From Structure to Function: Analysis of the First Monomeric Pyridoxal-5'-Phosphate-Dependent Transaminase from the Bacterium .

The first monomeric pyridoxal-5'-phosphate (PLP)-dependent transaminase from a marine, aromatic-compound-degrading, sulfate-reducing bacterium Tol2, has been studied using structural, kinetic, and spectral methods. The monomeric organization of the transaminase was confirmed by both gel filtration and crystallography. The PLP-dependent transaminase is of the fold type IV and deaminates D-alanine and ()-phenylethylamine in half-reactions.

Incorporation of pyridoxal-5'-phosphate into the apoenzyme: A structural study of D-amino acid transaminase from Haliscomenobacter hydrossis.

Pyridoxal-5'-phosphate (PLP)-dependent transaminases are key enzymes of amino acid metabolism in cells and remarkable biocatalysts of stereoselective amination for process chemistry applications. As cofactor-dependent enzymes, transaminases are prone to cofactor leakage. Here we discuss the holoenzyme-apoenzyme interconversion and the kinetics of PLP incorporation into the apo form of a PLP-dependent transaminase from Haliscomenobacter hydrossis.

Multifunctionality of arginine residues in the active sites of non-canonical d-amino acid transaminases.

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.

Expanded Substrate Specificity in D-Amino Acid Transaminases: A Case Study of Transaminase from .

Enzymes with expanded substrate specificity are good starting points for the design of biocatalysts for target reactions. However, the structural basis of the expanded substrate specificity is still elusive, especially in the superfamily of pyridoxal-5'-phosphate-dependent transaminases, which are characterized by a conserved organization of both the active site and functional dimer. Here, we analyze the structure-function relationships in a non-canonical D-amino acid transaminase from , which is active towards D-amino acids and primary ()-amines.

In search for structural targets for engineering d-amino acid transaminase: modulation of pH optimum and substrate specificity.

The development of biocatalysts requires reorganization of the enzyme's active site to facilitate the productive binding of the target substrate and improve turnover number at desired conditions. Pyridoxal-5'-phosphate (PLP) - dependent transaminases are highly efficient biocatalysts for asymmetric amination of ketones and keto acids. However, transaminases, being stereoselective enzymes, have a narrow substrate specificity due to the ordered structure of the active site and work only in neutral-alkaline media.

To the Understanding of Catalysis by D-Amino Acid Transaminases: A Case Study of the Enzyme from .

Pyridoxal-5'-phosphate (PLP)-dependent transaminases are highly efficient biocatalysts for stereoselective amination. D-amino acid transaminases can catalyze stereoselective transamination producing optically pure D-amino acids. The knowledge of substrate binding mode and substrate differentiation mechanism in D-amino acid transaminases comes down to the analysis of the transaminase from .

Mechanistic aspects of the transamination reactions catalyzed by D-amino acid transaminase from Haliscomenobacter hydrossis.

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.

The Uncommon Active Site of D-Amino Acid Transaminase from : Biochemical and Structural Insights into the New Enzyme.

Among industrially important pyridoxal-5'-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from (Halhy).

Probing the role of the residues in the active site of the transaminase from Thermobaculum terrenum.

Creating biocatalysts for (R)-selective amination effectively is highly desirable in organic synthesis. Despite noticeable progress in the engineering of (R)-amine activity in pyridoxal-5'-phosphate-dependent transaminases of fold type IV, the specialization of the activity is still an intuitive task, as there is poor understanding of sequence-structure-function relationships. In this study, we analyzed this relationship in transaminase from Thermobaculum terrenum, distinguished by expanded substrate specificity and activity in reactions with L-amino acids and (R)-(+)-1-phenylethylamine using α-ketoglutarate and pyruvate as amino acceptors.

Effects of pH and temperature on (S)-amine activity of transaminase from the cold-adapted bacterium Psychrobacter cryohalolentis.

(7R,8S)-diaminopelargonic acid transaminase from the cold-adapted Gram-negative bacterium Psychrobacter cryohalolentis (Pcryo361) is able to react with unnatural substrates including (S)-( +)-1-phenylethylamine, aldehydes and α-diketones. Additionally, Pcryo361 is active at 0-50 °C and retains up to 10% of the maximum activity at 0 °C. Here, we report a detailed study on the stability and low temperature activity of Pcryo361.

Structural insight into the substrate specificity of PLP fold type IV transaminases.

Pyridoxal-5'-phosphate-dependent transaminases of fold type IV (class IV) are promising enzymes for (R)-selective amination of organic compounds. Transaminases of fold type IV exhibit either strict (R)-selectivity or (S)-selectivity that is implemented within geometrically similar active sites of different amino acid compositions. Based on substrate specificity, class IV comprises three large families of transaminases: (S)-selective branched-chain L-amino acid aminotransferases and (R)-selective D-amino acid aminotransferases and (R)-amine:pyruvate transaminases.

Corrigendum: Thermostable Branched-Chain Amino Acid Transaminases From the Archaea and : Biochemical and Structural Characterization.

[This corrects the article DOI: 10.3389/fbioe.2019.

Functional characterization of PLP fold type IV transaminase with a mixed type of activity from Haliangium ochraceum.

Pyridoxal-5'-phosphate (PLP)-dependent transaminases are industrially important enzymes catalyzing the stereoselective amination of ketones and keto acids. Transaminases of PLP fold type IV are characterized by (R)- or (S)-stereoselective transfer of amino groups, depending on the substrate profile of the enzyme. PLP fold type IV transaminases include branched-chain amino acid transaminases (BCATs), D-amino acid transaminases and (R)-amine:pyruvate transaminases.

Thermostable Branched-Chain Amino Acid Transaminases From the Archaea and : Biochemical and Structural Characterization.

Two new thermophilic branched chain amino acid transaminases have been identified within the genomes of different hyper-thermophilic archaea, , and . These enzymes belong to the class IV of transaminases as defined by their structural fold. The enzymes have been cloned and over-expressed in and the recombinant enzymes have been characterized both biochemically and structurally.

Biochemical and structural insights into PLP fold type IV transaminase from Thermobaculum terrenum.

The high catalytic efficiency of enzymes under reaction conditions is one of the main goals in biocatalysis. Despite the dramatic progress in the development of more efficient biocatalysts by protein design, the search for natural enzymes with useful properties remains a promising strategy. The pyridoxal 5'-phosphate (PLP)-dependent transaminases represent a group of industrially important enzymes due to their ability to stereoselectively transfer amino groups between diverse substrates; however, the complex mechanism of substrate recognition and conversion makes the design of transaminases a challenging task.

Diaminopelargonic acid transaminase from Psychrobacter cryohalolentis is active towards (S)-(-)-1-phenylethylamine, aldehydes and α-diketones.

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.

Structural characterization of geranylgeranyl pyrophosphate synthase GACE1337 from the hyperthermophilic archaeon Geoglobus acetivorans.

A novel type 1 geranylgeranyl pyrophosphate synthase GACE1337 has been identified within the genome of a newly identified hyperthermophilic archaeon Geoglobus acetivorans. The enzyme has been cloned and over-expressed in Escherichia coli. The recombinant enzyme has been biochemically and structurally characterized.

Identification of branched-chain amino acid aminotransferases active towards (R)-(+)-1-phenylethylamine among PLP fold type IV transaminases.

New class IV transaminases with activity towards L-Leu, which is typical of branched-chain amino acid aminotransferases (BCAT), and with activity towards (R)-(+)-1-phenylethylamine ((R)-PEA), which is typical of (R)-selective (R)-amine:pyruvate transaminases, were identified by bioinformatics analysis, obtained in recombinant form, and analyzed. The values of catalytic activities in the reaction with L-Leu and (R)-PEA are comparable to those measured for characteristic transaminases with the corresponding specificity. Earlier, (R)-selective class IV transaminases were found to be active, apart from (R)-PEA, only with some other (R)-primary amines and D-amino acids.

NADP-Dependent Aldehyde Dehydrogenase from Archaeon : Structural and Functional Features.

We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. , AlDHPyr1147 catalyzes the irreversible oxidation of short aliphatic aldehydes at 60-85°С, and the affinity of AlDHPyr1147 to the NADP+ at 60°С is comparable to that for mesophilic analogues at 25°С. We determined the structures of the apo form of AlDHPyr1147 (3.

A Novel highly thermostable branched-chain amino acid aminotransferase from the crenarchaeon Vulcanisaeta moutnovskia.

A new fold-type IV branched-chain amino acid aminotransferase VMUT0738 from the hyperthermophilic Crenarchaeon Vulcanisaeta moutnovskia was successfully expressed in Escherichia coli. Purified VMUT0738 showed activity toward numerous aliphatic and aromatic l-amino acids and 2-oxo acids at optimal pH 8.0.

Experimental and computational studies on the unusual substrate specificity of branched-chain amino acid aminotransferase from Thermoproteus uzoniensis.

PLP-Dependent fold-type IV branched-chain amino acid aminotransferases (BCATs) from archaea have so far been poorly characterized. A new BCAT from the hyperthermophilic archaeon Thermoproteus uzoniensis (TUZN1299) has been studied. TUZN1299 was found to be highly active toward branched-chain amino acids (BCAAs), positively charged amino acids, l-methionine, l-threonine, l-homoserine, l-glutamine, as well as toward 2-oxobutyrate and keto analogs of BCAAs, whereas l-glutamate and α-ketoglutarate were not converted in the overall reaction.

First structure of archaeal branched-chain amino acid aminotransferase from Thermoproteus uzoniensis specific for L-amino acids and R-amines.

The gene TUZN1299 from the genome of the hyperthermophilic archaeon Thermoproteus uzoniensis encoding a new 32.8 kDa branched-chain amino acid aminotransferase (BCAT) was expressed in Escherichia coli. The recombinant protein TUZN1299 was purified to homogeneity in the PLP-bound form.

Intramolecular hydrogen bonding in the polyextremophilic short-chain dehydrogenase from the archaeon Thermococcus sibiricus and its close structural homologs.

The short-chain alcohol dehydrogenase from the archaeon Thermococcus sibiricus (TsAdh319) exhibits adaptation to different kinds of stress: high temperature, high salinity, and the presence of organic solvents and denaturants. Previously a comparison of TsAdh319 with close structural homologs revealed an abnormally large number of charged residues on the surface of TsAdh319 tetramer. We further focused on the analysis of hydrogen bonding of TsAdh319 and its structural homologs from thermophilic and mesophilic organisms as a structural factor of adaptation to extreme environment.

Sodium chloride-induced modulation of the activity and thermal stability of short-chain oxidoreductase from the archaeon Thermococcus sibiricus.

Recently, we have studied properties and structural features of the thermostable halotolerant alcohol dehydrogenase from archaeon Thermococcus sibiricus (TsAdh319). In the present work, the effect of sodium chloride on activity and thermostability was explored using circular dichroism, fluorescent spectroscopy, and differential scanning calorimetry. The activity of TsAdh319 increased in the presence of NaCl and remained at the elevated level up to 4 M of NaCl.