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Process intensification is crucial for industrial implementation of biocatalysis and can be achieved by continuous process operation in miniaturized reactors with efficiently immobilized biocatalysts, enabling their long-term use. Due to their extremely large surface-to-volume ratio, nanomaterials are promising supports for enzyme immobilization. In this work, different functionalized nanofibrous nonwoven membranes were embedded in a two-plate microreactor to enable immobilization of hexahistidine (His 6 )-tagged amine transaminases (ATAs) in flow. A membrane coated with Cu 2+ ions gave the best results regarding His 6 -tagged ATAs immobilization among the membranes tested yielding an immobilization yield of up to 95.3 % for the purified N-His 6 -ATA-wt enzyme. Moreover, an efficient one-step enzyme immobilization process from overproduced enzyme in Escherichia coli cell lysate was developed and yielded enzyme loads up to 1088 U mL -1 . High enzyme loads resulted in up to 80 % yields of acetophenone produced from 40 mM (S)-α-methylbenzylamine in less than 4 min using a continuously operated microreactor. Up to 81 % of the initial activity was maintained in a 5-day continuous microreactor operation with immobilized His 6 -tagged ATA constructs. The highest turnover number within the indicated time was 7.23·10 6 , which indicates that this immobilization approach using advanced material and reactor system is highly relevant for industrial implementation., Competing Interests: Declaration of Competing Interest On behalf of all the authors of a submission I hereby disclose no competing interest. Prof. Dr. Polona Žnidaršič-Plazl., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

(R)-selective transaminases have the potential to act as efficient biocatalysts for the synthesis of important pharmaceutical intermediates. However, their low catalytic efficiency and unfavorable equilibrium limit their industrial application. Seven (R)-selective transaminases were identified using homologous sequence mining. Beginning with the optimal candidate from Mycolicibacterium hippocampi, virtual mutagenesis and substrate tunnel engineering were performed to improve catalytic efficiency. The obtained variant, T282S/Q137E, exhibited 3.68-fold greater catalytic efficiency (k cat /K m ) than the wild-type enzyme. Using substrate fed-batch and air sweeping processes, effective conversion of 100 mM 4-hydroxy-2-butanone was achieved with a conversion rate of 93 % and an ee value > 99.9 %. This study provides a basis for mutation of (R)-selective transaminases and offers an efficient biocatalytic process for the asymmetric synthesis of (R)-3-aminobutanol., Competing Interests: Declaration of competing interest All authors disclosed no relevant relationships., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In this study, food-grade glutamine transaminase (TGase) was utilized for the green-catalyzed preparation of N-butyryl amino acids. For improving the reusability of the enzyme preparation, immobilized TG enzyme (94.23% immobilization rate) was prepared. Furthermore, the yield of N-butyryl phenylalanine (BP) synthesized by TGase was obtained as 20.73% by one-factor experiment. The BP synthesis yield of immobilized TGase was 95.03% of that of TGase and remained above 60% of the initial enzyme activity after five runs. The sensory evaluation and E-tongue results showed that the addition of BP significantly increased the umami, saltiness, and richness intensities of the samples, and decreased the intensities of sourness, bitterness, and aftertaste-B. The molecular docking results indicated that hydrogen bonding dominated the binding of BP to taste receptors in the taste presentation mechanism of BP. These results confirmed the potential of BP as a flavor enhancer with promising applications in the food industry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Transaminases (EC 2.6.1.X, TAs) are important biocatalysts in the synthesis of chiral amines, and have significant value in the field of medicine. However, TAs suffer from low enzyme activity and poor catalytic efficiency in the synthesis of chiral amines or non-natural amino acids, which hinders their industrial applications. In this study, a novel TA derived from Paracoccus pantotrophus (ppTA) that was investigated in our previous study was employed with a semi-rational design strategy to improve its enzyme activity to 2-ketobutyrate. By using homology modeling and molecular docking, four surrounding sites in the substrate-binding S pocket were selected as potential mutational sites. Through alanine scanning and saturation mutagenesis, the optimal mutant V153A with significantly improved enzyme activity was finally obtained, which was 578 % higher than that of the wild-type ppTA (WT). Furthermore, the mutant enzyme ppTA-V153A also exhibited slightly improved temperature and pH stability compared to WT. Subsequently, the mutant was used to convert 2-ketobutyrate for the preparation of L-2-aminobutyric acid (L-ABA). The mutant can tolerate 300 mM 2-ketobutyrate with a conversion rate of 74 %, which lays a solid foundation for the preparation of chiral amines., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study explores a combination of the concept of enantioselective enzymatic synthesis of β-chiral amines through transamination with in situ product crystallization (ISPC) to overcome product inhibition. Using 2-phenylpropanal as a readily available and easily racemizing substrate of choice, (R)-β-methylphenethylamine ((R)-2-phenylpropan-1-amine) concentrations of up to 250 mM and enantiomeric excesses of up to 99 % are achieved when using a commercially available transaminase from Ruegeria pomeroyi in a fed-batch based dynamic kinetic resolution reaction on preparative scale. The source of substrate decomposition during the reaction is also investigated and the resulting unwanted byproduct formation is successfully reduced to insignificant levels., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Biobased furans have emerged as chemical building blocks for the development of materials because of their diverse scaffolds and as they can be directly prepared from sugars. However, selective, efficient, and cost-effective scalable conversion of biobased furans remains elusive. Here, we report a robust transaminase (TA) from Shimia marina (SMTA) that enables the scalable amination of biobased furanaldehydes with high activity and broad substrate specificity. Crystallographic and mutagenesis analyses provide mechanistic insights and a structural basis for understanding SMTA, which enables a higher substrate conversion. The enzymatic cascade process established in this study allows one-pot synthesis of 2,5-bis(aminomethyl)furan (BAMF) and 5-(aminomethyl)furan-2-carboxylic acid from 5-hydroxymethylfurfural. The biosynthesis of various furfurylamines, including a one-pot cascade reaction for BAMF generation using whole cells, demonstrates their practical application in the pharmaceutical and polymer industries., (© 2024. The Author(s).)

In the field of chiral amine synthesis, ω-amine transaminase (ω-ATA) is one of the most established enzymes capable of asymmetric amination under optimal conditions. However, the applicability of ω-ATA toward more non-natural complex molecules remains limited due to its low transamination activity, thermostability, and narrow substrate scope. Here, by employing a combined approach of computational virtual screening strategy and combinatorial active-site saturation test/iterative saturation mutagenesis strategy, we have constructed the best variant M14C3-V5 (M14C3-V62A-V116S-E117I-L118I-V147F) with improved ω-ATA from Aspergillus terreus ( At ATA) activity and thermostability toward non-natural substrate 1-acetylnaphthalene, which is the ketone precursor for producing the intermediate ( R )-(+)-1-(1-naphthyl)ethylamine [( R )-NEA] of cinacalcet hydrochloride, showing activity enhancement of up to 3.4-fold compared to parent enzyme M14C3 ( At ATA-F115L-M150C-H210N-M280C-V149A-L182F-L187F). The computational tools YASARA, Discovery Studio, Amber, and FoldX were applied for predicting mutation hotspots based on substrate-enzyme binding free energies and to show the possible mechanism with features related to At ATA structure, catalytic activity, and stability in silico analyses. M14C3-V5 achieved 71.8% conversion toward 50 mM 1-acetylnaphthalene in a 50 mL preparative-scale reaction for preparing ( R )-NEA. Moreover, M14C3-V5 expanded the substrate scope toward aromatic ketone compounds. The generated virtual screening strategy based on the changes in binding free energies has successfully predicted the At ATA activity toward 1-acetylnaphthalene and related substrates. Together with experimental data, these approaches can serve as a gateway to explore desirable performances, expand enzyme-substrate scope, and accelerate biocatalysis.IMPORTANCEChiral amine is a crucial compound with many valuable applications. Their asymmetric synthesis employing ω-amine transaminases (ω-ATAs) is considered an attractive method. However, most ω-ATAs exhibit low activity and stability toward various non-natural substrates, which limits their industrial application. In this work, protein engineering strategy and computer-aided design are performed to evolve the activity and stability of ω-ATA from Aspergillus terreus toward non-natural substrates. After five rounds of mutations, the best variant, M14C3-V5, is obtained, showing better catalytic efficiency toward 1-acetylnaphthalene and higher thermostability than the original enzyme, M14C3. The robust combinational variant acquired displayed significant application value for pushing the asymmetric synthesis of aromatic chiral amines to a higher level., Competing Interests: The authors declare no conflict of interest.

ω-Transaminases (ω-TAs) are attractive biocatalysts asymmetrically catalyzing ketones to chiral amines. However, poor non-native catalytic activity and substrate promiscuity severely hamper its wide application in industrial production. Protein engineering efforts have generally focused on reshaping the substrate-binding pockets of ω-TAs. However, hotspots around the substrate tunnel as well as distant sites outside the pockets may also affect its activity. In this study, the ω-TA from Bacillus megaterium (BmeTA) was selected for engineering. The tunnel mutation Y164F synergy with distant mutation A245T which was acquired through a multiple sequence alignment showed improved soluble expression, a 3.7-fold higher specific activity and a 19.9-fold longer half-life at 45 °C. Molecule Dynamics simulation explains the mechanism of improved catalytic activity, enhanced thermostability and improved soluble expression of BmeTA Y164F/A245T (2 M). Finally, the resting cells of 2 M were used for biocatalytic processes. 450 mM of S-methoxyisopropylamine (S-MOIPA) was obtained with an ee value of 97.3 % and a conversion rate of 90 %, laying the foundation for its industrial production. Mutant 2 M was also found to be more advantageous in catalyzing the transamination of various ketones. These results demonstrated that sites that are far away from the active center also play an important role in the redesign of ω-TAs., (© 2024 Wiley-VCH GmbH.)

Transaminases are choice biocatalysts for the synthesis of chiral primary amines, including amino acids bearing contiguous stereocenters. In this study, we employ lysine as a "smart" amine donor in transaminase-catalyzed dynamic kinetic resolution reactions to access β-branched noncanonical arylalanines. Our mechanistic investigation demonstrates that, upon transamination, the lysine-derived ketone byproduct readily cyclizes to a six-membered imine, driving the equilibrium in the desired direction and thus alleviating the need to load superstoichiometric quantities of the amine donor or deploy a multienzyme cascade. Lysine also shows good overall compatibility with a panel of wild-type transaminases, a promising hint of its application as a smart donor more broadly. Indeed, by this approach, we furnished a broad scope of β-branched arylalanines, including some bearing hitherto intractable cyclopropyl and isopropyl substituents, with high yields and excellent selectivities.

Structure-function relationships are key to understanding enzyme mechanisms, controlling enzyme activities, and designing biocatalysts. Here, we investigate the functions of arginine residues in the active sites of pyridoxal-5'-phosphate (PLP)-dependent non-canonical d-amino acid transaminases, focusing on the analysis of a transaminase from Haliscomenobacter hydrossis. Our results show that the tandem of arginine residues R28* and R90, which form the conserved R-[RK] motif in non-canonical d-amino acid transaminases, not only facilitates effective substrate binding but also regulates the catalytic properties of PLP. Non-covalent interactions between residues R28*, R90, and Y147 strengthen the hydrogen bond between Y147 and PLP, thereby maintaining the reactivity of the cofactor. Next, the R90 residue contributes to the stability of the holoenzyme. Finally, the R90I substitution induces structural changes that lead to substrate promiscuity, as evidenced by the effective binding of substrates with and without the α-carboxylate group. This study sheds light on the structural determinants of the activity of non-canonical d-amino acid transaminases. Understanding the structural basis of the active site plasticity in the non-canonical transaminase from H. hydrossis, which is characterized by effective conversion of d-amino acids and α-keto acids, may help to tailor it for industrial applications., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Amino acid transferase is a family of enzymes used to catalyze and separate chiral amino acids. However, due to the low efficiency, by-products and reverse reactions occur in cascade reactions. Therefore, in the research, phenylglycine aminotransferase and aspartate aminotransferase were self-assembled in vitro by leucine zipper. The self-assembled enzyme system with d-phenylglycine and α-ketoglutarate as substrates were used for the chiral transformation reaction. By studying the enzyme combination, kinetic reaction stability and catalytic efficiency, it was found that the self-assembled enzyme showed improved stability and better affinity to the substrate than the control and achieved only ee value of 17.86% for the control at the substrate ratio was 1:2. In contrast, the self-assembled enzyme basically catalyzed the complete conversion of d-Phg to l-Phg, with the ee value as 99%. These results demonstrated the feasibility of the leucine zipper and the conversion of d-phenylglycine to the l-type by fusion enzyme., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Raman spectroscopy has been used to measure the concentration of a pharmaceutically relevant model amine intermediate for positive allosteric modulators of nicotinic acetylcholine receptor in a ω-transaminase-catalyzed conversion. A model based on a one-dimensional convolutional neural network was developed to translate raw data augmented Raman spectra directly into substrate concentrations, with which the conversion from ketone to amine by ω-transaminase could be determined over time. The model showed very good predictive capabilities, with R 2 values higher than 0.99 for the spectra included in the modeling and 0.964 for an independent dataset. However, the model could not extrapolate outside the concentrations specified by the model. The presented work shows the potential of Raman spectroscopy as a real-time monitoring tool for biocatalytic reactions., (© 2024 The Authors. Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

(R)-selective transaminases show promise as catalysts for the asymmetric synthesis of chiral amines, which are building blocks of various small molecule drugs. However, their application is limited by poor substrate acceptance and low catalytic efficiency. Here, a potential (R)-selective transaminase from Fodinicurvata sediminis (FsTA) was identified through a substrate truncating strategy, and used as starting point for enzyme engineering toward catalysis of 4-hydroxy-2-butanone, a substrate that poses challenges in catalysis. Molecular docking and dynamics simulations revealed Y90 as the key residue responsible for poor substrate binding. Starting from the variant (Y90F, mut1) with initial activity, FsTA was systematically modified to improve substrate-binding through active site reshaping and consensus sequence strategy, yielding three variants (H30R, V152K, and Y156F) with improved activity. A quadruple mutation variant H30R/Y90F/V152K/Y156F (mut4) was also found to show a 7.95-fold greater catalytic efficiency (k cat /K M ) than the initial variant mut1. Furthermore, mut4 also enhanced the thermostability of enzyme significantly, with the T m value increasing by 10 °C. This variant also exhibited significantly improved activity toward a series of ketones that are either not accepted or poorly accepted by the wild-type. This study provides a basis for the rational design of an active to creating variants that can accommodate novel substrates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

( S )-1-(2-fluorophenyl) ethylamine plays a crucial role as a chiral building block in pharmaceutical synthesis. ω-transaminases are widely recognized as environmentally friendly and efficient catalysts for the preparation of chiral amines. In this study, we isolated a novel ω-transaminase, Pf TA, from Pseudogulbenkiania ferrooxidans through gene mining in the NCBI database. By employing semi-rational design, we obtained a Y168R/R416Q variant with enhanced enzyme activity. This variant exhibited the ability to catalyze the synthesis of ( S )-1-(2-fluorophenyl) ethylamine from 2-fluorophenone, achieving a yield of 83.58% and an enantioselectivity exceeding 99% after a 10 h reaction. Compared to the wild type, the specific enzyme activity of the Y168R/R416Q variant reached 47.04 U/mg, which represents an increase of 11.65 times. Additionally, the catalytic efficiency, as measured by k cat / K m , was increased by 20.9 times. Molecular docking and structural simulation analysis revealed that the primary factor contributing to the improved catalytic efficiency is the expansion of the enzyme's active pocket and the alleviation of steric hindrance.

Primary hyperoxaluria type 1 (PH1) is a childhood-onset autosomal recessive disease, characterized by nephrocalcinosis, multiple recurrent urinary calcium oxalate stones, and a high risk of progressive kidney damage. PH1 is caused by inherent genetic defects of the alanine glyoxylate aminotransferase (AGXT) gene. The in vivo repair of disease-causing genes was exceedingly inefficient before the invention of base editors which can efficiently introduce precisely targeted base alterations without double-strand DNA breaks. Adenine base editor (ABE) can precisely convert A·T to G·C with the assistance of specific guide RNA. Here, we demonstrated that systemic delivery of dual adeno-associated virus encoding a split-ABE8e could artificially repair 13% of the pathogenic allele in Agxt Q84X rats, a model of PH1, alleviating the disease phenotype. Specifically, ABE treatment partially restored the expression of alanine-glyoxylate-aminotransferase (AGT), reduced endogenous oxalate synthesis and alleviated calcium oxalate crystal deposition. Western blot and immunohistochemistry confirmed that ABE8e treatment restored AGT protein expression in hepatocytes. Moreover, the precise editing efficiency in the liver remained stable six months after treatment. Thus, our findings provided a prospect of in vivo base editing as a personalized and precise medicine for PH1 by directly correcting the mutant Agxt gene., (Copyright © 2023 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Primary hyperoxaluria type I (PH1) is caused by deficient alanine:glyoxylate aminotransferase (AGT) activity. PH1-causing mutations in AGT lead to protein mistargeting and aggregation. Here, we use hydrogen-deuterium exchange (HDX) to characterize the wild-type (WT), the LM (a polymorphism frequent in PH1 patients) and the LM G170R (the most common mutation in PH1) variants of AGT. We provide the first experimental analysis of AGT structural dynamics, showing that stability is heterogeneous in the native state and providing a blueprint for frustrated regions with potentially functional relevance. The LM and LM G170R variants only show local destabilization. Enzymatic transamination of the pyridoxal 5-phosphate cofactor bound to AGT hardly affects stability. Our study, thus, supports that AGT misfolding is not caused by dramatic effects on structural dynamics., (© 2024 Federation of European Biochemical Societies.)

Methanobactin (Mbn) is a ribosomally synthesized and post-translationally modified peptide (RiPP) natural product that binds Cu(I) with high affinity. The copper-chelating thioamide/oxazolone groups in Mbn are installed on the precursor peptide MbnA by the core enzyme complex, MbnBC, which includes the multinuclear non-heme iron-dependent oxidase (MNIO) MbnB and its RiPP recognition element-containing partner protein MbnC. For the extensively characterized Mbn biosynthetic gene cluster (BGC) from the methanotroph Methylosinus trichosporium OB3b, the tailoring aminotransferase MbnN further modifies MbnA after leader sequence cleavage by an unknown mechanism. Here we detail methods to express and purify M. trichosporium OB3b MbnBC and MbnN along with protocols for assessing MbnA modification by MbnBC and MbnN aminotransferase activity. In addition, we describe crystallization and structure determination of MbnBC. These procedures can be adapted for other MNIOs and partner proteins encoded in Mbn and Mbn-like BGCs. Furthermore, these methods provide a first step toward in vitro biosynthesis of Mbns and related natural products as potential therapeutics., (Copyright © 2024. Published by Elsevier Inc.)

The application of (R)-ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by inadequate stereoselectivity and narrow substrate spectrum. Herein, an effective evolution strategy for (R)-ω-transaminase designing for the asymmetric synthesis of sitagliptin intermediate is presented. Since natural transaminases lack activity toward bulky prositagliptin ketone, transaminase scaffolds with catalytic machinery and activity toward the truncated prositagliptin ketone were firstly screened based on substrate walking principle. A transaminase chimera was established synchronously conferring catalytic activity and (R)-selectivity toward prositagliptin ketone through motif swapping, followed by stepwise evolution. The process resulted in a "best" engineered variant MwTA M8 , which exhibited 79.2-fold higher activity than the chimeric scaffold MwTA Mc . Structural analysis revealed that the heightened activity is mainly due to the enlarged and adaptive substrate pocket and tunnel. The novel (R)-transaminase exhibited unsatisfied industrial operation stability, which is expected to further modify the protein to enhance its tolerance to temperature, pH, and organic solvents to meet sustainable industrial demands. This study underscores a useful evolution strategy of engineering biocatalysts to confer new properties and functions on enzymes for synthesizing high-value drug intermediates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this work., (Copyright © 2023 Elsevier B.V. All rights reserved.)

An enzyme cascade was established previously consisting of a recycling system with an l-amino acid oxidase (hcLAAO4) and a catalase (hCAT) for different α-keto acid co-substrates of (S)-selective amine transaminases (ATAs) in kinetic resolutions of racemic amines. Only 1 mol % of the co-substrate was required and l-amino acids instead of α-keto acids could be applied. However, soluble enzymes cannot be reused easily. Immobilization of hcLAAO4, hCAT and the (S)-selective ATA from Vibrio fluvialis (ATA-Vfl) was addressed here. Immobilization of the enzymes together rather than on separate beads showed higher reaction rates most likely due to fast co-substrate channeling between ATA-Vfl and hcLAAO4 due to their close proximity. Co-immobilization allowed further reduction of the co-substrate amount to 0.1 mol % most likely due to a more efficient H 2 O 2 -removal caused by the stabilized hCAT and its proximity to hcLAAO4. Finally, the co-immobilized enzyme cascade was reused in 3 cycles of preparative kinetic resolutions to produce (R)-1-PEA with high enantiomeric purity (97.3 %ee). Further recycling was inefficient due to the instability of ATA-Vfl, while hcLAAO4 and hCAT revealed high stability. An engineered ATA-Vfl-8M was used in the co-immobilized enzyme cascade to produce (R)-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethanamine, an apremilast-intermediate, with a 1,000 fold lower input of the co-substrate., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

ω-transaminase (ω-TA) is a natural biocatalyst that has good application potential in the synthesis of chiral amines. However, the poor stability and low activity of ω-TA in the process of catalyzing unnatural substrates greatly hampers its application. To overcome these shortcomings, the thermostability of ( R )-ω-TA ( At TA) from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation. An optimal mutant At TA-E104D/A246V/R266Q (M3) with synchronously enhanced thermostability and activity was obtained. Compared with the wild- type (WT) enzyme, the half-life t 1/2 (35 ℃) of M3 was prolonged by 4.8-time (from 17.8 min to 102.7 min), and the half deactivation temperature ( T 10 50 ) was increased from 38.1 ℃ to 40.3 ℃. The catalytic efficiencies toward pyruvate and 1-( R )-phenylethylamine of M3 were 1.59- and 1.56-fold that of WT. Molecular dynamics simulation and molecular docking showed that the reinforced stability of α-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability. The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3. Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT, which further showed the application potential of M3 in the synthesis of chiral amines.

D-cycloserine inhibits pyridoxal-5'-phosphate (PLP)-dependent enzymes. Inhibition effect depend on organization of the active site and mechanism of the catalyzed reaction. D-cycloserine interacts with the PLP form of the enzyme similarly to the substrate (amino acid), and this interaction is predominantly reversible. Several products of the interaction of PLP with D-cycloserine are known. For some enzymes formation of a stable aromatic product - hydroxyisoxazole-pyridoxamine-5'-phosphate at certain pH - leads to irreversible inhibition. The aim of this work was to study the mechanism of D-cycloserine inhibition of the PLP-dependent D-amino acid transaminase from Haliscomenobacter hydrossis. Spectral methods revealed several products of interaction of D-cycloserine with PLP in the active site of transaminase: oxime between PLP and β-aminooxy-D-alanine, ketimine between pyridoxamine-5'-phosphate and cyclic form of D-cycloserine, and pyridoxamine-5'-phosphate. Formation of hydroxyisoxazole-pyridoxamine-5'-phosphate was not observed. 3D structure of the complex with D-cycloserine was obtained using X-ray diffraction analysis. In the active site of transaminase, a ketimine adduct between pyridoxamine-5'-phosphate and D-cycloserine in the cyclic form was found. Ketimine occupied two positions interacting with different active site residues via hydrogen bonds. Using kinetic and spectral methods we have shown that D-cycloserine inhibition is reversible, and activity of the inhibited transaminase from H. hydrossis could be restored by adding excess of keto substrate or excess of cofactor. The obtained results confirm reversibility of the inhibition by D-cycloserine and interconversion of various adducts of D-cycloserine and PLP.

ω-Transaminases (ω-TAs) are widely available for the production of chiral amines and unnatural amino acids. Herein, a rapid spectrophotometric method was developed for screening ω-TAs based on the colored products that can be generated from transamination reactions between aliphatic α-diketones and amino donors catalyzed by ω-TAs. The possible mechanism of the formation of the colored product was investigated according to LC-Q-TOF-MS analysis. Among seven diketones, 2,3-butanedione was selected as the most suitable amino acceptor for colorimetric screening of ω-TAs with high efficiency, high sensitivity, and low background interference. Meanwhile, the absorbance of the colored product generated by 2,3-butanedione catalyzed by ω-TAs in this method was linearly correlated with the results by HPLC analysis. This method was also confirmed to effectively screen ω-TA mutants with high activity towards isopropylamine., (© 2023. Springer-Verlag GmbH Germany, part of Springer Nature.)

Organisms from all kingdoms of life synthesize L-serine (L-Ser) from 3-phosphoglycerate through the phosphorylated pathway, a three-step diversion of glycolysis. Phosphoserine aminotransferase (PSAT) catalyzes the intermediate step, the pyridoxal 5'-phosphate-dependent transamination of 3-phosphohydroxypyruvate and L-glutamate to O-phosphoserine (OPS) and α-ketoglutarate. PSAT is particularly relevant in the central nervous system of mammals because L-Ser is the metabolic precursor of D-serine, cysteine, phospholipids, and nucleotides. Several mutations in the human psat gene have been linked to serine deficiency disorders, characterized by severe neurological symptoms. Furthermore, PSAT is overexpressed in many tumors and this overexpression has been associated with poor clinical outcomes. Here, we report the detailed functional and structural characterization of the recombinant human PSAT. The reaction catalyzed by PSAT is reversible, with an equilibrium constant of about 10, and the enzyme is very efficient, with a k cat /K m of 5.9 × 10 6  M -1  s -1 , thus contributing in driving the pathway towards the products despite the extremely unfavorable first step catalyzed by 3-phosphoglycerate dehydrogenase. The 3D X-ray crystal structure of PSAT was solved in the substrate-free as well as in the OPS-bound forms. Both structures contain eight protein molecules in the asymmetric unit, arranged in four dimers, with a bound cofactor in each subunit. In the substrate-free form, the active site of PSAT contains a sulfate ion that, in the substrate-bound form, is replaced by the phosphate group of OPS. Interestingly, fast crystal soaking used to produce the substrate-bound form allowed the trapping of different intermediates along the catalytic cycle., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Aminotransferases (ATs) catalyze pyridoxal 5'-phosphate-dependent transamination reactions between amino donor and keto acceptor substrates and play central roles in nitrogen metabolism of all organisms. ATs are involved in the biosynthesis and degradation of both proteinogenic and nonproteinogenic amino acids and also carry out a wide variety of functions in photorespiration, detoxification, and secondary metabolism. Despite the importance of ATs, their functionality is poorly understood as only a small fraction of putative ATs, predicted from DNA sequences, are associated with experimental data. Even for characterized ATs, the full spectrum of substrate specificity, among many potential substrates, has not been explored in most cases. This is largely due to the lack of suitable high-throughput assays that can screen for AT activity and specificity at scale. Here we present a new high-throughput platform for screening AT activity using bioconjugate chemistry and mass spectrometry imaging-based analysis. Detection of AT reaction products is achieved by forming an oxime linkage between the ketone groups of transaminated amino donors and a probe molecule that facilitates mass spectrometry-based analysis using nanostructure-initiator mass spectrometry or MALDI-mass spectrometry. As a proof-of-principle, we applied the newly established method and found that a previously uncharacterized Arabidopsis thaliana tryptophan AT-related protein 1 is a highly promiscuous enzyme that can utilize 13 amino acid donors and three keto acid acceptors. These results demonstrate that this oxime-mass spectrometry imaging AT assay enables high-throughput discovery and comprehensive characterization of AT enzymes, leading to an accurate understanding of the nitrogen metabolic network., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Published by Elsevier Inc.)

Histidinol-phosphate aminotransferase is the sixth protein (hence HISN6) in the histidine biosynthetic pathway in plants. HISN6 is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the reversible conversion of imidazole acetol phosphate into L-histidinol phosphate (HOLP). Here, we show that plant HISN6 enzymes are closely related to the orthologs from Chloroflexota. The studied example, HISN6 from Medicago truncatula (MtHISN6), exhibits a surprisingly high affinity for HOLP, which is much higher than reported for bacterial homologs. Moreover, unlike the latter, MtHISN6 does not transaminate phenylalanine. High-resolution crystal structures of MtHISN6 in the open and closed states, as well as the complex with HOLP and the apo structure without PLP, bring new insights into the enzyme dynamics, pointing at a particular role of a string-like fragment that oscillates near the active site and participates in the HOLP binding. When MtHISN6 is compared to bacterial orthologs with known structures, significant differences arise in or near the string region. The high affinity of MtHISN6 appears linked to the particularly tight active site cavity. Finally, a virtual screening against a library of over 1.3 mln compounds revealed three sites in the MtHISN6 structure with the potential to bind small molecules. Such compounds could be developed into herbicides inhibiting plant HISN6 enzymes absent in animals, which makes them a potential target for weed control agents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Transaminases capable of carrying out chiral selective transamination of 1-(3-methylphenyl)ethan-1-one to (1R)-(3-methylphenyl)ethan-1-amine were screened, and ATA-025 was the best enzyme, while dimethylsulfoxide (10% V/V) was the best co-solvent for said bioconversion. The variables such as enzyme loading, substrate loading, temperature, and pH for development of process displaying maximum conversion with good product formation and higher yield were optimized. The ambient processing conditions were 10% enzyme loading/50 g/L substrate loading/45 °C/pH 8.0, and 5% enzyme loading/36.78 g/L substrate loading/42.66 °C/pH 8.2 displaying maximum conversion 99.01 ± 2.47% and 96.115 ± 1.97%, and 76.93 ± 1.05% and 73.12 ± 1.04% yield with one factor at a time approach and numerical optimization with Box Behnken Design, respectively. In the final optimized reaction, ATA-025 showed the highest 99.22 ± 2.61% conversion, 49.55 g/L product formation, with an actual product recovery of 38.16 g corresponding to a product yield 77.03 ± 1.01% with respect to the product formed after reaction. The purity of recovered product (1R)-(3-methylphenyl)ethan-1-amine formed was ≥ 99% (RP-HPLC), and chiral purity ≥ 98.5% (Chiral-GC), and it was also confirmed and characterized with instrumental methods using boiling point, LC-MS, ATR-FTIR, and 1 H NMR. The findings of 'What If' studies performed by investigating timely progress of reaction on gram scale by drastically changing the process parameters revealed a substantial modification in process variables to achieve desired results. (1R)-(3-methylphenyl)ethan-1-amine synthesized by green, facile and novel enzymatic approach with an optimized process could be used for synthesis of different active pharma entities., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Pyridoxal-5'-phosphate-(PLP-) dependent D-amino acid transaminases (DAATs) catalyze stereoselective reversible transfer of the amino group between D-amino acids and keto acids. In vivo DAATs are commonly known to synthesize D-glutamate for cell wall peptidoglycans. Today DAATs meet increasing attention for application in the synthesis of D-amino acids, whereas little is known about the mechanism of substrate recognition and catalytic steps of the D-amino acids conversion by DAATs. In this work, the pre-steady-state kinetics of the half-reactions of DAAT from Haliscomenobacter hydrossis with D-glutamate, D-alanine, D-leucine, and D-phenylalanine was examined at two wavelengths, 416 and 330 nm, using a stopped-flow technique. Monophasic kinetics was observed with specific substrates D-glutamate and D-alanine, whereas half-reactions with D-leucine and D-phenylalanine exhibited biphasic kinetics. All half-reactions proceeded until the complete conversion of PLP due to the release of the pyridoxamine-5'-phosphate form of cofactor from the holoenzyme . Comparison of kinetic parameters of half-reactions and the overall transamination reactions for D-leucine, D-phenylalanine revealed the increase in the rates of deamination of these substrates in the overall reaction with α-ketoglutarate. In the overall transamination reaction, the catalytic turnover rates for D-leucine and D-phenylalanine increased by 260 and 60 times, correspondingly, comparing with the slowest step rate constants in the half-reactions. We suggested the activating effect by a specific substrate α-ketoglutarate in the overall transamination reaction. The study of half-reactions helped to quantify the specificity of DAAT from H. hydrossis for D-amino acids with different properties. The results obtained are the first detailed analysis of half-reactions catalyzed by DAAT., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Aromatic aminotransferases (Aro ATs) are pyridoxal-5-phosphate (PLP)-dependent enzymes that catalyze the transamination reactions of an aromatic amino acid (AAA) or a keto acid. Aro ATs are involved in biosynthesis or degradation of AAAs and play important functions in controlling the production of plant hormones and secondary metabolites, such as auxin, tocopherols, flavonoids, and lignin. Most Aro ATs show substrate promiscuity and can accept multiple aromatic and non-aromatic amino and keto acid substrates, which complicates and limits our understanding of their in planta functions. Considering the critical roles Aro ATs play in plant primary and secondary metabolism, it is important to accurately determine substrate specificity and kinetic properties of Aro ATs. This chapter describes various methodologies of protein expression, purification and enzymatic assays, which can be used for biochemical characterization of Aro ATs., (Copyright © 2023 Elsevier Inc. All rights reserved.)

ω-Transaminases (ω-TAs) show considerable potential for the synthesis of chiral amines. However, their low catalytic efficiency towards bulky substrates limits their application, and complicated catalytic mechanisms prevent precise enzyme design. Herein, we address this challenge using a mechanism-guided computational enzyme design strategy by reprograming the transition and ground states in key reaction steps. The common features among the three high-energy-barrier steps responsible for the low catalytic efficiency were revealed using quantum mechanics (QM). Five key residues were simultaneously tailored to stabilize the rate-limiting transition state with the aid of the Rosetta design. The 14 top-ranked variants showed 16.9-143-fold improved catalytic activity. The catalytic efficiency of the best variant, M9 (Q25F/M60W/W64F/I266A), was significantly increased, with a 1660-fold increase in k cat /K m and a 1.5-26.8-fold increase in turnover number (TON) towards various indanone derivatives., (© 2022 Wiley-VCH GmbH.)

Tuberculosis (TB) is one of the most dangerous infectious diseases and is caused by Mycobacterium bovis (Mb) and Mycobacterium tuberculosis (Mt). Branched-chain amino acid aminotransferases (BCATs) were reported to be the key enzyme for methionine synthesis in Mycobacterium. Blocking the methionine synthesis in Mycobacterium can inhibit the growth of Mycobacterium. Therefore, in silico screening of inhibitors can be a good way to develop a potential drug for treating TB. A pyridoxal 5'-phosphate (PLP)-form of Mycobacterium bovis branched-chain amino acid aminotransferases (MbBCAT), an active form of MbBCAT, was constructed manually for docking approximately 150 000 compounds and the free energy was calculated in Autodock Vina. The 10 compounds which had the highest affinity to MbBCAT were further evaluated for their inhibitory effects against MbBCAT. Within the selected compounds, compound 4 (ZINC12359007) was found to be the best inhibitor against MbBCAT with the inhibitory constant K i of 0·45 μmol l -1 and IC 50 of 2·37 μmol l -1 . Our work provides potential candidates to develop effective drugs to prevent TB since the well-known structural information would be beneficial in the structure-based modification and design., (© 2022 Society for Applied Microbiology.)

This study was to screen a strain with both branched-chain amino acid transaminase (BCAT) and protease activities for producing methyl-branched flavor compounds in a myofibrillar protein extract model. Lactic acid bacteria (LAB) was isolated from Jinhua ham and screened with BCAT activity by an electrochemical sensor and protease activity by the agar plate method. In the culture medium, strain L6 was selected with high utilization rate and characteristic metabolites content of branched-chain amino acids (BCAAs), and identified as Lactobacillus fermentum YZU-06 (L. fermentum). Compared with the previously reported L. plantarum, L. fermentum exhibited an excellent capacity of hydrolyzing myofibrillar protein with the higher contents of free amino acids, peptides and small molecular weight proteins. Moreover, L. fermentum group presented more BCAAs metabolites of 2-methylbutanal and 3-methylbutanal than that of L. plantarum group. In conclusion, L. fermentum YZU-06 is a promising starter culture to improve the flavor of fermented meat products., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Enzymology, the study of enzyme structures and reaction mechanisms can be considered a classical discipline. However, enzymes cannot be freely designed to catalyze desired reactions yet, and enzymology is by no means a complete science. I have long studied the reaction mechanisms of enzymes related to amino acid metabolism, such as aminotransferases and racemases, which depend on pyridoxal 5'-phosphate, a coenzyme form of vitamin B6. During these studies, I have often been reminded that enzymatic reactions are extremely sophisticated processes based on chemical principles and enzyme structures, and have often been amazed at the evolutionary mechanisms that bestowed them with such structures. In this review, I described the reaction mechanism of various pyridoxal enzymes especially related to d-amino acids metabolism, whose roles in mammals have recently attracted attention. I hope to convey some of the significance and interest in enzymology through this review., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Novel functions can emerge in an enzyme family while conserving catalytic mechanism, motif or fold. Pyridoxal 5'-phosphate-dependent enzymes have evolved into seven fold-types and catalyze diverse reactions using the same mechanism for the formation of external aldimine. Nucleotide sugar aminotransferases (which will be henceforth referred to as aminotransferases) belong to fold type I and mediate the biosynthesis of several monosaccharides. They use diverse substrates but are highly selective to the C3 or C4 carbon to which amine group is transferred. Profile hidden Markov models (HMMs) were able to identify aminotransferases but could not capture reaction specificity. A search for discriminating features led to the discovery of sequence motifs that are located near the pyranose binding site suggesting their role in imparting reaction specificity. Using a position weight matrix for this motif, we were able to assign reaction specificity to a large number of aminotransferases. Inferences from this analysis set way for future experiments that can shed light on mechanisms of functional diversification in nucleotide sugar aminotransferases of fold type I., (© 2022 Wiley Periodicals LLC.)

Ornithine δ-aminotransferase (Orn-AT) activity was detected for the enzyme annotated as a γ-aminobutyrate aminotransferase encoded by PH1423 gene from Pyrococcus horikoshii OT-3. Crystal structures of this novel archaeal ω-aminotransferase were determined for the enzyme in complex with pyridoxal 5'-phosphate (PLP), in complex with PLP and l-ornithine (l-Orn), and in complex with N-(5'-phosphopyridoxyl)-l-glutamate (PLP-l-Glu). Although the sequence identity was relatively low (28%), the main-chain coordinates of P. horikoshii Orn-AT monomer showed notable similarity to those of human Orn-AT. However, the residues recognizing the α-amino group of l-Orn differ between the two enzymes. In human Orn-AT, Tyr55 and Tyr85 recognize the α-amino group, whereas the side chains of Thr92* and Asp93*, which arise from a loop in the neighboring subunit, form hydrogen bonds with the α-amino group of the substrate in P. horikoshii enzyme. Site-directed mutagenesis suggested that Asp93* plays critical roles in maintaining high affinity for the substrate. This study provides new insight into the substrate binding of a novel type of Orn-AT. Moreover, the structure of the enzyme with the reaction-intermediate analogue PLP-l-Glu bound provides the first structural evidence for the "Glu switch" mechanism in the dual substrate specificity of Orn-AT., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The conformational landscape of a protein is constantly expanded by genetic variations that have a minimal impact on the function(s) while causing subtle effects on protein structure. The wider the conformational space sampled by these variants, the higher the probabilities to adapt to changes in environmental conditions. However, the probability that a single mutation may result in a pathogenic phenotype also increases. Here we present a paradigmatic example of how protein evolution balances structural stability and dynamics to maximize protein adaptability and preserve protein fitness. We took advantage of known genetic variations of human alanine:glyoxylate aminotransferase (AGT1), which is present as a common major allelic form (AGT-Ma) and a minor polymorphic form (AGT-Mi) expressed in 20% of Caucasian population. By integrating crystallographic studies and molecular dynamics simulations, we show that AGT-Ma is endowed with structurally unstable (frustrated) regions, which become disordered in AGT-Mi. An in-depth biochemical characterization of variants from an anticonsensus library, encompassing the frustrated regions, correlates this plasticity to a fitness window defined by AGT-Ma and AGT-Mi. Finally, co-immunoprecipitation analysis suggests that structural frustration in AGT1 could favor additional functions related to protein-protein interactions. These results expand our understanding of protein structural evolution by establishing that naturally occurring genetic variations tip the balance between stability and frustration to maximize the ensemble of conformations falling within a well-defined fitness window, thus expanding the adaptability potential of the protein., (© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

We examined the ability of two human cytosolic transaminases, aspartate aminotransferase (GOT1) and alanine aminotransferase (GPT), to transform their preferred substrates whilst discriminating against similar metabolites. This offers an opportunity to survey our current understanding of enzyme selectivity and specificity in a biological context. Substrate selectivity can be quantitated based on the ratio of the k cat / K M values for two alternative substrates (the 'discrimination index'). After assessing the advantages, implications and limits of this index, we analyzed the reactions of GOT1 and GPT with alternative substrates that are metabolically available and show limited structural differences with respect to the preferred substrates. The transaminases' observed selectivities were remarkably high. In particular, GOT1 reacted ~10 6 -fold less efficiently when the side-chain carboxylate of the 'physiological' substrates (aspartate and glutamate) was replaced by an amido group (asparagine and glutamine). This represents a current empirical limit of discrimination associated with this chemical difference. The structural basis of GOT1 selectivity was addressed through substrate docking simulations, which highlighted the importance of electrostatic interactions and proper substrate positioning in the active site. We briefly discuss the biological implications of these results and the possibility of using k cat / K M values to derive a global measure of enzyme specificity.

Transaminases have shown the ability to catalyze the amination of a series of aliphatic and (hetero)aromatic α,α-difluorinated ketones with high stereoselectivity, thus providing the corresponding β,β-difluoroamines in high isolated yields (55-82%) and excellent enantiomeric excess (>99%). It was also observed that these activated substrates could be quantitatively transformed by employing a small molar excess of the amine donor since this amination process was thermodynamically favored. Selected transformations could be scaled up to 500 mg, showing the robustness of this methodology.

Aspergillus fumigatus is a saprophytic ubiquitous fungus whose spores can trigger reactions such as allergic bronchopulmonary aspergillosis or the fatal invasive pulmonary aspergillosis. To survive in the lungs, the fungus must adapt to a hypoxic and nutritionally restrictive environment, exploiting the limited availability of aromatic amino acids (AAAs) in the best possible way, as mammals do not synthesize them. A key enzyme for AAAs catabolism in A. fumigatus is AroH, a pyridoxal 5'-phosphate-dependent aromatic aminotransferase. AroH was recently shown to display a broad substrate specificity, accepting L-kynurenine and α-aminoadipate as amino donors besides AAAs. Given its pivotal role in the adaptability of the fungus to nutrient conditions, AroH represents a potential target for the development of innovative therapies against A. fumigatus-related diseases. We have solved the crystal structure of Af-AroH at 2.4 Å resolution and gained new insight into the dynamics of the enzyme's active site, which appears to be crucial for the design of inhibitors. The conformational plasticity of the active site pocket is probably linked to the wide substrate specificity of AroH., (© 2021 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

Fatty amines represent an important class of commodity chemicals which have broad applicability in different industries. The synthesis of fatty amines starts from renewable sources such as vegetable oils or animal fats, but the process has multiple drawbacks that compromise the overall effectiveness and efficiency of the synthesis. Herein, we report a proof-of-concept biocatalytic alternative towards the synthesis of primary fatty amines from renewable triglycerides and oils. By coupling a lipase with a carboxylic acid reductase (CAR) and a transaminase (TA), we have accomplished the direct synthesis of multiple medium and long chain primary fatty amines in one pot with analytical yields as high as 97 %. We have also performed a 75 mL preparative scale reaction for the synthesis of laurylamine from trilaurin, obtaining 73 % isolated yield., (© 2021 Wiley-VCH GmbH.)

Shuttle catalysis has emerged as a useful methodology for the reversible transfer of small functional groups, such as CO and HCN, and goes far beyond transfer hydrogenation chemistry. While a biocatalytic hydrogen-borrowing methodology is well established, the biocatalytic borrowing of alternative functional groups has not yet been realized. Herein, we present a new concept of amine borrowing via biocatalytic shuttle catalysis, which has no counterpart in chemo-shuttle catalysis and allows efficient intermolecular amine shuttling to generate reactive intermediates in situ . By coupling this dynamic exchange with an irreversible downstream step to displace the reaction equilibrium in the forward direction, high conversion to target products can be achieved. We showcase the potential of this amine-borrowing methodology using a biocatalytic equivalent of both the Knorr-pyrrole synthesis and Pictet-Spengler reaction.

Amine transaminases (ATAs) are pyridoxal-5'-phosphate (PLP)-dependent enzymes that catalyze the transfer of an amino group from an amino donor to an aldehyde and/or ketone. In the past decade, the enzymatic reductive amination of prochiral ketones catalyzed by ATAs has attracted the attention of researchers, and more traditional chemical routes were replaced by enzymatic ones in industrial manufacturing. In the present work, the influence of the presence of an α,β-unsaturated system in a methylketone model substrate was investigated, using a set of five wild-type ATAs, the ( R )-selective from Aspergillus terreus (Atr-TA) and Mycobacterium vanbaalenii (Mva-TA), the ( S )-selective from Chromobacterium violaceum (Cvi-TA), Ruegeria pomeroyi (Rpo-TA), V. fluvialis (Vfl-TA) and an engineered variant of V. fluvialis (ATA-256 from Codexis). The high conversion rate (80 to 99%) and optical purity (78 to 99% ee ) of both ( R )- and ( S )-ATAs for the substrate 1-phenyl-3-butanone, using isopropylamine (IPA) as an amino donor, were observed. However, the double bond in the α,β-position of 4-phenylbut-3-en-2-one dramatically reduced wild-type ATA reactivity, leading to conversions of <10% (without affecting the enantioselectivity). In contrast, the commercially engineered V. fluvialis variant, ATA-256, still enabled an 87% conversion, yielding a corresponding amine with >99% ee . Computational docking simulations showed the differences in orientation and intermolecular interactions in the active sites, providing insights to rationalize the observed experimental results.

The link between branched-chain amino acids (BCAAs) and obesity has been known for decades but the functional role of BCAA metabolism in white adipose tissue (WAT) of obese individuals remains vague. Here, we show that mice with adipose tissue knockout of Bcat2, which converts BCAAs to branched-chain keto acids (BCKAs), are resistant to high-fat diet-induced obesity due to increased inguinal WAT browning and thermogenesis. Mechanistically, acetyl-CoA derived from BCKA suppresses WAT browning by acetylation of PR domain-containing protein 16 (PRDM16) at K915, disrupting the interaction between PRDM16 and peroxisome proliferator-activated receptor-γ (PPARγ) to maintain WAT characteristics. Depletion of BCKA-derived acetyl-CoA robustly prompts WAT browning and energy expenditure. In contrast, BCKA supplementation re-establishes high-fat diet-induced obesity in Bcat2 knockout mice. Moreover, telmisartan, an anti-hypertension drug, significantly represses Bcat2 activity via direct binding, resulting in enhanced WAT browning and reduced adiposity. Strikingly, BCKA supplementation reverses the lean phenotype conferred by telmisartan. Thus, we uncover the critical role of the BCAA-BCKA axis in WAT browning., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Despite the plethora of information on ( S )-selective amine transaminases, the ( R )-selective ones are still not well-studied; only a few structures are known to date, and their substrate scope is limited, apart from a few stellar works in the field. Herein, the structure of Luminiphilus syltensis ( R )-selective amine transaminase is elucidated to facilitate engineering towards variants active on bulkier substrates. The V37A variant exhibited increased activity towards 1-phenylpropylamine and to activity against 1-butylamine. In contrast, the S248 and T249 positions, located on the β-turn in the P-pocket, seem crucial for maintaining the activity of the enzyme.

The hyperthermophilic bacterium Thermotoga maritima peptidoglycan contains unusual d-lysine alongside typical d-alanine and d-glutamate. We previously identified lysine racemase and threonine dehydratase, but knowledge of d-amino acid metabolism remains limited. Herein, we identified and characterized T. maritima acetylornithine aminotransferase TM1785. The enzyme was most active towards acetyl-l-ornithine, but also utilized l-glutamate, l-ornithine and acetyl-l-lysine as amino donors, and 2-oxoglutarate was the preferred amino acceptor. TM1785 also displayed racemase activity towards four amino acids and lyase activity towards l-cysteine, but no dehydratase activity towards l-serine, l-threonine or corresponding d-amino acids. Catalytic efficiency (k cat /K m ) was highest for aminotransferase activity and lowest for racemase activity. TM1785 is a novel acetylornithine aminotransferase associated with l-arginine biosynthesis that possesses two additional distinct activities., (© 2021 Federation of European Biochemical Societies.)

ω-Transaminase (ω-TA) is an attractive biocatalyst for stereospecific preparation of amino acids and derivatives, but low catalytic efficiency and unfavorable substrate specificity hamper their industrial application. In this work, to obtain applicable (R)-ω-TA responsible for amination of α-keto acids substrates, the reactivities of eight previously synthesized ω-TAs toward pyruvate using (R)-α-methylbenzylamine ((R)-α-MBA) as amine donor were investigated, and Gibberella zeae TA (GzTA) with the highest (R)-TA activity and stereoselectivity was selected as starting scaffold for engineering. Site-directed mutagenesis around enzymatic active pocket and access tunnel identified three positive mutation sites, S214A, F113L, and V60A. Kinetic analysis synchronously with molecular docking revealed that these mutations afforded desirable alleviation of steric hindrance for pyruvate and α-MBA. Furthermore, the constructed single-, double-, and triple-mutant exhibited varying degrees of improved specificities toward bulkier α-keto acids. Using 2-oxo-2-phenylacetic acid (1d) as substrate, the conversion rate of triple-mutant F113L/V60A/S214A increased by 3.8-fold relative to that of wide-type GzTA. This study provided a practical engineering strategy for improving catalytic efficiency and substrate specificity of (R)-ω-TA. The obtained experience shed light on creating more industrial ω-TAs mutants that can accommodate structurally diverse substrates., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Acanthamoeba polyphaga Mimivirus, a complex virus that infects amoeba, was first reported in 2003. It is now known that its DNA genome encodes for nearly 1,000 proteins including enzymes that are required for the biosynthesis of the unusual sugar 4-amino-4,6-dideoxy-d-glucose, also known as d-viosamine. As observed in some bacteria, the pathway for the production of this sugar initiates with a nucleotide-linked sugar, which in the Mimivirus is thought to be UDP-d-glucose. The enzyme required for the installment of the amino group at the C-4' position of the pyranosyl moiety is encoded in the Mimivirus by the L136 gene. Here, we describe a structural and functional analysis of this pyridoxal 5'-phosphate-dependent enzyme, referred to as L136. For this analysis, three high-resolution X-ray structures were determined: the wildtype enzyme/pyridoxamine 5'-phosphate/dTDP complex and the site-directed mutant variant K185A in the presence of either UDP-4-amino-4,6-dideoxy-d-glucose or dTDP-4-amino-4,6-dideoxy-d-glucose. Additionally, the kinetic parameters of the enzyme utilizing either UDP-d-glucose or dTDP-d-glucose were measured and demonstrated that L136 is efficient with both substrates. This is in sharp contrast to the structurally related DesI from Streptomyces venezuelae, whose three-dimensional architecture was previously reported by this laboratory. As determined in this investigation, DesI shows a profound preference in its catalytic efficiency for the dTDP-linked sugar substrate. This difference can be explained in part by a hydrophobic patch in DesI that is missing in L136. Notably, the structure of L136 reported here represents the first three-dimensional model for a virally encoded PLP-dependent enzyme and thus provides new information on sugar aminotransferases in general., (© 2021 The Protein Society.)

Peptides are important compounds with broad applications in many areas. Asymmetric transamination of α-keto amides can provide an efficient strategy to synthesize peptides, however, the process has not been well developed yet and still remains a great challenge in both enzymatic and catalytic chemistry. For biological transamination, the high activity is attributed to manifold structural and electronic factors of transaminases. Based on the concept of multiple imitation of transaminases, here we report N-quaternized axially chiral pyridoxamines 1 for enantioselective transamination of α-keto amides, to produce various peptides in good yields with excellent enantio- and diastereoselectivities. The reaction is especially attractive for the synthesis of peptides made of unnatural amino acids since it doesn't need great efforts to make chiral unnatural amino acids before amide bond formation., (© 2021. The Author(s).)

Artificial intelligence technologies such as machine learning have been applied to protein engineering, with unique advantages in protein structure, function prediction, catalytic activity, and other issues in recent years. Screening better mutants is still a bottleneck in protein engineering. In this paper, a new sequence-activity relationship method was analyzed for its application in improving the thermal stability of Aspergillus terreus (R)- ω -selective amine transaminase. The experimental data from 6 single-point mutated enzymes were used as a learning dataset to build models and predict the thermostability of 2 6 mutants. Based on digital signal processing (DSP), this method digitized the amino acid sequence of proteins by fast Fourier transform (FFT) and then established the best model applying partial least squares regression (PLSR) to screen out all possible mutants, especially those with high performance. In protein engineering, the innovative sequence activity relationship (ISAR) method can make a reasonable prediction using limited experimental data and significantly reduce the experimental cost. The half-life ( T 1/2 ) of (R)- ω -transaminase was fitted with the amino acid sequence by the ISAR algorithm, resulting in an R 2 of 0.8929 and a cvRMSE of 4.89. At the same time, the mutants with higher T 1/2 than the existing ones were predicted, laying the groundwork for better (R)- ω -transaminase in the later stage. The ISAR algorithm is expected to provide a new technique for protein evolution and screening., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Li-li Jia et al.)

β-Branched noncanonical amino acids are valuable molecules in modern drug development efforts. However, they are still challenging to prepare due to the need to set multiple stereocenters in a stereoselective fashion, and contemporary methods for the synthesis of such compounds often rely on the use of rare-transition-metal catalysts with designer ligands. Herein, we report a highly diastereo- and enantioselective biocatalytic transamination method to prepare a broad range of aromatic β-branched α-amino acids. Mechanistic studies show that the transformation proceeds through dynamic kinetic resolution that is unique to the optimal enzyme. To highlight its utility and practicality, the biocatalytic reaction was applied to the synthesis of several sp 3 -rich cyclic fragments and the first total synthesis of jomthonic acid A., (© 2021 Wiley-VCH GmbH.)

Apremilast is an important active pharmaceutical ingredient that relies on a resolution to produce the key chiral amine intermediate. To provide a new catalytic and enzymatic process for Apremilast, we performed the directed evolution of the amine transaminase fromVibriofluvialis. Six rounds of evolution resulted in the VF-8M-E variant with > 400-fold increase specific activity over the wildtype enzyme. A homology model of VF-8M-E was built and a molecular docking study was performed to explain the increase in activity. The purified VF-8M-E was successfully applied to produce the key chiral amine intermediate in enantiopure form and 49% conversion via a kinetic resolution, representing a new enzymatic access towards Apremilast., (Copyright © 2021 Elsevier Ltd. All rights reserved.)