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Aminotransferases (ATs) are an ancient enzyme family that play central roles in core nitrogen metabolism, essential to all organisms. However, many of the AT enzyme functions remain poorly defined, limiting our fundamental understanding of the nitrogen metabolic networks that exist in different organisms. Here, we traced the deep evolutionary history of the AT family by analyzing AT enzymes from 90 species spanning the tree of life (ToL). We found that each organism has maintained a relatively small and constant number of ATs. Mapping the distribution of ATs across the ToL uncovered that many essential AT reactions are carried out by taxon-specific AT enzymes due to wide-spread nonorthologous gene displacements. This complex evolutionary history explains the difficulty of homology-based AT functional prediction. Biochemical characterization of diverse aromatic ATs further revealed their broad substrate specificity, unlike other core metabolic enzymes that evolved to catalyze specific reactions today. Interestingly, however, we found that these AT enzymes that diverged over billion years share common signatures of multisubstrate specificity by employing different nonconserved active site residues. These findings illustrate that AT family enzymes had leveraged their inherent substrate promiscuity to maintain a small yet distinct set of multifunctional AT enzymes in different taxa. This evolutionary history of versatile ATs likely contributed to the establishment of robust and diverse nitrogen metabolic networks that exist throughout the ToL. The study provides a critical foundation to systematically determine diverse AT functions and underlying nitrogen metabolic networks across the ToL. [ABSTRACT FROM AUTHOR]

Terpenoids are one of the largest class of natural products with diverse structures and activities. This enormous diversity is embedded in enzymes called terpene synthases (TSs), which generate diverse terpene skeletons via sophisticated cyclization cascades. In addition to the many highly selective TSs, there are many promiscuous TSs that accept multiple prenyl substrates, or even noncanonical ones, with 6, 7, 8, 11, and 16 carbon atoms, synthesized via chemical approaches, C-methyltransferases, or engineered lepidopteran mevalonate pathways. The substrate promiscuity of TSs not only expands the structural diversity of terpenes but also highlights their potential for the discovery of novel terpenoids via combinatorial biosynthesis. In this review, we focus on the current knowledge on multisubstrate terpene synthases (MSTSs) and highlight their potential applications.

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Family 1 UDP‐glycosyltransferases (UGTs) are known to glycosylate multiple secondary plant metabolites and have been extensively studied. The increased availability of plant genome resources allows the identification of wide gene families, both functional and organizational. In this investigation, two MpUGT isoforms were cloned and functionally characterized from liverworts marchantia polymorpha and had high glycosylation activity against several flavonoids. MpUGT735A2 protein, in particular, tolerates a wide spectrum of substrates (flavonols, flavanones, flavones, stilbenes, bibenzyls, dihydrochalcone, phenylpropanoids, xanthones, and isoflavones). Overexpression of MpUGT735A2 and MpUGT743A1 in Arabidopsis thaliana enhances the accumulation of 3‐O‐glycosylated flavonol (kaempferol 3‐O‐glucoside‐7‐O‐rhamnose), consistent with its in vitro enzymatic activity. Docking and mutagenesis techniques were applied to identify the structural and functional properties of MpUGT735A2 with promiscuous substrates. Mutation of Pro87 to Ser, or Gln88 to Val, substantially altered the regioselectivity for luteolin glycosylation, predominantly from the 3′‐O‐ to the 7‐O‐position. The results were elucidated by focusing on the novel biocatalysts designed for producing therapeutic flavonoids. This investigation provides an approach to modulate MpUGT735A2 as a candidate gene for diverse glycosylation catalysis and a tool to design GTs with new substrate specificities for biomedical applications. [ABSTRACT FROM AUTHOR]

Wood-decaying fungi are the major decomposers of plant litter. Heavy sequencing efforts on genomes of wood-decaying fungi have recently been made due to the interest in their lignocellulolytic enzymes; however, most parts of their proteomes remain uncharted. We hypothesized that wood-decaying fungi would possess promiscuous enzymes for detoxifying antifungal phytochemicals remaining in the dead plant bodies, which can be useful biocatalysts. We designed a computational mass spectrometry–based untargeted metabolomics pipeline for the phenotyping of biotransformation and applied it to 264 fungal cultures supplemented with antifungal plant phenolics. The analysis identified the occurrence of diverse reactivities by the tested fungal species. Among those, we focused on O- xylosylation of multiple phenolics by one of the species tested, Lentinus bruma- lis. By integrating the metabolic phenotyping results with publicly available genome sequences and transcriptome analysis, a UDP-glycosyltransferase designated UGT66A1 was identified and validated as an enzyme catalyzing O-xylosylation with broad sub- strate specificity. We anticipate that our analytical workflow will accelerate the further characterization of fungal enzymes as promising biocatalysts. [ABSTRACT FROM AUTHOR]

Fungal epidithiodiketopiperazines (ETPs) possess large structural diversity and complexity due to modifications of the cyclodipeptide skeleton. Elucidation of the biosynthetic pathway of pretrichodermamide A (1) in Trichoderma hypoxylon revealed a flexible catalytic machinery of multiple enzymes for generating ETP diversity. Seven tailoring enzymes encoded by the tda cluster are involved in 1 biosynthesis, that is, four P450s TdaB and TdaQ for 1,2‐oxazine formation, TdaI for C7′‐hydroxylation, and TdaG for C4, C5‐epoxidation, two methyltransferases TdaH for C6′‐ and TdaO for C7′‐O‐methylation, and a reductase TdaD for furan opening. Gene deletions led to the identification of 25 novel ETPs, including 20 shunt products, indicating the catalytic promiscuity of Tda enzymes. Particularly, TdaG and TdaD accept various substrates and catalyze regiospecific reactions at different stages of 1 biosynthesis. Our study not only uncovers a hidden library of ETP alkaloids, but also helps to understand the hidden chemical diversity of natural products by pathway manipulation. [ABSTRACT FROM AUTHOR]

High concentrations of electrophilic lipid alkenals formed during oxidative stress are implicated in cytotoxicity and disease. However, low concentrations of alkenals are required to induce antioxidative stress responses. An established clearance pathway for lipid alkenals includes conjugation to glutathione (GSH) via Michael addition, which is catalyzed mainly by glutathione transferase isoform A4 (GSTA4-4). Based on the ability of GSTs to catalyze hydrolysis or retro-Michael addition of GSH conjugates, and the antioxidant function of low concentrations of lipid alkenals, we hypothesize that GSTA4-4 contributes a homeostatic role in lipid metabolism. Enzymatic kinetic parameters for retro-Michael addition with trans-2-Nonenal (NE) reveal the chemical competence of GSTA4-4 in this putative role. The forward GSTA4-4-catalyzed Michael addition occurs with the rapid exchange of the C2 proton of NE in D2O as observed by NMR. The isotope exchange was completely dependent on the presence of GSH. The overall commitment to catalysis, or the ratio of first order kcat,f for 'forward' Michael addition to the first order kcat,ex for H/D exchange is remarkably low, approximately 3:1. This behavior is consistent with the possibility that GSTA4-4 is a regulatory enzyme that contributes to steady-state levels of lipid alkenals, rather than a strict 'one way' detoxication enzyme. [ABSTRACT FROM AUTHOR]

SUMMARY: Hydroxycinnamic acid derivatives (HADs) such as chicoric acid are the main active ingredients of purple coneflower (Echinacea purpurea (L.) Moench). Recently, the biosynthesis of chicoric acid catalyzed by both BAHD and serine carboxypeptidase‐like (SCPL) acyltransferases has been elucidated. However, the diversity of HADs in purple coneflower and their biosynthesis pathways remain unclear. Here, through an alignment of extract ion chromatograms of potential fragments, tentative HADs, such as phaselic acid and chicoric acid analogs (deoxy‐ and methyl‐), were found in purple coneflower. Phylogenetic analyses based on the full‐length transcriptome were conducted to explore additional BAHD and SCPL acyltransferases that might be involved in HAD biosynthesis. One BAHD, conserved among Echinacea species, showed weak hydroxycinnamoyl‐CoA:tartaric acid hydroxycinnamoyl transferase (HTT) activity. Further results indicated that this BAHD was hydroxycinnamoyl‐CoA:malic acid hydroxycinnamoyl transferase (HMT), which catalyzes the biosynthesis of phaselic acid. Although no potential isozyme was found for chicoric acid synthase (CAS), CAS itself showed acyl acceptor promiscuity and catalyzed the biosynthesis of deoxychicoric acid and methylchicoric acid. Overall, we identified additional HADs and depicted their biosynthesis network in purple coneflower. The substrate promiscuity of identified acyltransferases diversifies HADs, indicating the complexity of plant secondary metabolism. [ABSTRACT FROM AUTHOR]

High concentrations of electrophilic lipid alkenals formed during oxidative stress are implicated in cytotoxicity and disease. However, low concentrations of alkenals are required to induce antioxidative stress responses. An established clearance pathway for lipid alkenals includes conjugation to glutathione (GSH) via Michael addition, which is catalyzed mainly by glutathione transferase isoform A4 (GSTA4-4). Based on the ability of GSTs to catalyze hydrolysis or retro-Michael addition of GSH conjugates, and the antioxidant function of low concentrations of lipid alkenals, we hypothesize that GSTA4-4 contributes a homeostatic role in lipid metabolism. Enzymatic kinetic parameters for retro-Michael addition with trans-2-Nonenal (NE) reveal the chemical competence of GSTA4-4 in this putative role. The forward GSTA4-4-catalyzed Michael addition occurs with the rapid exchange of the C2 proton of NE in D2O as observed by NMR. The isotope exchange was completely dependent on the presence of GSH. The overall commitment to catalysis, or the ratio of first order kcat,f for ‘forward’ Michael addition to the first order kcat,ex for H/D exchange is remarkably low, approximately 3:1. This behavior is consistent with the possibility that GSTA4-4 is a regulatory enzyme that contributes to steady-state levels of lipid alkenals, rather than a strict ‘one way’ detoxication enzyme.

Serotonin is a phylogenetically ancient compound found in animals, plants, and some bacteria. In eukaryotes, serotonin is synthesized from the aromatic amino acid tryptophan via the key enzymes aromatic amino acid hydroxylase (AAAH) and aromatic amino acid decarboxylase (AAAD). Serotonin is also an intermediate in the melatonin biosynthetic pathway and is involved in several vital functions. In humans, serotonin is produced in the gut and in the brain, is critical in the regulation of multiple body functions, and its depletion has been implicated in multiple neurological disorders including depression and Alzheimer's disease, as well as other peripheral conditions namely irritable bowel syndrome and fibromyalgia. The serotonin biosynthetic pathway is well described in eukaryotes, but very little is known about this pathway in bacteria. Evidence points to similar pathways since eukaryote-like AAAH and AAAD (and their genes) have been identified in multiple bacteria, even though serotonin production has not yet been detected in most species. Although data on bacterial tryptophan decarboxylase genes are very limited and no bacterial tryptophan hydroxylase genes have been identified to date, evidence suggests that serotonin production in bacteria might occur through different AAAH and AAAD. Substrate promiscuity in these enzymes has been previously reported and seems to be the key aspect in bacterial serotonin synthesis. Considering the human gut microbiota as a potential source of serotonin, further investigation on its biosynthetic pathways in microbes might lead to important discoveries, which may ultimately foster the development of new therapeutic strategies to treat serotonin depletion-related disorders in humans. [ABSTRACT FROM AUTHOR]

Serotonin is a phylogenetically ancient compound found in animals, plants, and some bacteria. In eukaryotes, serotonin is synthesized from the aromatic amino acid tryptophan via the key enzymes aromatic amino acid hydroxylase (AAAH) and aromatic amino acid decarboxylase (AAAD). Serotonin is also an intermediate in the melatonin biosynthetic pathway and is involved in several vital functions. In humans, serotonin is produced in the gut and in the brain, is critical in the regulation of multiple body functions, and its depletion has been implicated in multiple neurological disorders including depression and Alzheimer’s disease, as well as other peripheral conditions namely irritable bowel syndrome and fibromyalgia. The serotonin biosynthetic pathway is well described in eukaryotes, but very little is known about this pathway in bacteria. Evidence points to similar pathways since eukaryote-like AAAH and AAAD (and their genes) have been identified in multiple bacteria, even though serotonin production has not yet been detected in most species. Although data on bacterial tryptophan decarboxylase genes are very limited and no bacterial tryptophan hydroxylase genes have been identified to date, evidence suggests that serotonin production in bacteria might occur through different AAAH and AAAD. Substrate promiscuity in these enzymes has been previously reported and seems to be the key aspect in bacterial serotonin synthesis. Considering the human gut microbiota as a potential source of serotonin, further investigation on its biosynthetic pathways in microbes might lead to important discoveries, which may ultimately foster the development of new therapeutic strategies to treat serotonin depletion-related disorders in humans.

N-methylated tryptamines, such as the hallucinogenic natural products, psilocybin and N,N-dimethyltryptamine (DMT), are gaining interest from the medical community due to their potential as next generation treatments for mental health disorders. The clinical relevance of these compounds has driven scientists to develop biosynthetic production routes to a number of tryptamine drug candidates, and efforts are ongoing to expand and further develop these biosynthetic capabilities. To that end, we have further characterized the substrate preferences of two enzymes involved in tryptamine biosynthesis: TrpM, a tryptophan N-methyltransferase from Psilocybe serbica, and PsiD, the gateway decarboxylase of the psilocybin biosynthesis pathway. Here, we show that TrpM can N-methylate the non-native amino acid substrate, 4-hydroxytryptophan, a key intermediate in the Escherichia coli-based recombinant psilocybin biosynthesis pathway. However, the ability to incorporate TrpM into a functional psilocybin biosynthesis pathway was thwarted by PsiD's inability to use N,N-dimethyl-4-hydroxytryptophan as substrate, under the culturing conditions tested, despite demonstrating activity on N-methylated and 4-hydroxylated tryptophan derivatives individually. Taken together, this work expands upon the known substrates for TrpM and PsiD, further increasing the diversity of tryptamine biosynthetic products., (© 2024 The Author(s). Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

Acyclic terpenes, commonly found in plants, are of high physiological importance and commercial value, and their diversity was controlled by different terpene synthases. During the screen of sesquiterpene synthases from Tripterygium wilfordii , we observed that Ses- Tw TPS1-1 and Ses- Tw TPS2 promiscuously accepted GPP, FPP, and GGPP to produce corresponding terpene alcohols (linalool/nerolidol/geranyllinalool). The Ses- Tw TPS1-2, Ses- Tw TPS3, and Ses- Tw TPS4 also showed unusual substrate promiscuity by catalyzing GGPP or GPP in addition to FPP as substrate. Furthermore, key residues for the generation of diterpene product, (E , E)-geranyllinalool, were screened depending on mutagenesis studies. The functional analysis of Ses- Tw TPS1-1:V199I and Ses- Tw TPS1-2:I199V showed that Val in 199 site assisted the produce of diterpene product geranyllinalool by enzyme mutation studies, which indicated that subtle differences away from the active site could alter the product outcome. Moreover, an engineered sesquiterpene high-yielding yeast that produced 162 mg/L nerolidol in shake flask conditions was constructed to quickly identify the function of sesquiterpene synthases in vivo and develop potential applications in microbial fermentation. Our functional characterization of acyclic sesquiterpene synthases will give some insights into the substrate promiscuity of diverse acyclic terpene synthases and provide key residues for expanding the product portfolio. [ABSTRACT FROM AUTHOR]

Enzyme promiscuity toward substrates has been discussed in evolutionary terms as providing the flexibility to adapt to novel environments. In the present work, we describe an approach toward exploring such enzyme promiscuity in the space of a metabolic network. This approach leverages genome-scale models, which have been widely used for predicting growth phenotypes in various environments or following a genetic perturbation; however, these predictions occasionally fail. Failed predictions of gene essentiality offer an opportunity for targeting biological discovery, suggesting the presence of unknown underground pathways stemming from enzymatic cross-reactivity. We demonstrate a workflow that couples constraint-based modeling and bioinformatic tools with KO strain analysis and adaptive laboratory evolution for the purpose of predicting promiscuity at the genome scale. Three cases of genes that are incorrectly predicted as essential in Escherichia coli--aspC, argD, and gltA--are examined, and isozyme functions are uncovered for each to a different extent. Seven isozyme functions based on genetic and transcriptional evidence are suggested between the genes aspC and tyrB, argD and astC, gabT and puuE, and gltA and prpC. This study demonstrates how a targeted model-driven approach to discovery can systematically fill knowledge gaps, characterize underground metabolism, and elucidate regulatory mechanisms of adaptation in response to gene KO perturbations.

Despite the success of some nitrilases in industrial applications, there is a constant demand to broaden the catalog of these hydrolases, especially robust ones with high operational stability. By using the criteria of thermoresistance to screen a collection of candidate enzymes heterologously expressed in Escherichia coli, the enzyme Nitphym from the mesophilic organism Paraburkholderia phymatum was selected and further characterized. Its quick and efficient purification by heat treatment is of major interest for large-scale applications. The purified nitrilase displayed a high thermostability with 90% of remaining activity after 2 days at 30°C and a half-life of 18 h at 60°C, together with a broad pH range of 5.5–8.5. Its high resistance to various miscible cosolvents and tolerance to high substrate loadings enabled the quantitative conversion of 65.5 g⋅L–1 of 3-phenylpropionitrile into 3-phenylpropionic acid at 50°C in 8 h at low enzyme loadings of 0.5 g⋅L–1, with an isolated yield of 90%. This study highlights that thermophilic organisms are not the only source of industrially relevant thermostable enzymes and extends the scope of efficient nitrilases for the hydrolysis of a wide range of nitriles, especially trans-cinnamonitrile, terephthalonitrile, cyanopyridines, and 3-phenylpropionitrile.

Bacterial lipolytic enzymes of family IV are homologs of the mammalian hormone‐sensitive lipases (HSL) and have been successfully used for various biotechnological applications. The broad substrate specificity and ability for enantio‐, regio‐, and stereoselective hydrolysis are remarkable features of enzymes from this class. Many crystal structures are available for esterases and lipases, but structures of enzyme–substrate or enzyme–inhibitor complexes are less frequent although important to understand the molecular basis of enzyme–substrate interaction and to rationalize biochemical enzyme characteristics. Here, we report on the structures of a novel family IV esterase isolated from a metagenomic screen, which shows a broad substrate specificity. We solved the crystal structures in the apo form and with a bound substrate analogue at 1.35 and 1.81 Å resolution, respectively. This enzyme named PtEst1 hydrolyzed more than 60 out 96 structurally different ester substrates thus being substrate promiscuous. Its broad substrate specificity is in accord with a large active site cavity, which is covered by an α‐helical cap domain. The substrate analogue methyl 4‐methylumbelliferyl hexylphosphonate was rapidly hydrolyzed by the enzyme leading to a complete inactivation caused by covalent binding of phosphinic acid to the catalytic serine. Interestingly, the alcohol leaving group 4‐methylumbelliferone was found remaining in the active site cavity, and additionally, a complete inhibitor molecule was found at the cap domain next to the entrance of the substrate tunnel. This unique situation allowed gaining valuable insights into the role of the cap domain for enzyme–substrate interaction of esterases belonging to family IV. Database: Structural data of PtEst1 are available in the worldwide protein data bank (https://www.rcsb.org) under the accession codes: 6Z68 (apo‐PtEst1) and 6Z69 (PtEst1‐inhibitor complex). [ABSTRACT FROM AUTHOR]

Abstract Background The family of D-isomer specific 2-hydroxyacid dehydrogenases (2HADHs) contains a wide range of oxidoreductases with various metabolic roles as well as biotechnological applications. Despite a vast amount of biochemical and structural data for various representatives of the family, the long and complex evolution and broad sequence diversity hinder functional annotations for uncharacterized members. Results We report an in-depth phylogenetic analysis, followed by mapping of available biochemical and structural data on the reconstructed phylogenetic tree. The analysis suggests that some subfamilies comprising enzymes with similar yet broad substrate specificity profiles diverged early in the evolution of 2HADHs. Based on the phylogenetic tree, we present a revised classification of the family that comprises 22 subfamilies, including 13 new subfamilies not studied biochemically. We summarize characteristics of the nine biochemically studied subfamilies by aggregating all available sequence, biochemical, and structural data, providing comprehensive descriptions of the active site, cofactor-binding residues, and potential roles of specific structural regions in substrate recognition. In addition, we concisely present our analysis as an online 2HADH enzymes knowledgebase. Conclusions The knowledgebase enables navigation over the 2HADHs classification, search through collected data, and functional predictions of uncharacterized 2HADHs. Future characterization of the new subfamilies may result in discoveries of enzymes with novel metabolic roles and with properties beneficial for biotechnological applications.

Genome mining of microbial natural products enables chemists not only to discover the bioactive molecules with novel skeletons, but also to identify the enzymes that catalyze diverse chemical reactions. Exploring the substrate promiscuity and catalytic mechanism of those biosynthetic enzymes facilitates the development of potential biocatalysts. SfaB is an acyl adenylate‐forming enzyme that adenylates a unique building block, 3‐isocyanobutanoic acid, in the biosynthetic pathway of the diisonitrile natural product SF2768 produced by Streptomyces thioluteus, and this AMP‐ligase was demonstrated to accept a broad range of short‐chain fatty acids (SCFAs). Herein, we repurpose SfaB to catalyze amidation or thioesterification between those SCFAs and various amine or thiol nucleophiles, thereby providing an alternative enzymatic approach to prepare the corresponding amides and thioesters in vitro. [ABSTRACT FROM AUTHOR]

Terpenoids are one of the largest class of natural products with diverse structures and activities. This enormous diversity is embedded in enzymes called terpene synthases (TSs), which generate diverse terpene skeletons via sophisticated cyclization cascades. In addition to the many highly selective TSs, there are many promiscuous TSs that accept multiple prenyl substrates, or even noncanonical ones, with 6, 7, 8, 11, and 16 carbon atoms, synthesized via chemical approaches, C -methyltransferases, or engineered lepidopteran mevalonate pathways. The substrate promiscuity of TSs not only expands the structural diversity of terpenes but also highlights their potential for the discovery of novel terpenoids via combinatorial biosynthesis. In this review, we focus on the current knowledge on multisubstrate terpene synthases (MSTSs) and highlight their potential applications., (Copyright © 2024, Li and Tao.)

Nylon 5 and nylon 6,5 are recently explored as new commercial polyamides, of which the monomer includes δ-valerolactam. In this study, a novel catalytic activity of lysine 2-monooxygenase (DavB) was explored to produce δ-valerolactam from l-pipecolic acid (L-PA), functioning as oxidative decarboxylase on a cyclic compound. Recombinant Escherichia coli BS01 strain expressing DavB from Pseudomonas putida could synthesize δ-valerolactam from l-pipecolic acid with a concentration of 90.3 mg/L. Through the co-expression of recombinant apoptosis-inducing protein (rAIP) from Scomber japonicus, glucose dehydrogenase (GDH) from Bacillus subtilis, Δ1-piperideine-2-carboxylae reductase (DpkA) from P. putida and lysine permease (LysP) from E. coli with DavB, δ-valerolactam was produced with the highest concentration of 242 mg/L. α-Dioxygenases (αDox) from Oryza sativa could act as a similar catalyst on l-pipecolic acid. A novel δ-valerolactam synthesis pathway was constructed entirely via microbial conversion from feedstock lysine in this study. Our system has great potential in the development of a bio-nylon production process. Key points: • DavB performs as an oxidative decarboxylase on L-PA with substrate promiscuity. • Strain with rAIP, GDH, DpkA, LysP, and DavB coexpression could produce δ-valerolactam. • This is the first time to obtain valerolactam entirely via biosynthesis from lysine. [ABSTRACT FROM AUTHOR]

Cold-active enzymes increase their catalytic efficiency at low-temperature, introducing structural flexibility at or near the active sites. Inevitably, this feat seems to be accompanied by lower thermal stability. These characteristics have made cold-active enzymes into attractive targets for the industrial applications, since they could reduce the energy cost in the reaction, attenuate side-reactions, and simply be inactivated. In addition, the increased structural flexibility could result in broad substrate specificity for various non-native substrates, which is called substrate promiscuity. In this perspective, we deal with a less addressed aspect of cold-active enzymes, substrate promiscuity, which has enormous potential for semi-synthesis or enzymatic modification of fine chemicals and drugs. Further structural and directed-evolutional studies on substrate promiscuity of cold-active enzymes will provide a new workhorse in white biotechnology. [ABSTRACT FROM AUTHOR]

NotF from Aspergillus sp. MF297-2 and BrePT from Aspergillus versicolor catalyze a reverse C2-prenylation of brevianamide F in the biosynthetic pathway of brevianamides and notoamides. NotF was reported to use only brevianamide F as substrate while BrePT demonstrated broad substrate promiscuity. With high identity at amino acid level, it is interesting to reinvestigate the catalytic activities of these two prenyltransferases in vitro toward 14 cyclodipeptides. Product identification of the in vitro assays by MS proved that NotF and BrePT share similar catalytic ability and substrate promiscuity. [ABSTRACT FROM AUTHOR]

Hydroxysteroid dehydrogenases (HSDHs) are valuable biocatalysts for the regio‐ and stereoselective modification of steroids, bile acids and other steroid derivatives. In this work, we investigated the substrate promiscuity of this highly selective class of enzymes. In order to reach this goal, a preliminary search of HSDH homologues in in‐house or public available (meta)genomes was carried out. Eight novel NAD(H)‐dependent HSDHs, showing either 7α‐, 7β‐, or 12α‐HSDH activity, and including, for the first time, enzymes from extremophilic microorganisms, were identified, recombinantly produced, and characterized. Among the novel HSDHs, four highly active (up to 92 U mg−1) NAD(H)‐dependent 7β‐HSDHs showing negligible similarity towards previously described 7β‐HSDHs, were discovered. These enzymes, along with previously characterized HSDHs, were tested as biocatalysts for the stereoselective reduction of a panel of substrates including two α‐ketoesters of pharmaceutical interest and selected ketones that partially resemble the structural features of steroids. All the reactions were coupled with a suitable cofactor regeneration system. Regarding the α‐ketoesters, nearly all of the tested HSDHs showed a good activity toward the selected substrates, yielding the reduced α‐hydroxyester with up to 99% conversions and enantiomeric excesses. On the other hand, only the 7β‐HSDHs from Collinsella aerofaciens and Clostridium absonum showed appreciable activity toward more complex ketones, i. e., (±)‐trans‐1‐decalone, but with interesting as well as different selectivity. [ABSTRACT FROM AUTHOR]

Promiscuity is the coincidental ability of an enzyme to catalyze its native reaction and additional reactions that are not biological functions in the same active site. Promiscuity plays a central role in enzyme evolution and is thus a useful property for protein and metabolic engineering. This review examines enzyme evolution holistically, beginning with evaluating biochemical support for four enzyme evolution models. As expected, there is strong biochemical support for the subfunctionalization and innovation–amplification–divergence models, in which promiscuity is a central feature. In many cases, however, enzyme evolution is more complex than the models indicate, suggesting much is yet to be learned about selective pressures on enzyme function. A complete understanding of enzyme evolution must also explain the ability of metabolic networks to integrate new enzyme activities. Hidden within metabolic networks are underground metabolic pathways constructed from promiscuous activities. We discuss efforts to determine the diversity and pervasiveness of underground metabolism. Remarkably, several studies have discovered that some metabolic defects can be repaired via multiple underground routes. In prokaryotes, metabolic innovation is driven by connecting enzymes acquired by horizontal gene transfer (HGT) into the metabolic network. Thus, we end the review by discussing how the combination of promiscuity and HGT contribute to evolution of metabolism in prokaryotes. Future studies investigating the contribution of promiscuity to enzyme and metabolic evolution will need to integrate deeper probes into the influence of evolution on protein biophysics, enzymology, and metabolism with more complex and realistic evolutionary models. Enzymes: lactate dehydrogenase (EC 1.1.1.27), malate dehydrogenase (EC 1.1.1.37), OSBS (EC 4.2.1.113), HisA (EC 5.3.1.16), TrpF, PriA (EC 5.3.1.24), R‐mandelonitrile lyase (EC 4.1.2.10), Maleylacetate reductase (EC 1.3.1.32). [ABSTRACT FROM AUTHOR]

Enzyme promiscuity can be defined as the capability of enzymes to catalyse side reaction in addition to its main reaction. The side reaction of an enzyme is termed as promiscuous or sometimes as the "darker" side of enzyme cross-reactivity/specificity. This unique property of enzyme allows organisms to adapt under varying environmental conditions. Promiscuous enzymes can modify their catalytic activities with altered substrates and can adjust their catalytic and kinetic mechanisms according to substrate properties. This group of enzymes evolved from ancestral proteins found in primitive organisms like archaea that survive under extreme environmental conditions. Such ancestral proteins possessed the potential to catalyse a wide range of reactions at low levels, hence create families or superfamilies of highly specialized enzymes. Further, some enzymes were identified which have non-catalytic functions in addition to their major catalytic activities. These enzymes are referred to as moonlighting enzymes. The study of these enzymes will provide important information regarding enzyme evolution and will help in optimizing protein engineering applications. [ABSTRACT FROM AUTHOR]

• Identification of glycosyltransferases from Bacillus subtilis converting HQNO. • Kinetic parameters of YjiC, YdhE and Yojk towards HQNO and UDP-glucose. • Identification of glucosylated plant and Pseudomona s derived antimicrobials. • Comparison of NDP-glucose and aglycon promiscuities. Glycosylation of natural and synthetic products can alter the physical, chemical and pharmacological properties of the aglycon. Conversion of 2-heptyl-1-hydroxyquinolin-4-one (HQNO), a potent respiratory inhibitor produced by Pseudomonas aeruginosa , to the less toxic 2-heptyl-1-(β-D-glucopyranosydyl)-quinolin-4-one, was recently demonstrated for Bacillus subtilis strain 168. In this study, we compared the genomes of several Bacillus spp. to identify candidate enzymes for HQNO glucosylation. All three (putative) UDP-glycosyltransferases (GT) of B. subtili s 168 tested, YjiC, YdhE and YojK, were capable of HQNO glucosylation, with YjiC showing the highest turnover rate (k cat) of 4.6 s−1, and YdhE exhibiting the lowest K m value for HQNO of 9.1 μM. All three GT predominantly utilized UDP-glucose, but YdhE was similarly active with TDP-glucose. Among the aglycons tested, HQNO was the preferred substrate of all three GT, but they also showed activities toward the P. aeruginosa exoproducts pyocyanin, 2-heptyl-3-hydroxyquinolin-4(1 H)-one (the Pseudomonas quinolone signal) and 2,4-dihydroxyquinoline, the plant derived antimicrobials vanillin and quercetin, and the macrolide antibiotic tylosin A. Our results underline the promiscuity and substrate flexibility of YjiC, YdhE and YojK, and suggest a physiological role in natural toxin resistance of B. subtilis. Especially YdhE appears to be an attractive biocatalyst for the glycoengineering of natural products. [ABSTRACT FROM AUTHOR]

CotA laccases are multicopper oxidases known for promiscuously oxidizing a broad range of substrates. However, studying substrate promiscuity is limited by the complexity of electron transfer (ET) between substrates and laccases. Here, a systematic analysis of factors affecting ET including electron donor acceptor coupling (Η DA), driving force (ΔG) and reorganization energy (λ) was done. Catalysis rates of syringic acid (SA), syringaldehyde (SAD) and acetosyringone (AS) (kcat(SAD) > kcat(SA) > kcat(AS)) are not entirely dependent on the ability to form phenol radicals indicated by ΔG and λ calculated by Density Functional Theory (SA < SAD ≈ AS). In determined CotA/SA and CotA/SAD structures, SA and SAD bound at 3.9 and 3.7 Å away from T1 Cu coordinating His419 ensuring a similar Η DA. Abilities of substrate to form phenol radicals could mainly account for difference between kcat(SAD) and kcat(SA). Furthermore, substrate pocket is solvent exposed at the para site of substrate's phenol hydroxyl, which would destabilize binding of AS in the same orientation and position resulting in low kcat. Our results indicated shallow partially covered binding site with propensity of amino acids distribution might help CotA discriminate lignin-phenol derivatives. These findings give new insights for developing specific catalysts for industrial application. [ABSTRACT FROM AUTHOR]

The addition of water to non‐activated carbon–carbon double bonds catalyzed by fatty acid hydratases (FAHYs) allows for highly regio‐ and stereoselective oxyfunctionalization of renewable oil feedstock. So far, the applicability of FAHYs has been limited to free fatty acids, mainly owing to the requirement of a carboxylate function for substrate recognition and binding. Herein, we describe for the first time the hydration of oleic acid (OA) derivatives lacking this free carboxylate by the oleate hydratase from Elizabethkingia meningoseptica (OhyA). Molecular docking of OA to the OhyA 3D‐structure and a sequence alignment uncovered conserved amino acid residues at the entrance of the substrate channel as target positions for enzyme engineering. Exchange of selected amino acids gave rise to OhyA variants which showed up to an 18‐fold improved conversion of OA derivatives, while retaining the excellent regio‐ and stereoselectivity in the olefin hydration reaction. [ABSTRACT FROM AUTHOR]

Over several decades, glucosinolates have become a model system for the study of specialized metabolic diversity in plants. The near-complete identification of biosynthetic enzymes, regulators, and transporters has provided support for the role of gene duplication and subsequent changes in gene expression, protein function, and substrate specificity as the evolutionary bases of glucosinolate diversity. Here, we provide examples of how whole-genome duplications, gene rearrangements, and substrate promiscuity potentiated the evolution of glucosinolate biosynthetic enzymes, regulators, and transporters by natural selection. This in turn may have led to the repeated evolution of glucosinolate metabolism and diversity in higher plants. [ABSTRACT FROM AUTHOR]

The substrate specificity of enzymes is bound to be imperfect, because of unavoidable physicochemical limits. In extant metabolic enzymes, furthermore, such limits are seldom approached, suggesting that the degree of specificity of these enzymes, on average, is much lower than could be attained. During biological evolution, the activity of a single enzyme with available alternative substrates may be preserved to a significant or even substantial level for different reasons – for example when the alternative reaction contributes to fitness, or when its undesirable products are nevertheless dispatched by metabolite repair enzymes. In turn, the widespread occurrence of promiscuous reactions is a consistent source of metabolic 'messiness', from which both liabilities and opportunities ensue in the evolution of metabolic systems. Highlights Substrate specificity cannot be absolute and is inherently limited. The maximum capacity to discriminate between alternative substrates can be relatively low, and in any case it is seldom approached owing to evolutionary constraints. In some enzymes of primary metabolism, substrate promiscuity is favored, although this may interfere with high flux and efficient regulation. In other cases, enzymes acting on alternative substrates generate toxic or useless products; however, these can be destroyed or recycled by repair enzymes. The limited substrate specificity of enzymes often results in the production of non-standard metabolites which contribute to the complexity of the metabolome. Substrate promiscuity helps to fuel an 'underground' network of reactions which may represent a basis for further evolution and diversification of metabolism. [ABSTRACT FROM AUTHOR]

Substrate and reaction promiscuity is a remarkable property of some enzymes and facilitates the adaptation to new metabolic demands in the evolutionary process. Substrate promiscuity is also a basis for protein engineering for biocatalysis. However, molecular principles of enzyme promiscuity are not well understood. Even for the widely studied PLP-dependent transaminases of class III, the reliable prediction of the biocatalytically important amine transaminase activity is still difficult if the desired activity is unrelated to the natural activity. Here, we show that 7,8-diaminopelargonic acid transaminase (synthase), previously considered to be highly specific, is able to convert (S)-(-)-1-phenylethylamine and a number of aldehydes and diketones. We were able to characterize the (S)-amine transaminase activity of 7,8-diaminopelargonic acid transaminase from Psychrobacter cryohalolentis (Pcryo361) and analyzed the three-dimensional structure of the enzyme. New substrate specificity for α-diketones was observed, though only a weak activity towards pyruvate was found. We examined the organization of the active site and binding modes of S-adenosyl-L-methionine and (S)-(-)-1-phenylethylamine using X-ray analysis and molecular docking. We suggest that the Pcryo361 affinity towards (S)-(-)-1-phenylethylamine arises from the recognition of the hydrophobic parts of the specific substrates, S-adenosyl-L-methionine and 7-keto-8-aminopelargonic acid, and from the flexibility of the active site. Our results support the observation that the conversion of amines is a promiscuous activity of many transaminases of class III and is independent from their natural function. The analysis of amine transaminase activity from among various transaminases will help to make the sequence-function prediction for biocatalysis more reliable. [ABSTRACT FROM AUTHOR]

Cold-active enzymes increase their catalytic efficiency at low-temperature, introducing structural flexibility at or near the active sites. Inevitably, this feat seems to be accompanied by lower thermal stability. These characteristics have made cold-active enzymes into attractive targets for the industrial applications, since they could reduce the energy cost in the reaction, attenuate side-reactions, and simply be inactivated. In addition, the increased structural flexibility could result in broad substrate specificity for various non-native substrates, which is called substrate promiscuity. In this perspective, we deal with a less addressed aspect of cold-active enzymes, substrate promiscuity, which has enormous potential for semi-synthesis or enzymatic modification of fine chemicals and drugs. Further structural and directed-evolutional studies on substrate promiscuity of cold-active enzymes will provide a new workhorse in white biotechnology.

Cytochrome P450 3A4 (CYP3A4) metabolizes a wide range of drugs and toxins. Interactions of CYP3A4 with ligands are difficult to predict due to promiscuity and conformational flexibility. To better understand CYP3A4 conformational responses to ligands we use hydrogen deuterium exchange mass spectrometry (HDX-MS) to investigate the effect of ligands on nanodisc-embedded CYP3A4. For a subset of CYP3A4-ligand complexes, differences in the low-frequency modes derived by principal component analyses of molecular dynamics trajectories mirrored the HDX-MS results. The effects of ligands are distributed to flexible elements of CYP3A4 between stretches of secondary structure. The largest effects occur in the F- and G-helices, where most ligands increase the flexibility of the F-helix and connecting loops and decrease the flexibility of the C-term of the G-helix. Most ligands affect the E -F-G, C D and H I regions of the protein. Ligand-dependent differences are observed in the A"-A' loop, B C region, E -helix, K-β1 region, proximal loop, and C-term loop. Correlated HDX responses were observed in the C D region and the C-term of the G-helix that were most pronounced for Type II ligands. Collectively, the HDX and molecular dynamics results suggest that CYP3A4 accommodates diverse binding partners by propagating local backbone fluctuations from the binding site onto the flexible regions of the enzyme via long-range interactions that are differentially modulated by ligands. In contrast to the paradigm wherein ligands decrease protein dynamics at their binding site, a wide range of ligands modestly increase CYP3A4 dynamics throughout the protein including effects remote from the active site. Superimposition of average structures of CYP3A4 from GaMD simulations of the ligand-free CYP3A4 (tan), CYP3A4-Azimulin complex (blue), and CYP3A4-Ritonavir complex (pink) illustrating the differences in the packing of the F-helix and E -F loop against the E-helix. MD and H/DX mass spectrometry confirm regions uniquely or universally affected by various ligands. [Display omitted] • CYP3A4 is a highly promiscuous detoxication enzyme. • H/DX MS reveals CYP3A4 ligands cause globally distributed changes in dynamics. • Results are supported by MD simulations and principle component analysis. • Ligands induce an increase in local dynamics far from the binding site. [ABSTRACT FROM AUTHOR]

Plant NADPH-dependent glyoxylate/succinic semialdehyde reductases 1 and 2 (cytosolic GLYR1 and plastidial/mitochondrial GLYR2) are considered to be of particular importance under abiotic stress conditions. Here, the apple (Malus × domestica Borkh.) and rice (Oryza sativa L.) GLYR1s and GLYR2s were characterized and their kinetic properties were compared to those of previously characterized GLYRs from Arabidopsis thaliana [L.] Heynh. The purified recombinant GLYRs had an affinity for glyoxylate and succinic semialdehyde, respectively, in the low micromolar and millimolar ranges, and were inhibited by NADP+. Comparison of the GLYR activity in cell-free extracts from wild-type Arabidopsis and a glyr1 knockout mutant revealed that approximately 85 and 15% of the cellular GLYR activity is cytosolic and plastidial/mitochondrial, respectively. Recovery of GLYR activity in purified mitochondria from the Arabidopsis glyr1 mutant, free from cytosolic GLYR1 or plastidial GLYR2 contamination, provided additional support for the targeting of GLYR2 to mitochondria, as well as plastids. The growth of plantlets or roots of various Arabidopsis lines with altered GLYR activity responded differentially to succinic semialdehyde or glyoxylate under chilling conditions. Taken together, these findings highlight the potential regulation of highly conserved plant GLYRs by NADPH/NADP+ ratios in planta, and their roles in the reduction of toxic aldehydes in plants subjected to chilling stress.

The substrate specificity of enzymes is crucial to control the fate of metabolites to different pathways. However, there is growing evidence that many enzymes can catalyze alternative reactions. This promiscuous behavior has important implications in protein evolution and the acquisition of new functions. The question is how the undesirable outcomes of in vivo promiscuity can be prevented. ADP-glucose pyrophosphorylase from Escherichia coli is an example of an enzyme that needs to select the correct substrate from a broad spectrum of alternatives. This selection will guide the flow of carbohydrate metabolism toward the synthesis of reserve polysaccharides. Here, we show that the allosteric activator fructose-1,6-bisphosphate plays a role in such selection by increasing the catalytic efficiency of the enzyme toward the use of ATP rather than other nucleotides. In the presence of fructose-1,6-bisphosphate, the kcat/S0.5 for ATP was near ~600-fold higher that other nucleotides, whereas in the absence of activator was only ~3-fold higher. We propose that the allosteric regulation of certain enzymes is an evolutionary mechanism of adaptation for the selection of specific substrates.

Plant NADPH-dependent glyoxylate/succinic semialdehyde reductases 1 and 2 (cytosolic GLYR1 and plastidial/mitochondrial GLYR2) are considered to be of particular importance under abiotic stress conditions. Here, the apple (Malus × domestica Borkh.) and rice (Oryza sativa L.) GLYR1s and GLYR2s were characterized and their kinetic properties were compared to those of previously characterized GLYRs from Arabidopsis thaliana [L.] Heynh. The purified recombinant GLYRs had an affinity for glyoxylate and succinic semialdehyde, respectively, in the low micromolar and millimolar ranges, and were inhibited by NADP+. Comparison of the GLYR activity in cell-free extracts from wild-type Arabidopsis and a glyr1 knockout mutant revealed that approximately 85 and 15% of the cellular GLYR activity is cytosolic and plastidial/mitochondrial, respectively. Recovery of GLYR activity in purified mitochondria from the Arabidopsis glyr1 mutant, free from cytosolic GLYR1 or plastidial GLYR2 contamination, provided additional support for the targeting of GLYR2 to mitochondria, as well as plastids. The growth of plantlets or roots of various Arabidopsis lines with altered GLYR activity responded differentially to succinic semialdehyde or glyoxylate under chilling conditions. Taken together, these findings highlight the potential regulation of highly conserved plant GLYRs by NADPH/NADP+ ratios in planta, and their roles in the reduction of toxic aldehydes in plants subjected to chilling stress. [ABSTRACT FROM AUTHOR]

Glycosylated small molecules are often bioactive and obtained mainly via microbial biotransformation especially by fungi. However, no responsible glycosylation gene/enzyme has yet been uncovered in a filamentous fungus. We report here the first identification of a phenolic glycosyltransferase MhGT1 from Mucor hiemalis. The substrate promiscuity of the new phenolic O-glycosyltransferase was explored by using phenols from Traditional Chinese Medicinal herbs as substrates. MhGT1 exhibited robust capabilities for the regio- and stereospecific O-glycosylation of 72 structurally diverse drug-like scaffolds and sterols with uridine diphosphate (UDP) glucose as a sugar donor. Unprecedentedly, MhGT1 showed higher regiospecificities and activities for prenylated phenols than for their non-prenylated analogues. Computational modelling of MhGT1 uncovered a truncated N-terminal domain of the enzyme consisting of hydrophobic and charged amino acid residues which contributed to the broad substrate scope and regiospecificity towards prenylated compounds. Our findings expand the ways to obtain new glycosyltransferases and also effectively apply the enzymatic approach to obtain glycosylated compounds in drug discovery. [ABSTRACT FROM AUTHOR]

Summary Gene-directed enzyme-prodrug therapy (GDEPT) is a promising anti-cancer strategy. However, inadequate prodrugs, inefficient prodrug activation, and a lack of non-invasive imaging capabilities have hindered clinical progression. To address these issues, we used a high-throughput Escherichia coli platform to evolve the multifunctional nitroreductase E. coli NfsA for improved activation of a promising next-generation prodrug, PR-104A, as well as clinically relevant nitro-masked positron emission tomography-imaging probes EF5 and HX4, thereby addressing a critical and unmet need for non-invasive bioimaging in nitroreductase GDEPT. The evolved variant performed better in E. coli than in human cells, suggesting optimal usefulness in bacterial rather than viral GDEPT vectors, and highlighting the influence of intracellular environs on enzyme function and the shaping of promiscuous enzyme activities within the “black box” of in vivo evolution. We provide evidence that the dominant contribution to improved PR-104A activity was enhanced affinity for the prodrug over-competing intracellular substrates. [ABSTRACT FROM AUTHOR]

Nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) represents the main subtype of the NPP family of nucleotide hydrolyzing enzymes. The ecto-enzyme hydrolyzes structurally diverse substrates and has recently been proposed as a drug target for immuno-oncology. To get more insights into the nature of the promiscuity of NPP1, we investigated its substrate preferences employing a broad range of natural nucleotides including ATP, UTP, diadenosine tetraphosphate (AP 4 A), cAMP, and cyclic guanosine-(2′,5′)-monophosphate-adenosine-(3″,5″)-monophosphate (2′,3″-cGAMP), as well as the artificial substrate p -nitrophenyl 5′-thymidine monophosphate ( p -Nph-5′-TMP). Despite their diverse structures, all substrates were converted to nucleoside 5′-monophosphates; 2′,3″-cGAMP yielded exclusively the nucleoside 5′-monophosphates AMP and GMP. In contrast, 3′,3″-bridged cyclic dinucleotides were not hydrolyzed. ATP was the most efficiently hydrolyzed substrate of NPP1, followed by AP 4 A and 2′,3″-cGAMP. UTP, cAMP and p -Nph-5′-TMP were much poorer substrates. A homology model of the human NPP1 was built based on the X-ray structure of its mouse orthologue. Docking studies were performed based on previously published mutagenesis data to rationalize the interactions of the different substrates and to explain the enzyme's preferences. The results provide an improved understanding of the interactions of NPP1 with its diverse substrates and will contribute to the validation of NPP1 as a drug target. [ABSTRACT FROM AUTHOR]

Wood-decaying fungi are the major decomposers of plant litter. Heavy sequencing efforts on genomes of wood-decaying fungi have recently been made due to the interest in their lignocellulolytic enzymes; however, most parts of their proteomes remain uncharted. We hypothesized that wood-decaying fungi would possess promiscuous enzymes for detoxifying antifungal phytochemicals remaining in the dead plant bodies, which can be useful biocatalysts. We designed a computational mass spectrometry-based untargeted metabolomics pipeline for the phenotyping of biotransformation and applied it to 264 fungal cultures supplemented with antifungal plant phenolics. The analysis identified the occurrence of diverse reactivities by the tested fungal species. Among those, we focused on O -xylosylation of multiple phenolics by one of the species tested, Lentinus brumalis . By integrating the metabolic phenotyping results with publicly available genome sequences and transcriptome analysis, a UDP-glycosyltransferase designated UGT66A1 was identified and validated as an enzyme catalyzing O -xylosylation with broad substrate specificity. We anticipate that our analytical workflow will accelerate the further characterization of fungal enzymes as promising biocatalysts.

Amide N -methylation is important for the activity and permeability of bioactive compounds but can be challenging to perform selectively. The broad-spectrum antimicrobial natural products thiolutin and holomycin differ only by an N -methyl group at the endocyclic amide of thiolutin, but only thiolutin exhibits antifungal activity. The enzyme responsible for amide N- -methylation in thiolutin biosynthesis has remained elusive. Here, we identified and characterized the amide N -methyltransferase DtpM that is encoded >400 kb outside of the thiolutin gene cluster. DtpM catalyzes efficient conversion of holomycin to thiolutin, exhibits broad substrate scope toward dithiolopyrrolones, and has high thermal stability. In addition, sequence similarity network analysis suggests DtpM is more closely related to phenol O -methyltransferases than some amide methyltransferases. This study expands the limited examples of amide N -methyltransferases and may facilitate chemoenzymatic synthesis of diverse dithiolopyrrolone compounds as potential therapeutics.

The presence of latent activities in enzymes is posited to underlie the natural evolution of new catalytic functions. However, the prevalence and extent of such substrate and catalytic ambiguity in evolved enzymes is difficult to address experimentally given the order-of-magnitude difference in the activities for native and, sometimes, promiscuous substrate/s. Further, such latent functions are of special interest when the activities concerned do not fall into the domain of substrate promiscuity. In the present study, we show a special case of such latent enzyme activity by demonstrating the presence of two mechanistically distinct reactions catalysed by the catalytic domain of receptor protein tyrosine phosphatase isoform δ (PTPRδ). The primary catalytic activity involves the hydrolysis of a phosphomonoester bond (C—O—P) with high catalytic efficiency, whereas the secondary activity is the hydrolysis of a glycosidic bond (C—O—C) with poorer catalytic efficiency. This enzyme also displays substrate promiscuity by hydrolysing diester bonds while being highly discriminative for its monoester substrates. To confirm these activities, we also demonstrated their presence on the catalytic domain of protein tyrosine phosphatase Ω (PTPRΩ), a homologue of PTPRδ. Studies on the rate, metal-ion dependence, pH dependence and inhibition of the respective activities showed that they are markedly different. This is the first study that demonstrates a novel sugar hydrolase and diesterase activity for the phosphatase domain (PD) of PTPRδ and PTPRΩ. This work has significant implications for both understanding the evolution of enzymatic activity and the possible physiological role of this new chemistry. Our findings suggest that the genome might harbour a wealth of such alternative latent enzyme activities in the same protein domain that renders our knowledge of metabolic networks incomplete. [ABSTRACT FROM AUTHOR]

Secondary multidrug efflux transporters play a key role in the bacterial resistance phenotype. One of the major questions concerns the polyspecific recognition of substrates by these efflux pumps. To understand the molecular basis of this promiscuous recognition, we compared the substrate specificity of the well-studied Escherichia coli small multidrug resistance protein EmrE with that of the poorly studied Acinetobacter baumannii homologue AbeS. The latter drug/H + antiporter is a 109-amino-acid membrane protein with predicted four transmembrane helices. It effectively confers resistance toward ethidium, acriflavine and benzalkonium in an E . coli ΔemrEΔmdfA background. Purified AbeS and the substrate-specific hyperactive variant A16G bind tetraphenylphosphonium with nanomolar affinity and exhibit electrogenic transport of 1-methyl-4-phenylpyridinium after reconstitution into liposomes. A16G hyperactivity was apparent toward acriflavine and ethidium, resulting in 7- to 10-fold higher normalized IC 50 values, respectively, but not toward substrates 1-methyl-4-phenylpyridinium and benzalkonium. Substitution of Y3 and A42 with Ala or Ser, respectively, also displayed a substrate-dependent phenotype, as these variants were strongly affected in their properties to confer resistance against acriflavine and ethidium, but not against benzalkonium. The size and planarity of the conjugated aromatic moieties appear to be a critical and subtle criterion for substrate recognition by these transporters. Rather moderate changes in the property of side chains postulated to be part of the substrate binding site result in a large phenotypical difference. These observations provide indications for the molecular basis of specificity within the binding pocket of polyspecific transporters. [ABSTRACT FROM AUTHOR]

Glycosylation determines the complexity and diversity of plant natural products. To characterize fruit ripeningrelated UDP-dependent glycosyltransferases (UGTs) functionally in strawberry, we mined the publicly available Fragaria vesca genome sequence and found 199 putative UGT genes. Candidate UGTs whose expression levels were strongly up-regulated during fruit ripening were cloned from F.×ananassa and six were successfully expressed in Escherichia coli and biochemically characterized. UGT75T1 showed very strict substrate specificity and glucosylated only galangin out of 33 compounds. The other recombinant enzymes exhibited broad substrate tolerance, accepting numerous flavonoids, hydroxycoumarins, naphthols and the plant hormone, (+)-S-abscisic acid (ABA). UGT71W2 showed the highest activity towards 1-naphthol, while UGT71A33, UGT71A34a/b and UGT71A35 preferred 3- hydroxycoumarin and formed 3- and 7-O-glucosides as well as a diglucoside from flavonols. Screening of a strawberry physiological aglycone library identified kaempferol, quercetin, ABA and three unknown natural compounds as putative in planta substrates of UGT71A33, UGT71A34a and UGT71W2. Metabolite analyses of RNA interference (RNAi)- mediated silenced fruits demonstrated that UGT71W2 contributes to the glycosylation of flavonols, xenobiotics and, to a minor extent, of ABA, in planta. The study showed that both specialist and generalist UGTs were expressed during strawberry fruit ripening and the latter were probably not restricted to only one function in plants. [ABSTRACT FROM AUTHOR]

• Functional characterization of five different UGDH isoforms in G. uralensis. • Enzyme kinetics study of the isoforms. • Substrate promiscuity exploration of the isoforms. • Molecular docking, MD-Simulation, and binding free energy calculation. Uridine diphosphate glucose dehydrogenase (UGDH) has a crucial role in the synthesis of UDP-glucuronic acid (UDPGA), which is the important sugar donor in the glucuronosylation of bioactive triterpene saponins in the traditional Chinese medicine Glycyrrhiza uralensis Fisch. UGDHs usually exist as isoforms in plants, but knowledge of their unique binding sites and patterns remains largely incomplete. Here, we mined five UGDH isoforms in G. uralensis transcriptome that catalyze the oxidation of UDP-glucose (UDPG) to UDPGA and had different catalytic efficiencies. Molecular modeling and molecular dynamics simulation revealed the potential catalytic residues for the catalytic efficiencies of the isoforms. Moreover, the UGDH isoforms exhibited substrate promiscuity by catalyzing two structurally diverse UDPG derivatives, which was further demonstrated by docking studies. This work gives new insights into the UGDH isoforms involved in the biosynthesis of glucuronides in G. uralensis , and extends the understanding of the versatility of UGDHs. [ABSTRACT FROM AUTHOR]