who repTraditionally, in some regions of Mexico, MNF has been mixed with other flours, such as corn flour ; foods sIn addition, we report the mineral content of Maya nut flour by XRF for the first time in the literature. At least 15 minerals were detected in the samples evaluated, but the most abundant minerals are shown in Water content and Aw are two important factors affecting food safety  as valueThe water absorption capacity (WAC) and oil absorption capacity (OAC) of MNF are also shown in All the MNF we analyzed had higher WAC (189.39% to 220%) than the WAC in whole wheat flour (103.36%), amaranth flour (136.64%), soybean flour 202.26%), lupine flour (155.17%), and corn starch (106.05%) reported by Liu et al. %, lupine.OAC facilitates emulsification and is another critical attribute in ingredients intended to be incorporated into formulations with high fat content . OAC valL* (lightness) ranged from 82.44 ± 0.27 (in F45) to 58.12 ± 1.08 (in CF). CF’s a*  and b*  values were significantly higher than the values of all the MNF. Both F45 and F60 showed lower values of redness and yellowness; F45′s redness value was 2.04 ± 0.05, and its yellowness value was 17.60 ± 0.04, whereas F60′s redness value was 3.38 ± 1.06 and yellowness value was 17.46 ± 0.76. A darkening of flours obtained from roasted grains and cereals has been observed previously [a* and b* values in CF flour could be attributed to Maillard reactions caused by chemical reactions between sugars and proteins in the Maya nut during the drying process [eviously . The inc process ; for thiThe size of the granules was heterogeneous, with an average diameter ranging from 7 to 10 μm. Lindeboom, Chang, and Tyler (2004)  categoriThe change in amylose’s structure could have altered and transformed the oval structure of the starch granule into an amorphous shape; alternatively, the whole starch granule may have collapsed . RegardlHay Moo-Huchin et al.  mention −1 corresponding to the stretching vibrations of the hydroxyl groups (–OH) that contributed to the inter and intramolecular interactions of the hydroxyl group OH–, which is a particular characteristic of the starch structure. An intense band at 2920 cm−1 is attributable to stretching vibrations of the C–H bonds [The effect of the different drying methods on the changes in the molecular properties of the obtained flours was determined by a spectroscopic analysis of FTIR bands, and the results can be seen in –H bonds .−1. The bands at 1400 cm−1 and 1300 cm−1 correspond to C–H bonds. Starch’s fingerprint peaks are at 1150 cm−1 and 1056 cm−1; peaks at 1019 cm−1 are attributed to the vibrations of the C–O–C glucose bonds, and bands at 928 cm−1, 927 cm−1, 856 cm−1, 758 cm−1, and 678 cm−1 correspond to the pyranose ring [Water absorbed by starch is observed at 1620 cmose ring .We studied the antioxidant activity (AA) of the MNF because of the importance of looking for new sources of antioxidants for their multiple health benefits as well as their ability to be used as natural preservatives in food . Table 6The first polyphenolic compounds identified in the Maya nut were epicatechin, hydroxybenzoates, gallic, p-hydroxybenzoic, vanillic acids, hydroxycinnamates, and caffeic and p-coumaric acids. These polyphenolic compounds may contribute to the antioxidant activity of the seeds .In this study, three common tests were performed to measure the AA of Maya nut flours. Significant differences exist between treatments in antioxidant activity . The DPPIn the three methods we evaluated, CF has a higher TPC, which leads to a higher AA than the TPC and AA of the other flours. Another cause of this increase in AA could be the generation of other antioxidant compounds during the drying process. Maya nut has emerged recently as a commercial process, and it is common for roasting to be used similarly to the method used for coffee beans. Most of the natural antioxidants available in foods are unstable and sensitive to intense and prolonged heat treatment, which can cause their loss and their biological activity. However, little or no change in the content and activity of these compounds has been observed, though this may vary depending on the food matrix . FurtherThe fiber content in cookies made with 60% NF is higher than the fiber content in cookies made with CF. With the substitution of non-conventional flours in the production of functional cookies, the amount of fiber increases and favors the amount of available carbohydrates; this can contribute to a more adequate energy supply in the body and ameliorate consumers’ health .On the other hand, the protein content is directly affected as the flour concentration increases from 0% to 40% in NF cookies (13.5 g/100 g at 40% substitution). For CF cookies, protein content decreases with increasing concentration of CF (5 g/100 g at 60% substitution). Protein content could be affected by the treatment given to the commercial sample since thermal processes can directly affect the protein content; heat treatment can reduce the amount of protein by destroying some amino acids, which modifies the quality of the protein composition of the cookies.The carbohydrate concentration shows a significant increase as the amount of NF increases. This can be observed in CF cookies, which obtained the highest content (71.9 g/100 g) with 60% substitution; while using NF, the highest value of total carbohydrates occurs with 20% substitution of Maya nut flour (68.4 g/100 g).Fat content in the cookies substituted with NF showed a constant increase depending on the substitution percentage, so the highest concentration was observed in the 60% substitution. The opposite was seen when CF was substituted in the cookies, so the higher fat concentration occurred in the 20% substitution. Fat content is of substantial relevance in cookie doughs, mainly in the rheological factors; the presence of a greater amount of fats helps to improve handling in elaborating cookies, although little has been published on the subject.Henrique-Belmiro et al.  evaluateImproving the protein and fiber content in a popular snack, such as cookies, could have a substantial positive effect on its consumers . Other fAs discussed earlier, an important component of the Maya nut is its antioxidants. Therefore, to evaluate the stability of this antioxidant capacity when baking cookies, ABTS and DPPH tests were carried out. The results are shown in It should be noted that the AA in the cookies can be affected by other ingredients since MNF is not the only source of antioxidants. In this work, wheat flour was substituted with two types of MNF. Although MNF has a higher AA, it was evident that its optimal increase was with the replacement of wheat flour with only 20% of MNF; a higher substitution of MNF did not increase the AA of the cookies; instead, AA was maintained or decreased it, as in the case of the NF. A decrease in AA may also be caused by the antioxidant compounds degrading due to the baking temperatures, since these compounds might be unstable under these conditions .The F20 cookies scored the highest values for aroma (4.15) and flavor (3.92); the panelists expressed a preference for the cookie with Maya nut over the cookie with wheat flour (control). However, the panelists scored wheat cookies with the highest mark (4.2) for the texture attribute. The texture attribute is an essential factor in the quality of foods since it influences the final acceptability of the product . This prMNF proved to be an enriching product for food formulations. It can improve the nutritional profile of cookies by increasing the protein content and improve its functional characteristics, such as antioxidant activity, through a 20% substitution of MNF for wheat flour. Few differences were observed in the physicochemical characteristics of the MNF since there was little impact on the structural and molecular characteristics of the starch granules. However, the results of this work demonstrated that the drying methods used here significantly affected the TPC and the AA. Nevertheless, we developed a cookie rich in antioxidants that could ameliorate the damage caused by free radicals in the body and prevent them from causing damage to other cells; this could improve its consumer’s health. In addition, increasing the knowledge about undervalued and little-used products such as Maya nut, along with evaluating the physical, chemical, nutritional, and functional characteristics of ingredients, opens the opportunity to consider them in the development of new food formulations.


Here, we find multiple metal sulfides CuInSnS4 octahedral nanocrystal with exposed (1 1 1) plane for the selectively photocatalytic CO2 reduction to methane. Still, the product is switched to carbon monoxide on the corresponding individual metal sulfides In2S3, SnS2, and Cu2S. Unlike the common metal or defects as active sites, the non-metal sulfur atom in CuInSnS4 is revealed to be the adsorption center for responding to the selectivity of CH4 products. The carbon atom of CO2 adsorbed on the electron-poor sulfur atom of CuInSnS4 is favorable for stabilizing the intermediates and thus promotes the conversion of CO2 to CH4. Both the activity and selectivity of CH4 products over the pristine CuInSnS4 nanocrystal can be further improved by the modification of with various co-catalysts to enhance the separation of the photogenerated charge carrier. This work provides a non-metal active site to determine the conversion and selectivity of photocatalytic CO2 reduction.The active center for the adsorption and activation of carbon dioxide plays a vital role in the conversion and product selectivity of photocatalytic CO The product selectivity of photocatalytic carbon dioxide reduction from carbon monoxide to methane is determined by the active center from metal to sulfur site in metal sulfides. Non-metal sulfur in CuInSnS4 octahedral nanocrystal acts as carbon dioxide activation center for switching selectivity to methane. Various products of CO2 reduction from photocatalytic multi-electron processes, including CO (two electrons), HCOOH (two electrons), HCHO (four electrons), CH3OH (six electrons), and CH4 (eight electrons), have been produced by a great variety of photocatalysts8. Achieving both high selectivity and high conversion for photocatalytic CO2 reduction is highly desirable in the field of photocatalysis research. However, efficient photoreduction of CO2 is very challenging, both in terms of chemical thermodynamics and kinetics, due to the highly stable structure of CO2 and the involvement of multiple proton-coupled electron transfer11. Additionally, the regulation of product selectivity in photocatalytic CO2 conversion remains an unknown challenge.The solar-energy-driven photocatalytic conversion of CO2 is prerequisite for efficient CO2 conversion and product selectivity. Constructing an active center of catalysts for the adsorption and activation of CO2 is an efficient solution to improve CO2 conversion efficiency and product selectivity14. Various metal-free photocatalysts were reported for CO2 reduction19, typically such as covalent organic frameworks, graphitic carbon nitride, elemental phosphorus, boron nitride, and silicon carbide24. These metal-free photocatalysts have non-metallic sites as the adsorption and activation sites of CO2 molecules and thus photocatalytical CO2 reduction28. However, for metal oxide or sulfide photocatalysts, many studies suggest that metal components or defects on photocatalysts play a crucial role as primary sites in the adsorption and activation of CO2 and thus affect product selectivity29. Zhou et al. reported that the S vacancy or Cd vacancy CdS with single Au atom deposition for CO2 adsorption is different30. CO2 prefers to physically adsorb on single Au atoms of Au/CdS1−x and photoreduction into CO, while CO2 is more likely to chemically bond on the Cd vacancies of Au/Cd1−xS, resulting in a remarkable CO and CH4 generation rate on Au/Cd1−xS. He et al. synthesized a ZnIn2S4 nanosheet photocatalyst with abundant Zn vacancies31, where CO2 can be efficiently adsorbed on Zn vacancies to form CO2− species and highly selective photoreduction into CO. Yu et al. designed a Cu3SnS4 photocatalyst with S vacancies to increase ratios of Cu (I/II) for CO2 photoreduction32. The formed Cu (I) acts as adsorption sites for CO2, conducive to further hydrogenation of CO intermediate into CH4. Xie et al. showed that the defect-state CuIn5S8 ultrathin nanosheets have low-coordination Cu and In sites for CO2 adsorption to form highly stable Cu-C-O-In intermediates, which tend to obtain 100% CH4 selectivity33. Xu et al. designed a Co-Ni-P NH/BP catalyst with bimetallic sites to form a highly stable Co-O-C-Ni intermediate for the selective photoreduction of CO2 to CH434. However, the complex structures of defects on photocatalyst make it only a plausible correlation between defect structures and product selectivity. Some research shows that the adsorbed interaction between CO2 and metal sites is relatively weak since the formed metal-C bonds are weaker than the highly stable C=O bonds in CO2. This leads to the easy cleaving of metal-C bonds during the reaction process, hindering the deep reduction of CO2 into hydrocarbons30. Obviously, the non-metal sites on metal sulfide photocatalysts are very rarely considered the primary active center for the adsorption and activation of CO2.It has been well understood for the photocatalytic process that the identification of the active centers of catalysts for the adsorption and activation of CO4 octahedral nanocrystal and corresponding individual metal sulfides In2S3, SnS2, and Cu2S, through a simple one-step hydrothermal method. The CuInSnS4 nanocrystal is thermodynamically favorable to activate CO2 and leads to a switch of main products from CO to CH4 with a yield of 6.53 μL h−1 for the visible-light-driven CO2 reduction with H2O vapor without the assistance of any noble metal cocatalysts. In contrast, individual metal sulfides can only produce CO. We reveal that different adsorption configurations of CO2 on metal sulfides lead to different products in CO2 photoreduction. The non-metal sulfur atom in the prepared multiple metal sulfides CuInSnS4 octahedron nanocrystal is thermodynamically favorable to activate CO2 and leads to a switch of main products to CH4, as compared with the common individual metal sulfides In2S3, SnS2 and Cu2S with metal center as active sites to form CO products. CO2 is revealed to be adsorbed on the S atom center of CuInSnS4 to form an S-C-O-In structural unit, which is more conducive to protonation and leads to the efficient photocatalytic yield of CH4. Thus, we provide an insight into the role of non-metal center of photocatalyst in determining the conversion and selectivity of photocatalytic CO2 reduction. Although the pristine CuInSnS4 only exhibits a yield of CH4 evolution of 6.53 μL h−1 (corresponding to 5.83 μmol h−1 g−1), the activity and selectivity of CH4 evolution on CuInSnS4 can be significantly improved by modifying with cocatalysts such as Pt, CoO, NiO, and Co(OH)2. We believe that this knowledge can contribute to the development of more efficient and selective photocatalysts for CO2 reduction in the future.Here, we have successfully prepared multiple metal sulfides, including CuInSnS4 nanocrystals belong to the cubic spinel structure (JCPDS No. 29-0548), as revealed by the X-ray diffraction pattern , SnS2 with a hexagonal crystal phase (JCPDS No. 23-0677), and Cu2S with a cubic crystal phase were also prepared (JCPDS No. 02-1284)  crystal plane but also has a single crystal structure  crystal faces. The specific surface area of the CuInSnS4 sample is about 24.1 m2 g−1, while that of the prepared In2S3, SnS2, and Cu2S are 26.8 m2 g−1, 37.6 m2 g−1, and 3.6 m2 g−1, respectively  between sulfur and metal atoms in CuInSnS4 is slightly larger than in monometallic sulfide39. Moreover, the S atom in CuInSnS4 has a higher binding energy than that of monometallic sulfides, indicating an electron-deficient state of the S atoms in CuInSnS4 compared to monometallic sulfides. This electron-deficient state of the S atom in CuInSnS4 can serve as the reaction site for CO2 adsorption and activation.X-ray photoelectron spectroscopy was used to compare the electronic states of the obtained sample. The Cuely Fig. . This denS2 Fig. . The pos2 reduction performance of the samples was evaluated in a customized sealed quartz glass vessel, in a gas-solid reaction system, with a small amount of water vapor in a CO2 atmosphere, under the irradiation of a 300 W Xe lamp with a 420 nm cutoff wavelength filter  are not detected. Figure 2 performance of the samples under visible-light irradiation. For single metal sulfides In2S3, Cu2S, and SnS2, CO is the main product with a yield rate of less than 3.2 μL h−1 from photocatalytic CO2 reduction, while the multi-electron transfer product CH4 is hardly formed. Whereas, the CuInSnS4 sample shows excellent photocatalytic CO2 reduction performance, yielding CH4 as the main product besides a slight amount of CO and H2 evolution. The hydrothermal temperature has no obvious impact on the CO2 reduction performance of CuInSnS4 . Neither crystal structural transformation nor absorption behavior changes are found in the XRD pattern and ultraviolet–visible diffuse reflectance spectra for the CuInSnS4 sample after photocatalytic reaction  with a selectivity of 67.3%, the activity and selectivity of CH4 evolution on CuInSnS4 can be improved by coupling with semiconductor photocatalysts or noble metals as cocatalysts. We evaluated the photocatalytic performance of CuInSnS4 modified with Pt, CoO, NiO, Co(OH)2, and dual co-catalysts Pt and Co(OH)2, for the CO2 reduction reaction. The composition and chemical states of Pt, CoO, NiO, Co(OH)2 cocatalysts are well verified by XRD patterns and XPS spectra 2. The activity of CH4 evolution on the modified CuInSnS4 photocatalysts surpasses the majority of the reported photocatalysts for CO2 reduction up to now. Particularly, the incorporation of Co(OH)2 as a co-catalyst significantly enhances the CO2 photoreduction activity of the CuInSnS4 photocatalyst. As the Co(OH)2 loading increases, the photoreduction activity of CO2 exhibits a characteristic volcanic pattern. With 5%Co(OH)2 loading onto CuInSnS4, the production rates for CH4 and CO, respectively, reach 145.45 and 32.32 μmol h−1 g−1, corresponding to a CH4 selectivity of 81.8%. The generation rates of CH4 and CO are 25 times and 13 times that of pure CuInSnS4, respectively. Furthermore, when CuInSnS4 is modified with a dual co-catalyst of 5% Co(OH)2 as an oxidation co-catalyst and 1%Pt as a reduction co-catalyst, CH4 production reaches 195.60 μmol h−1 g−1, along with 22.00 μmol h−1 g−1 of CO, and a CH4 selectivity of 89.9%. Photoelectrochemical characterization was employed to assess the separation efficiency of photogenerated carriers on the modified CuInSnS4, as shown in Supplementary Fig. 4 samples modified with the co-catalyst exhibit a higher photocurrent signal and a smaller electrochemical impedance radius as compared to the parent CuInSnS4 sample. The photocurrent increases sequentially in the order of CuInSnS4 < 5%Co(OH)2/CuInSnS4 ≈ 1%Pt/CuInSnS4 < 5%Co(OH)2/CuInSnS4/1%Pt, indicating that the modification of the dual co-catalyst improves photoelectric carrier separation and migration compared to the single co-catalyst. The NiO and CoO cocatalysts also improve the separation efficiency and migration rate of the photogenerated carriers of CuInSnS4 photocatalyst. The decreasing order of electrochemical resistance radius is CuInSnS4 > 1%Pt/CuInSnS4 ≈ 5%Co(OH)2/CuInSnS4 > 5%Co(OH)2/CuInSnS4/1%Pt, consistent with the photocatalytic activity trend. Additionally, both 10%NiO/CuInSnS4 and 10%CoO/CuInSnS4 exhibit smaller electrochemical resistance radii than pure CuInSnS4, confirming that the cocatalyst promotes the photogenerated charge separation and migration. Therefore, the modification of CuInSnS4 with various cocatalysts to enhance the separation of photogenerated carriers is related to the activity and selectivity of CH4 products. The apparent quantum yield is calculated by measuring the yield of CH4 and CO in 5%Co(OH)2/CuInSnS4/1%Pt under monochromatic light at 400 nm. Supplementary Fig. 4 and CO are 0.16% and 0.01%, respectively. Based on the above analysis, we believe that CuInSnS4 nano-single crystal photocatalysts through further optimization design of the different contents and types of cocatalyst modification can be more efficient and selective for CO2 reduction in the future.Although the pristine CuInSnS4 is calculated to be 787.5 nm, which corresponds to a band gap energy of 1.57 eV. For comparison, the absorption band edges of single metal sulfide Cu2S, SnS2, and In2S3 are 747.0 nm, 552.7 nm, and 641.6 nm, corresponding to the band gap of 1.66 eV, 2.24 eV, and 1.93 eV, respectively. Moreover, the valence band potential of CuInSnS4 is determined to be 0.50 V from the valence band XPS spectra  compared to the single metal sulfide under visible-light irradiation  crystal plane of the In2S3 sample and the (1 1 1) crystal plane of the CuInSnS4 sample were selected as models, and all atoms on the crystal planes were considered as potential sites for CO2 adsorption activation  crystal plane leads to inconsistent changes in two C=O lengths. The length of the C-O2 bond is 1.43 Å, equal to the ordinary C-O (1.43 Å) single bond, while the length of C-O1 is shortened to 1.19 Å, close to the length of C-O (1.12 Å) in a CO molecule. This asymmetric activation is more likely to cause the rupture of C-O2, thereby preferentially producing CO on In2S3. In the case of the CuInSnS4 (1 1 1) crystal plane, both C-O bonds are similar in length, measuring 1.26 ± 0.02 Å. They are longer than the C-O bond (1.16 Å) in a free CO2 molecule, indicating that the bond energy of two C=O bonds of the activated CO2 is simultaneously weakened. The calculation of the charge density difference reveals the difference in electronic structure and electron flow resulting from the interaction of CO2 with the surface atoms of In2S3 and CuInSnS4. On the In2S3 surface, there is extensive charge depletion for the C-O2 and S–O2 bonds, which implies that these chemical bonds are weakened. In contrast, CO2 exhibits a wide charge accumulation region on CuInSnS4, leading to the formation of a strong C-S and In-O chemical bond. This strong interaction is beneficial for the firm adsorption of CO2 on the CuInSnS4 surface, promoting the further deep reduction reaction. The Bader charges analysis further confirms that there is more charge transfer between the CuInSnS4 surface and CO2 molecules adsorbed on it. The surface of In2S3 and CuInSnS4 loses 0.28e and 0.32e, respectively, after CO2 adsorption  plane. With the addition of protons and electrons, the removal of the H2O molecule results in the breaking of the chemical bond between O atoms of intermediates and In atoms on CuInSnS4. Moreover, unlike most metal sulfide photocatalysts, the hydrogenation of CO2 adsorbed on the CuInSnS4 surface to COOH* is an easy step, requiring only a potential energy barrier of 0.075 eV33, as shown in Fig. 2 atom of CO2 molecules in the S-C-O-In adsorption configuration of CO2 on the CuInSnS4 surface to facilitate the addition of protons, thus lowering the formation energy of COOH* intermediates. However, converting COOH* to CO is an endothermic reaction that must overcome an energy barrier of 0.46 eV. On the other hand, the continuous hydrogenation of COOH* intermediates to produce HCOOH* is an exothermic reaction, promoting the hydrogenation of CO2. The formation of H2CO* intermediates is the rate-limiting step for further hydrogenation processes, but the hydrogenation of H2CO* to H3CO* and finally to CH4 is thermodynamically spontaneous. Therefore, CuInSnS4 can achieve high selectivity for CH4 products. Additionally, the adsorption energies of each intermediate product can explain the high CH4 selectivity. Supplementary Fig. 4, CH3OH, and HCOOH on the CuInSnS4 surface are −0.17, −0.61, and −0.67 eV, respectively, with CH4 having the highest adsorption energy. This indicates that CH4 products are most easily desorbed from the CuInSnS4 surface, which is one of the reasons why the CuInSnS4 photocatalyst has high selectivity for the photoreduction of CO2 into CH4.The stable configuration of CO2 on the CuSnInS4 surface is proposed in Fig. 2 adsorbing on the catalyst surface to form the unique S-C-O-In structural unit. This process weakens the C=O double bond in the CO2 molecule while non-metallic S atoms serve as adsorption sites, ensuring a strong bond to the C atom of CO2. This benefits the continuous reduction of CO2 molecules into COOH*, HCO*, H3CO* intermediates, and ultimately into CH4 on the catalyst surface through the assistance of photogenerated electrons and protons. Lastly, the low adsorption energy of CH4 on the catalyst surface facilitates its quick release, completing the full photocatalytic cycle reaction.The photoreduction mechanism of CO4 nano-single-crystal photocatalyst with exposed (1 1 1) facets is successfully prepared by a simple one-step hydrothermal reaction. Under visible-light irradiation, the CuSnInS4 nano-single crystal photocatalyzes the conversion of CO2 and H2O into main products of CH4 with a generation rate of 6.53 μL h−1, significantly higher than that of single metal sulfides . The electron-poor center sulfur atom on the CuSnInS4 (1 1 1) crystal plane acts as the site for CO2 adsorption and activation, which leads to the activation of the two symmetrical C=O double bonds of CO2 molecule to form a stable S-C-O-In transition state. This induces CH4 generation via the conversion route of COOH*→HCOOH*→H2CO*→H3CO*→CH4*. However, the asymmetric activation of CO2 by monometallic sulfides is more likely to result in the cleavage of individual C-O bonds in the CO2 molecule, leading to the preferential photoreduction of CO2 to CO. This work provides a distinctive understanding of catalysts for CO2 adsorption and activation for the CO2 selective conversion to help the conversion of CO2 resources into high-value-added products.In summary, a CuSnInS4 nanocrystal photocatalyst with the cubic crystal structure. The detailed operation process is as follows. Firstly, 1 mmol of CuCl, 1 mmol of SnCI4·5H2O, and 1 mmol of InCI3·4H2O were added to 40 mL of deionized water to form a solution under vigorous stirring. Then, 5 mmol of TAA was dissolved in the above-mixed solution and stirred at room temperature for 30 min. Finally, the resulting mixed solution was transferred to a 50 mL Teflon-lined autoclave and sealed into a stainless steel tank for hydrothermal reaction. The hydrothermal temperature is controlled at 160, 180, and 200 °C for 24 hours. After the reaction, the product was collected and washed with deionized water, and dried under vacuum at 60 °C. The obtained samples were labeled CuInSnS4 (160 °C), CuInSnS4 (180 °C), and CuInSnS4 (200 °C) according to the reaction temperature. The detailed preparation processes of CuInSnS4 modified Pt, CoO, NiO, and Co(OH)2 cocatalysts can be seen in supplementary information.A simple one-step hydrothermal method was used to synthesize CuInSnS−1. Scanning electron microscopy  was used to observe the surface morphology of the catalyst. The element composition and ratio of the sample are detected by EDS. The apparent morphology and high-resolution TEM image of the catalyst were tested by transmission electron microscope . At the same time, SAED and element mapping images of the catalyst were obtained in the TEM measurement mode. A UV-VIS-NIR Spectrophotometer (Cary 500) was used to obtain the catalyst UV-VIS-NIR DRS, in which BaSO4 was used as a standard sample for 100% light transmission. The Micromeritics 3020 M physical adsorption instrument was used to obtain the nitrogen adsorption and desorption curves of different catalysts. The specific surface area of each catalyst was calculated from the type of nitrogen adsorption and desorption curves. The catalyst and dried potassium bromide were evenly ground, and 20 mg was weighed and pressed into slices, then placed in a quartz infrared tube for a carbon dioxide adsorption infrared test. In situ infrared spectra measurements were performed using a Fourier-transform infrared spectrometer (Nicolet iS50 FT-IR Spectrometer) equipped with a mercury cadmium telluride detector  was used to analyze 13CH4 and 13CO. Electron paramagnetic resonance spectroscopic measurements were performed at room temperature using a Bruker A300 EPR spectrometer.X-ray diffractometer  was used to analyze the crystal structure of the catalyst. The XRD test range is 10°−80°, and the scan rate is 10° min2 reduction tests. Firstly, 50 mg of catalyst was dispersed in 5 mL of deionized water and sonicated for 10 min. Then the catalyst was dropped into a watch glass with a diameter of 3 cm and dried at 80 °C. Subsequently, the dried catalyst was placed in a quartz reactor with a volume of ~250 cm3, and then high-purity CO2 gas (99.999%) was introduced to replace all the air. The flow rate of carbon dioxide gas was ~100 mL/min and lasted for 1 h. Finally, 50 μL deionized water was injected into the quartz reactor from the rubber stopper through a gas chromatography liquid syringe , and the reactor was heated with a hair dryer to evaporate the water into water vapor. As a result, water is present in the form of water vapor throughout the reaction. The reactor was placed under a xenon lamp for photocatalytic reaction, and the current of the xenon lamp was 16 A. After 1 h of illumination, 0.5 mL of gas was extracted from the reactor with a 1 mL gas chromatograph gas syringe and injected into the gas chromatograph for product analysis and detection. Among them, H2, O2, and N2 are detected by a thermal conductivity detector. CH4 was detected by the flame ionization detector. CO passes through the flame ionization detector after being transformed by the nickel reformer. The product was qualitatively and quantitatively analyzed by gas chromatography retention time and appearance standard curve method.300 W xenon lamp  was equipped with a 420 nm cutoff wavelength filter as a light source that simulates visible light for photocatalytic CO54. Generalized gradient approximation in the scheme of Perdew-Bueke-Ernzerhof was used for the exchange-correlation functional54. The PBEsol exchange-correlation function was adopted in the optimization calculations. Grimme’s DFT-D3 scheme was used to describe the long-range vdW interactions54. The cutoff energy of the basis function was 420 eV. For the CuSnInS4 (1 1 1) crystal plane, a 2 × 2 × 1 supercell with a four-layer slab was constructed, and only the top two layers were allowed to relax. A vacuum region of 12 Å was set above the surface for periodic boundary conditions, and dipole correction was also applied. Gamma-centered 1 × 1 × 1 grid k-points were used for the interface. Geometry relaxation was performed until the energy, and atomic forces converged to be smaller than 10−5 eV and 0.03 eV/Å. Charge transfers were calculated using the Bader charge analysis55.The density functional theory calculations were performed using the VASP code with the projected augmented wave method2 reduction reaction elementary steps can be determined correspondingly by using the computational hydrogen electrode model40.The free energy of each reaction intermediate was determined by: G = E + ZPE–TS. The electronic energy was directly obtained from DFT calculations. The zero-point energy and entropy correction  were computed from vibration analysis according to standard methods. The adsorption-free energy of the adsorbates can be calculated using: ∆Gads = ∆Eads + ∆ZPE–T∆S, where ∆Eads is the adsorption energy of the adsorbates, and ∆ZPE and ∆S are the difference between ZPE and S, respectively. After the adsorption-free energies of the adsorbates are obtained, the reaction-free energies of COSupplementary InformationPeer Review FileSource data


Ultraviolet light generates cyclobutane pyrimidine dimer (CPD) and pyrimidine 6−4 pyrimidone (6−4PP) photoproducts that cause skin malignancies if not repaired by nucleotide excision repair (NER). While the faster repair of the more distorting 6–4PPs is attributed mainly to more efficient recognition by XPC, the XPD lesion verification helicase may play a role, as it directly scans the damaged DNA strand. With extensive molecular dynamics simulations of XPD-bound single-strand DNA containing each lesion outside the entry pore of XPD, we elucidate strikingly different verification processes for these two lesions that have very different topologies. The open book-like CPD thymines are sterically blocked from pore entry and preferably entrapped by sensors that are outside the pore; however, the near-perpendicular 6−4PP thymines can enter, accompanied by a displacement of the Arch domain toward the lesion, which is thereby tightly accommodated within the pore. This trapped 6−4PP may inhibit XPD helicase activity to foster lesion verification by locking the Arch to other domains. Furthermore, the movement of the Arch domain, only in the case of 6−4PP, may trigger signaling to the XPG nuclease for subsequent lesion incision by fostering direct contact between the Arch domain and XPG, and thereby facilitating repair of 6−4PP.  Exposure of human skin to ultraviolet (UV) light causes damage to DNA by converting two adjacent thymines into pyrimidine dimers, generating mutagenic and carcinogenic UV-induced photolesions, the cyclobutane pyrimidine dimer (CPD) and the pyrimidine (6−4) pyrimidone (6−4PP) photoproducts  Figure , which aTwo NER pathways eliminate the photolesions: transcription coupled NER (TC-NER) and global genomic NER (GG-NER), which differ primarily in how the lesion is initially recognized; however, they recruit the same transcription factor IIH (TFIIH) complex for lesion verification, and then converge to a common set of NER factors that form the pre-incision complex for the subsequent dual incisions, repair and ligation ,15,36–38The NER susceptibilities of diverse lesions vary greatly depending on their chemical structures, stereochemistry, physical size, conformations and base sequence context . The CPDHence, the focus of our work is to uncover whether XPD utilizes different strategies to verify the 6−4PP and CPD lesions and consider the possible impact on the different NER efficiencies of the two lesions. We therefore explored the conformational and dynamic possibilities of CPD and 6−4PP outside the entry pore of XPD. The entry pore, formed by the Arch domain together with the FeS and the ATPase lobe 1 domains Figure , is deliWe have previously generated a cryo-EM-based (PBD ID 6RO4 ) model otoward the ATPase lobe 1 helix (residues 215−221), giving a model of the XPD complexed with ssDNA containing a 6−4PP lesion (XPD-64PP-R) and a CPD lesion (XPD-CPD-R)  Figure . FurtherL Figure . These mL Figure  to permiet al., AMBER 2018, University of California, San Francisco, 2018). To explore the conformational space of the flexible DNA extension containing the lesion, we carried out ten ∼8 μs production runs (10 × 8 μs) for each model of XPD complexed with lesion-containing DNA, resulting in a total of 20 simulations for each lesion. All MD simulations began with minimization and equilibration steps that would mitigate any strains that might be present in the initial models, ensuring the systems are equilibrated during the production step. Details concerning force field, the routine MD protocol and structural analyses are given in the All XPD-ssDNA systems were subject to energy minimization, equilibration and ∼8 μs production MD using AMBER18  and 5′-base (5T) ; it adopIn all 20 simulations, our results revealed that the bases of CPD are completely inhibited from entering the pore due to their bulky topology . The CPDIn one of 20 simulations for XPD-CPD, the CPD initially positioned outside the pore undergoes a large displacement, mainly reflected in phosphodiester backbone translocation (see S1 in In 4 out 20 simulations (i.e. 20% of the population), our MD results showed that 6−4PP initially positioned outside the pore enters into the unoccupied space within the pore via translocation of its modified bases Figure ; howeverWhen the bases of the 6−4PP lesion are translocated into the pore, they form close contacts with the Arch domain and the 5′-phosphate group is anchored stably by the FeS residues R196 and/or R192, together locking the Arch domain to the FeS domain to form a closed, less flexible pore. Notably, in one of these four simulations which revealed the base translocation into the pore, we observed a significant displacement of the Arch domain from its initial state: it is shifted toward the ATPase lobe 1 and the FeS domains as well as the 3′-DNA , resultiSixteen out of 20 simulations revealed blockage of the 6–4PP lesion from entry into the unoccupied space within the pore . Unlike et al. showed that covalently crosslinking the Arch domain to the FeS domain in a closed state inactivates the helicase activity of XPD ; in both cases this occurs mainly via entrapment of the modified bases by lesion sensors, thereby supporting the function of such sensors identified in other studies , and thet al. reported that DNA unwinding by XPD was 20-fold faster in the presence of XPG than in its absence (et al. (et al. (We speculate that such a distinct displacement of the Arch domain that traps 6−4PP tightly within the tunnel may facilitate the repair of 6−4PP. In this tightly ‘trapped in the pore’ state, the perpendicular thymines of 6−4PP are clamped tightly between the Arch and the ATPase lobe1 domains and its backbone is anchored stably with the FeS domain. As a result, this trapped-in 6–4PP locks the Arch domain to the other two domains; this would inhibit the XPD helicase activity, as it would be unable to undergo the conformational change between the Arch and the FeS domains necessary to translocate DNA, as suggested by experimental studies ,80. The  absence . A recen (et al.  elucidat (et al. . Our fin (et al.  which sh (et al. ,65,66.In conclusion, we have obtained for the first time dynamic molecular characterizations of the differences in XPD’s inhibition by two topologically different UV lesions, CPD and 6−4PP via very extensive conformational sampling with long molecular dynamics simulations. We delineate lesion-dependent differences in atomistic and dynamic functioning of XPD in nucleotide excision repair, especially the roles of the Arch dynamics and lesion sensors in the dynamic mechanism of lesion verification. Furthermore, the presence of XPG has been shown to stimulate the XPD translocation activity , and XPGgkad974_Supplemental_FilesClick here for additional data file.


Removal of calcium from the cytoplasm is essential for signalling and for relaxation of skeletal muscle and heart. There are three genes and over a dozen isoforms of SERCA in mammals. These can be potentially influenced by small membrane peptides, also called regulins. The discovery of micropeptides has increased in recent years, mostly because of the small ORFs found in long RNAs, annotated formerly as noncoding (lncRNAs). Several excellent works have analysed the mechanism of interaction of micropeptides with each other and with the best known SERCA1a (fast muscle) and SERCA2a  isoforms. However, the array of tissue and developmental expressions of these potential regulators raises the question of interaction with other SERCAs. For example, the most abundant calcium pump in neonatal and regenerating skeletal muscle, SERCA1b has never been looked at with scrutiny to determine whether it is influenced by micropeptides. Further details might be interesting on the interaction of these peptides with the less studied SERCA1b isoform.Calcium is a major signalling bivalent cation within the cell. Compartmentalization is essential for regulation of calcium mediated processes. A number of players contribute to intracellular handling of calcium, among them are the sarco/endoplasmic reticulum calcium ATP-ases (SERCAs). These molecules function in the membrane of ER/SR pumping Ca The calcium handling machinery pervasively affects cellular functions ,2. Its v+/K+ membrane pump [+/K+ ATPase has more than one subunit, SERCA is a single protein of about 110 KDa having 10 or 11 transmembrane domains (except the truncated isoforms) [Ca2+) and maximal ATPase activity. Their expression displays a myriad of patterns in various tissues and developmental conditions [The name of this protein does not entirely indicate its main function: it pumps calcium ions from the sarcoplasm into the sarcoplasmic reticulum of muscle cells or from the cytoplasm into the endoplasmic reticulum of non-muscle cells. Lowering the cytoplasmic calcium concentrations can regulate a wide range of signalling processes. Decreasing sarcoplasmic calcium level makes all kinds of muscle relax. The energy required for the pumping of two calcium ions is provided by the hydrolysis of one ATP molecule into ADP . SERCA iane pump . While tnditions . SERCA1 nditions . SERCA1b2+ among these pumps. There is a SERCA2c isoform created by a small intron intrusion between exon 21 and 22 of the SERCA2a message. This results in frameshift and a truncated C-terminal of the protein which is expressed in monocyte differentiation [The other major pump, SERCA2 also has two main isoforms, 2a and 2b . SERCA2antiation . One morntiation , howeverntiation .2+ affinity. This and the localization of SERCA3 in small intracellular membrane vesicles and dense tubules closely link it to the process of store operated calcium entry (SOCE) rendered to the calcium entering unit (CEU). CEU includes Orai channels and ER/SR interacting molecule STIM  . These iiewed in ). The exiewed in .2+ transport cycle is as follows: The E1 state binds two cytosolic calcium ions one after the other from the cytoplasm in the affinity site formed by M4, M5, M6 and M8 helices, meanwhile it binds ATP in the N domain [2+ pulls the P domain to bend [Most of the three-dimensional structural knowledge of SERCA comes from the crystallization of the rabbit SERCA1a isoform  because N domain ,21. The  to bend ,21,22. T to bend ,21,22. C to bend ,21,22. T to bend ,21,22 an to bend ,20. Afte to bend ,24. The  to bend ,26. It i to bend ,20,25,262+-ATP it bends toward the A domain. The A domain turns and phosphorylates the P domain, while the two halves of the P domain bend toward each other. The calcium ions are bound by complex deformations of four transmembrane domains . The first Ca2+ binds to M8 and the second to the induced conformation of the M4 domain. The SERCA conformations described so far show rigid states and do not represent the entire conformational landscape [On the canonical structural model of SERCA1 the three cytoplasmic domains are depicted from C to N-terminal direction, in N-P-A order. The N domain sits atop the P domain while the A domain is held up at the most N-terminal part by the M1, M2 and M3 domains. During the transport cycle the three cytoplasmic domains move rigidly. When the N domain binds Mgandscape . Interesandscape ,28,29.Ca2+) have been extensively tested in microsomes (self-forming vesicles) for the different isoforms. SERCA1a has virtually identical calcium affinity but a slightly higher maxi-mum calcium uptake and ATPase activity than SERCA2a [The maximum activity (Vmax) (ATPase turnover rate) and calcium affinity  by protein kinase A (PKA) in response to adrenergic stimulation. The other parts are the hinge domain and the transmembrane domain [2+ binding sites of the pump. This explains why the Ca2+ affinity is decreased by dephosphorylated PLN without altering the transport velocity at low Ca2+ [2+ binding site of SERCA and dissociates. The phosphorylated monomers form pentamers, which have less access to the Ca2+ binding site, therefore a dynamic balance between monomers and pentamers will maintain reversible inhibition of SERCA [16) has another phosphorylation site (Thr17) that suits for calmodulin dependent kinase II and Akt. However mostly the Ser16 site is regarded as the main regulator of cardiac function. The Thr17 appears more like a tool for tuning and terminating physiological need or in pathological conditions [2+ cycling in cardiomyocytes [The name means an easy receiver of phosphate because PLN was found phosphorylated in cardiac SR when putative mediators of sympathetic stimulation were sought ,38. Targtermates . Phosphoe domain   has 31 amino acids and it is homologous to PLN. However, it consists of a significantly shorter cytoplasmic domain , a smaller transmembrane domain (~8–26 vs. ~31–49) and a highly conserved longer luminal tail (~27–31 vs. ~50–52) . It has  for PLN ,55. The  for PLN . The cry for PLN . Both SL for PLN ,56, on t for PLN . The lev for PLN . If thisIn respect of regulation, less is known about SLN than PLN. The Thr(5) residue in the cytosolic domain can be a subject of phosphorylation by CAMKII or serine threonine kinase 16 ,60. This2+ uptake rate by decreasing Ca2+ affinity of the SERCA1a pump similar to the other two micropeptides, PLN and SLN. To much surprise, the MLN transcript was abundantly expressed in every tested skeletal muscle of the hind limb and also in diaphragm and tongue of adult mice, therefore it was also co-expressed with SERCA1. However, the co-expression with SERCA2a was much weaker and found only in soleus and diaphragm, since mice in general have less slow-twitch fibres expressing this pump than fast-twitch fibres which express SERCA1a. As it has been reported previously, SLN or PLN were hardly expressed in skeletal muscles of mouse and rat in which SERCA1a or SERCA2a were abundant [Advancement in genome-wide studies suggested that hundreds of micropeptides are encoded in long RNAs (lncRNA) that have been misannotated as non-coding ones . Bioinfoabundant . This imabundant ). The reabundant . MLN theabundant ,65. Of n2+ affinity of SERCA [MLN like PLN and SLN binds to the inhibitory groove of SERCA . Howeverof SERCA . Howeverof SERCA . This is2+ is elevated the E1P and E2P states accumulate in the population of SERCA and this is the state when DWORF gradually outcompetes PLN. The increased fraction of PLN monomers then may have the chance to incorporate into pentamers. On the other hand, at low calcium DWORF binding is decreased and PLN monomers increasingly bind to SERCA which accumulates in the E1-ATP state. However, this process is rate-limited by the dissociation of PLN monomers from pentamers. The computer model in [2+ is high and this supports the increase in SERCA activity. This activity is further increased by DWORF binding to E1P and E2P, the rate limiting steps in the catalytic cycle, and by the tendency of releasing PLN monomers. The further inhibition at low Ca2+ and the stimulation at high Ca2+ can enhance the efficiency of energy use because the ATP consumption is conserved until the calcium transport is most effective and needed. Another interesting observation is, that PLN can be trapped in pentamers because of the increasing phosphorylation by PKA and CaMKII in response to adrenergic stimulation at a faster heart rate and at high Ca2+ [Dwarf Open Reading Frame (DWORF) was also discovered in a lncRNA by the same group as MLN . The murmodel in  suggesteigh Ca2+ . This miigh Ca2+ . Recentligh Ca2+ .2+ channel [2+ dysregulation [DWORF, already from its discovery tended to receive more attention than other regulins. This might be since it is expressed in heart and regulates SR calcium uptake that is pivotal in normal cardiac function. Administering synthetized rat DWORF peptide of 33–50% purity via the perfusion solution to normal isolated perfused rat hearts and hearts undergoing ischaemia/reperfusion injury increased ventricular contractile functions and reversed a Rho-kinase inhibitor-induced coronary vasodilator effect . The aut channel . Overexpgulation . A promigulation .Ca2+ similar to the effect of MLN. The authors note [2+ binding affinity. The other regulin, ELN, which might also be co-expressed with SERCA3 [2+ funnel, causing membrane perturbation and increasing the turnover rate of SERCA [The same group which discovered MLN and DWORF also found two other micropeptides in bioinformatics screening, each with a single transmembrane domain and regulating SERCA . One of ors note  that purors note . Thus, tors note ,75, mighh SERCA3 ). Althouh SERCA3 , their ch SERCA3 . It is ih SERCA3 . It is rh SERCA3  envisageh SERCA3 . The cryof SERCA ,79.+/K+ ATPase pump. However, one of the ER calcium pumps appeared to have its own self-regulatory domain. Removing exon 22 with a stop codon during splicing, the SERCA2 transcript is translated into SERCA2b isoform that is ubiquitously present in every tissue [2+-affinity). This showed M11 is an active part in regulation of SERCA2b even without the luminal extension sequence. A phylogenetic analysis showed that M11 is the most conserved part of the SERCA2b tail. Because of the similarities with the β subunit of the Na+/K+ ATPase pump, a model was proposed in which M11 interacts with M7 and M10 inducing a helix bending in M7 as a genuine regulator of the calcium pump [2+ affinity stage [2+-E1-ATP stage. Thus, the ATP is situated properly to the phosphorylation site, but actual phosphorylation does not happen until the two transmembrane Ca2+ binding sites are fully occupied. This prevents unproductive phosphorylation of the pump [Small transmembrane micropeptides are often considered as a regulatory subunit of SERCA similar to the β subunit of the Nay tissue . This puy tissue ,81  receive more regulation than the less expressed ones. Although the regulins are different from each other  they can2+ transport and calcium homeostasis. This complexity involves a balance between (self-)interactions of micropeptides and their influence on SERCA isoforms. Most of the knowledge has been collected about interactions with SERCA1a so contacts with the other isoforms might be expected to reveal additional information. The reported co-expressions of SERCA isoforms with regulins (summarized in Discovering the transmembrane micropeptides revealed a scenario with more regulation of sarco/endoplasmic Ca