Influence of Water and Enzyme on the Post-Transition State Bifurcation of NgnD-Catalyzed Ambimodal [6+4]/[4+2] Cycloaddition.

The enzyme NgnD catalyzes an ambimodal cycloaddition that bifurcates to [6+4]- and [4+2]-adducts. Both products have been isolated in experiments, but it remains unknown how enzyme and water influence the bifurcation selectivity at the femtosecond time scale. Here, we study the impact of water and enzyme on the post-transition state bifurcation of NgnD-catalyzed [6+4]/[4+2] cycloaddition by integrating quantum mechanics/molecular mechanics quasiclassical dynamics simulations and biochemical assays. The ratio of [6+4]/[4+2] products significantly differs in the gas phase, water, and enzyme. Biochemical assays were employed to validate computational predictions. The study informs how water and enzyme affect the bifurcation selectivity through perturbation of the reaction dynamics in the femtosecond time scale, revealing the fundamental roles of condensed media in dynamically controlling the chemical selectivity for biosynthetic reactions.

Production of the Carboxylate Reductase from Nocardia otitidiscaviarum in a Soluble, Active Form for in†vitro Applications.

Accessing aldehydes from carboxylate moieties is often a challenging task. In this regard, carboxylate reductases (CARs) are promising catalysts provided by nature that are able to accomplish this task in just one step, avoiding over-reduction to the alcohol product. However, the heterologous expression of CARs can be quite difficult due to the excessive formation of insoluble protein, thus hindering further characterization and application of the enzyme. Here, the heterologous production of the carboxylate reductase from Nocardia otitidiscaviarum (NoCAR) was optimized by a combination of i) optimized cultivation conditions, ii) post-translational modification with a phosphopantetheinyl transferase and iii) selection of an appropriate expression strain. Especially, the selection of Escherichia coli tuner cells as host had a strong effect on the final 110-fold increase in the specific activity of NoCAR. This highly active NoCAR was used to reduce sodium benzoate to benzaldehyde, and it was successfully assembled with an in vitro regeneration of ATP and NADPH, being capable of reducing about 30 mM sodium benzoate with high selectivity in only 2 h of reaction.

Identification of a secreted superoxide dismutase (SOD) from Nocardia seriolae which induces apoptosis in fathead minnow (FHM) cells.

Fish nocardiosis is a chronic systemic granulomatous disease, and Nocardia seriolae is the main pathogen. The pathogenesis and virulence factors of N. seriolae are not fully understood. Secreted superoxide dismutase (SOD) may be a virulence factor found by a comparative bioinformatics analysis of the whole genome sequence of N. seriolae and the virulence factor database (VFDB). In order to determine the subcellular localization and study the preliminary function of SOD from N. seriolae (NsSOD), gene cloning, secreted protein identification, subcellular localization in fish cells, and apoptosis detection of NsSOD were carried out in this study. Subcellular localization research revealed that NsSOD-GFP fusion proteins were evenly distributed in the cytoplasm. Furthermore, apoptotic bodies were observed in the transfected FHM cells by the overexpression of protein NsSOD. Then, assays of mitochondrial membrane potential (ΔΨm) value, caspase-3 activity and apoptosis-related genes (Bax, Bid, Bad and Bcl-2) mRNA expression were conducted. The results showed that ΔΨm was decreased, and caspase-3 was significantly activated. The mRNA expression of the Bad gene showed significant up-regulated expression at 24 h.p.t., while Bid and Bax genes showed significant up-regulated expression at 72 and 96 h.p.t. and anti-apoptotic gene (Bcl-2) was down-regulated in NsSOD overexpressed cells. Taken together, the results indicated that the protein NsSOD might be involved in apoptosis regulation. This study may lay the foundations for further studies on the function of NsSOD and promote the understanding of the virulence factors and the pathogenic mechanisms of N. seriolae.

Global analysis of adenylate-forming enzymes reveals ß-lactone biosynthesis pathway in pathogenic Nocardia.

Enzymes that cleave ATP to activate carboxylic acids play essential roles in primary and secondary metabolism in all domains of life. Class I adenylate-forming enzymes share a conserved structural fold but act on a wide range of substrates to catalyze reactions involved in bioluminescence, nonribosomal peptide biosynthesis, fatty acid activation, and ß-lactone formation. Despite their metabolic importance, the substrates and functions of the vast majority of adenylate-forming enzymes are unknown without tools available to accurately predict them. Given the crucial roles of adenylate-forming enzymes in biosynthesis, this also severely limits our ability to predict natural product structures from biosynthetic gene clusters. Here we used machine learning to predict adenylate-forming enzyme function and substrate specificity from protein sequences. We built a web-based predictive tool and used it to comprehensively map the biochemical diversity of adenylate-forming enzymes across >50,000 candidate biosynthetic gene clusters in bacterial, fungal, and plant genomes. Ancestral phylogenetic reconstruction and sequence similarity networking of enzymes from these clusters suggested divergent evolution of the adenylate-forming superfamily from a core enzyme scaffold most related to contemporary CoA ligases toward more specialized functions including ß-lactone synthetases. Our classifier predicted ß-lactone synthetases in uncharacterized biosynthetic gene clusters conserved in >90 different strains of Nocardia. To test our prediction, we purified a candidate ß-lactone synthetase from Nocardia brasiliensis and reconstituted the biosynthetic pathway in vitro to link the gene cluster to the ß-lactone natural product, nocardiolactone. We anticipate that our machine learning approach will aid in functional classification of enzymes and advance natural product discovery.

Biochemical characterization of the Nocardia lactamdurans ACV synthetase.

The L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine synthetase (ACVS) is a nonribosomal peptide synthetase (NRPS) that fulfills a crucial role in the synthesis of ß-lactams. Although some of the enzymological aspects of this enzyme have been elucidated, its large size, at over 400 kDa, has hampered heterologous expression and stable purification attempts. Here we have successfully overexpressed the Nocardia lactamdurans ACVS in E. coli HM0079. The protein was purified to homogeneity and characterized for tripeptide formation with a focus on the substrate specificity of the three modules. The first L-α-aminoadipic acid-activating module is highly specific, whereas the modules for L-cysteine and L-valine are more promiscuous. Engineering of the first module of ACVS confirmed the strict specificity observed towards its substrate, which can be understood in terms of the non-canonical peptide bond position.

Identification of a cell-wall peptidase (NlpC/P60) from Nocardia seriolae which induces apoptosis in fathead minnow cells.

Nocardia seriolae, a Gram-positive bacterium, is the main pathogen of fish nocardiosis. Protein NlpC/P60 is a cell-wall peptidase and a potential virulence factor of N. seriolae. Subcellular localization research revealed that both NlpC/P60-GFP and NlpC/P60Δsig-GFP fusion proteins were evenly distributed in the whole cell of fathead minnow (FHM) cells. Furthermore, typical apoptotic features, such as nuclear pyrosis and apoptotic bodies, were observed in the transfected FHM cells and grouper spleen cells by the overexpression of protein NlpC/P60. Then, quantitative assays of mitochondrial membrane potential (ΔΨm) value, caspase-3 activity and apoptosis-related gene (Bax, BNIP3, TNF1 and TNF6) mRNA expression were conducted. The results showed that ΔΨm was decreased, caspase-3 was significantly activated, and the mRNA expression of pro-apoptotic genes (Bax and BNIP3) and tumour necrosis factors (TNF1 and TNF6) was up-regulated in NlpC/P60-overexpressed cells. Taken together, the results indicated that the protein NlpC/P60 of N. seriolae might involve in apoptosis regulation. This study may lay the foundation for further study on the function of N. seriolae NlpC/P60 and promote the understanding of the virulence factors and pathogenic mechanism of N. seriolae.

Knockout of secondary alcohol dehydrogenase in Nocardia cholesterolicum NRRL 5767 by CRISPR/Cas9 genome editing technology.

Nocardia cholesterolicum NRRL 5767 is well-known for its ability to convert oleic acid to 10-hydroxystearic acid (~88%, w/w) and 10-ketostearic acid (~11%, w/w). Conversion of oleic acid to 10-hydroxystearic acid and then to 10-ketostearic acid has been proposed to be catalyzed by oleate hydratase and secondary alcohol dehydrogenase, respectively. Hydroxy fatty acids are value-added with many industrial applications. The objective of this study was to improve the Nocardia cholesterolicum NRRL5767 strain by CRISPR/Cas9 genome editing technology to knockout the secondary alcohol dehydrogenase gene, thus blocking the conversion of 10-hydroxystearic acid to 10-ketostearic acid. The improved strain would produce 10-hydroxystearic acid solely from oleic acid. Such improvement would enhance the production of 10-hydroxystearic acid by eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. Here, we report: (1) Molecular cloning and characterization of two functional recombinant oleate hydratase isozymes and a functional recombinant secondary alcohol dehydrogenase from Nocardia cholesterolicum NRRL5767. Existence of two oleate hydratase isozymes may explain the high conversion yield of 10-hydroxystearic acid from oleic acid. (2) Construction of a CRISPR/Cas9/sgRNA chimeric plasmid that specifically targeted the secondary alcohol dehydrogenase gene by Golden Gate Assembly. (3) Transformation of the chimeric plasmid into Nocardia cholesterolicum NRRL 5767 by electroporation and screening of secondary alcohol dehydrogenase knockout mutants. Two mutants were validated by their lack of secondary alcohol dehydrogenase activity at the protein level and mutation at the targeted 5' coding region and the 5' upstream at the DNA level. The knockout mutants offer improvements by converting added oleic acid to solely 10-hydroxystearic acid, thus eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. To the best of our knowledge, we report the first successful knockout of a target gene in the Nocardia species using CRISPR/Cas9/sgRNA-mediated genome editing technology.

Characterization of the latex clearing protein of the poly(cis-1,4-isoprene) and poly(trans-1,4-isoprene) degrading bacterium Nocardia nova SH22a.

Nocardia nova SH22a is an actinobacterium capable of degrading the polyisoprenes poly(cis-1,4-isoprene) and poly(trans-1,4-isoprene). Sequencing and annotating the genome of this strain led to the identification of a single gene coding for the key enzyme for the degradation of rubber: the latex clearing protein (Lcp). In this study, we showed that LcpSH22a-contrary to other already characterized rubber cleaving enzymes-is responsible for the initial cleavage of both polyisoprene isomers. For this purpose, lcpSH22a was heterologously expressed in an Escherichia coli strain and purified with a functional His6- or Strep-tag. Applying liquid chromatography electrospray ionization time-of-flight mass spectrometry (LC/ESI-ToF-MS) and a spectrophotometric pyridine hemochrome assay, heme b was identified as a cofactor. Furthermore, heme-associated iron was identified using total reflection X-ray fluorescence (TXRF) analysis and inhibition tests. The enzyme's temperature and pH optima at 30°C and 7, respectively, were determined using an oxygen consumption assay. Cleavage of poly(cis-1,4-isoprene) and poly(trans-1,4-isoprene) by the oxygenase was confirmed via detection of carbonyl functional groups containing cleavage products, using Schiff's reagent and electrospray ionization mass spectrometry (ESI-MS).

Inhibition Activity of Avibactam against Nocardia farcinica ß-Lactamase FARIFM10152.

Nocardia farcinica, one of the most frequent pathogenic species responsible for nocardiosis, is characterized by frequent brain involvement and resistance to ß-lactams mediated by a class A ß-lactamase. Kinetic parameters for hydrolysis of various ß-lactams by FARIFM10152 from strain IFM 10152 were determined by spectrophotometry revealing a high catalytic activity (kcat/Km ) for amoxicillin, aztreonam, and nitrocefin. For cephems, kcat/Km was lower but remained greater than 104 M-1 s-1 A low catalytic activity was observed for meropenem, imipenem, and ceftazidime hydrolysis. FARIFM10152 inhibition by avibactam and clavulanate was compared using nitrocefin as a reporter substrate. FARIFM10152 was efficaciously inhibited by avibactam with a carbamoylation rate constant (k2/Ki ) of (1.7 ± 0.3) × 104 M-1 s-1 The 50% effective concentrations (EC50s) of avibactam and clavulanate were 0.060 ± 0.007 µM and 0.28 ± 0.06 µM, respectively. Amoxicillin, cefotaxime, imipenem, and meropenem MICs were measured for ten clinical strains in the presence of avibactam and clavulanate. At 4 µg/ml, avibactam and clavulanate restored amoxicillin susceptibility in all but one of the tested strains but had no effect on the MICs of cefotaxime, imipenem, and meropenem. At 0.4 µg/ml, amoxicillin susceptibility (MIC ≤ 8 µg/ml) was restored for 9 out of 10 strains by avibactam but only for 4 out of 10 strains by clavulanate. Together, these results indicate that avibactam was at least as potent as clavulanate, suggesting that the amoxicillin-avibactam combination could be considered as an option for the rescue treatment of N. farcinica infections if clavulanate cannot be used.

Structure of a bound peptide phosphonate reveals the mechanism of nocardicin bifunctional thioesterase epimerase-hydrolase half-reactions.

Nonribosomal peptide synthetases (NRPSs) underlie the biosynthesis of many natural products that have important medicinal utility. Protection of the NRPS peptide products from proteolysis is critical to these pathways and is often achieved by structural modification, principally the introduction of D-amino acid residues into the elongating peptide. These amino acids are generally formed in situ from their L-stereoisomers by epimerization domains or dual-function condensation/epimerization domains. In singular contrast, the thioesterase domain of nocardicin biosynthesis mediates both the effectively complete L- to D-epimerization of its C-terminal amino acid residue (≥100:1) and hydrolytic product release. We report herein high-resolution crystal structures of the nocardicin thioesterase domain in ligand-free form and reacted with a structurally precise fluorophosphonate substrate mimic that identify the complete peptide binding pocket to accommodate both stereoisomers. These structures combined with additional functional studies provide detailed mechanistic insight into this unique dual-function NRPS domain.

Diterpene Biosynthesis in Actinomycetes: Studies on Cattleyene Synthase and Phomopsene Synthase.

Three diterpene synthases from actinomycetes have been studied. The first enzyme from Streptomyces cattleya produced the novel compound cattleyene. The other two enzymes from Nocardia testacea and Nocardia rhamnosiphila were identified as phomopsene synthases. The cyclisation mechanism of cattleyene synthase and the EIMS fragmentation mechanism of its product were extensively studied by incubation experiments with isotopically labelled precursors. Oxidative transformations expanded the chemical space of these unique diterpenes.

Characterization of Carboxylic Acid Reductases for Biocatalytic Synthesis of Industrial Chemicals.

Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids into aldehydes, which can serve as common biosynthetic precursors to many industrial chemicals. This work presents the systematic biochemical characterization of five carboxylic acid reductases from different microorganisms, including two known and three new ones, by using a panel of short-chain dicarboxylic acids and hydroxy acids, which are common cellular metabolites. All enzymes displayed broad substrate specificities. Higher catalytic efficiencies were observed when the carbon chain length, either of the dicarboxylates or of the terminal hydroxy acids, was increased from C2 to C6 . In addition, when substrates of the same carbon chain length are compared, carboxylic acid reductases favor hydroxy acids over dicarboxylates as their substrates. Whole-cell bioconversions of eleven carboxylic acid substrates into the corresponding alcohols were investigated by coupling the CAR activity with that of an aldehyde reductase in Escherichia coli hosts. Alcohol products were obtained in yields ranging from 0.5 % to 71 %. The de novo stereospecific biosynthesis of propane-1,2-diol enantiomer was successfully demonstrated with use of CARs as the key pathway enzymes. E. coli strains accumulated 7.0 mm (R)-1,2-PDO (1.0 % yield) or 9.6 mm (S)-1,2-PDO (1.4 % yield) from glucose. This study consolidates carboxylic acid reductases as promising enzymes for sustainable synthesis of industrial chemicals.

Structural Evidence for Rifampicin Monooxygenase Inactivating Rifampicin by Cleaving Its Ansa-Bridge.

Rifampicin monooxygenase (RIFMO) decreases the potency of rifampicin (RIF) by converting it to oxidative products. Further decomposition of RIF has been observed in bacteria producing RIFMO and contributes to RIFMO-mediated drug resistance. Here we report the first crystal structure of RIFMO in complex with the hydroxylated RIF product. The 2.10 Å resolution structure reveals a breach of the ansa aliphatic chain of RIF between naphthoquinone C2 and amide N1. Our data suggest that RIFMO catalyzes the hydroxylation of RIF at the C2 atom followed by cleavage of the ansa linkage, which leads to inactivation of the antibiotic by preventing key contacts with the RNA polymerase target.

Subcellular localization and function study of a secreted phospholipase C from Nocardia seriolae.

Fish nocardiosis is a chronic systemic granulomatous disease, and Nocardia seriolae is the main pathogen that causes it. The pathogenesis and virulence factors of N. seriolae are not fully understood. A phospholipase C (PLC), which is likely to be a secreted protein targeting host cell mitochondria, was found by a bioinformatics analysis of the whole genome sequence of N. seriolae. In order to determine the subcellular localization and study the preliminary function of PLC from N. seriolae (NsPLC), in this study gene cloning, secreted protein identification, subcellular localization in host cells and apoptosis detection of NsPLC were carried out. Mass spectrometry analysis of extracellular products from N. seriolae showed that NsPLC was a secreted protein. Subcellular localization of NsPLC-GFP fusion protein in fathead minnow (FHM) cells revealed that the green fluorescence exhibited a punctate distribution near the nucleus and did not co-localize with mitochondria. In addition, an apoptosis assay suggested that apoptosis was induced in FHM cells by the overexpression of NsPLC. This study may lay the foundations for further studies on the function of NsPLC and promote the understanding of the virulence factors and pathogenic mechanism of N. seriolae.

De novo biosynthesis of trans-cinnamic acid derivatives in Saccharomyces cerevisiae.

The production of natural aroma compounds is an expanding field within the branch of white biotechnology. Three aromatic compounds of interest are cinnamaldehyde, the typical cinnamon aroma that has applications in agriculture and medical sciences, as well as cinnamyl alcohol and hydrocinnamyl alcohol, which have applications in the cosmetic industry. Current production methods, which rely on extraction from plant materials or chemical synthesis, are associated with drawbacks regarding scalability, production time, and environmental impact. These considerations make the development of a sustainable microbial-based production highly desirable. Through steps of rational metabolic engineering, we engineered the yeast Saccharomyces cerevisiae as a microbial host to produce trans-cinnamic acid derivatives cinnamaldehyde, cinnamyl alcohol, and hydrocinnamyl alcohol, from externally added trans-cinnamic acid or de novo from glucose as a carbon source. We show that the desired products can be de novo synthesized in S. cerevisiae via the heterologous overexpression of the genes encoding phenylalanine ammonia lyase 2 from Arabidopsis thaliana (AtPAL2), aryl carboxylic acid reductase (acar) from Nocardia sp., and phosphopantetheinyl transferase (entD) from Escherichia coli, together with endogenous alcohol dehydrogenases. This study provides a proof of concept and a strain that can be further optimized for production of high-value aromatic compounds.

Mechanism of Rifampicin Inactivation in Nocardia farcinica.

A novel mechanism of rifampicin (Rif) resistance has recently been reported in Nocardia farcinica. This new mechanism involves the activity of rifampicin monooxygenase (RifMO), a flavin-dependent monooxygenase that catalyzes the hydroxylation of Rif, which is the first step in the degradation pathway. Recombinant RifMO was overexpressed and purified for biochemical analysis. Kinetic characterization revealed that Rif binding is necessary for effective FAD reduction. RifMO exhibits only a 3-fold coenzyme preference for NADPH over NADH. RifMO catalyzes the incorporation of a single oxygen atom forming an unstable intermediate that eventually is converted to 2'-N-hydroxy-4-oxo-Rif. Stable C4a-hydroperoxyflavin was not detected by rapid kinetics methods, which is consistent with only 30% of the activated oxygen leading to product formation. These findings represent the first reported detailed biochemical characterization of a flavin-monooxygenase involved in antibiotic resistance.

The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase.

Rifampicin monooxygenase (RIFMO) catalyzes the N-hydroxylation of the natural product antibiotic rifampicin (RIF) to 2'-N-hydroxy-4-oxo-rifampicin, a metabolite with much lower antimicrobial activity. RIFMO shares moderate sequence similarity with well characterized flavoprotein monooxygenases, but the protein has not been isolated and characterized at the molecular level. Herein, we report crystal structures of RIFMO from Nocardia farcinica, the determination of the oligomeric state in solution with small angle x-ray scattering, and the spectrophotometric characterization of substrate binding. The structure identifies RIFMO as a class A flavoprotein monooxygenase and is similar in fold and quaternary structure to MtmOIV and OxyS, which are enzymes in the mithramycin and oxytetracycline biosynthetic pathways, respectively. RIFMO is distinguished from other class A flavoprotein monooxygenases by its unique middle domain, which is involved in binding RIF. Small angle x-ray scattering analysis shows that RIFMO dimerizes via the FAD-binding domain to form a bell-shaped homodimer in solution with a maximal dimension of 110 Å. RIF binding was monitored using absorbance at 525 nm to determine a dissociation constant of 13 µm Steady-state oxygen consumption assays show that NADPH efficiently reduces the FAD only when RIF is present, implying that RIF binds before NADPH in the catalytic scheme. The 1.8 Å resolution structure of RIFMO complexed with RIF represents the precatalytic conformation that occurs before formation of the ternary E-RIF-NADPH complex. The RIF naphthoquinone blocks access to the FAD N5 atom, implying that large conformational changes are required for NADPH to reduce the FAD. A model for these conformational changes is proposed.

Identification of structural determinants of NAD(P)H selectivity and lysine binding in lysine N(6)-monooxygenase.

l-lysine (l-Lys) N(6)-monooxygenase (NbtG), from Nocardia farcinica, is a flavin-dependent enzyme that catalyzes the hydroxylation of l-Lys in the presence of oxygen and NAD(P)H in the biosynthetic pathway of the siderophore nocobactin. NbtG displays only a 3-fold preference for NADPH over NADH, different from well-characterized related enzymes, which are highly selective for NADPH. The structure of NbtG with bound NAD(P)(+) or l-Lys is currently not available. Herein, we present a mutagenesis study targeting M239, R301, and E216. These amino acids are conserved and located in either the NAD(P)H binding domain or the l-Lys binding pocket. M239R resulted in high production of hydrogen peroxide and little hydroxylation with no change in coenzyme selectivity. R301A caused a 300-fold decrease on kcat/Km value with NADPH but no change with NADH. E216Q increased the Km value for l-Lys by 30-fold with very little change on the kcat value or in the binding of NAD(P)H. These results suggest that R301 plays a major role in NADPH selectivity by interacting with the 2'-phosphate of the adenine-ribose moiety of NADPH, while E216 plays a role in l-Lys binding.

Synthesis of chiral 2-alkanols from n-alkanes by a P. putida whole-cell biocatalyst.

The cytochrome P450 monooxygenase CYP154A8 from Nocardia farcinica was previously found to catalyze hydroxylation of linear alkanes (C7 -C9 ) with a high regio- and stereoselectivity. The objective of this study was to integrate CYP154A8 along with suitable redox partners into a whole-cell system for the production of chiral 2-alkanols starting from alkanes. Both recombinant Escherichia coli and Pseudomonas putida whole-cell biocatalysts tested for this purpose showed the ability to produce chiral alkanols, but a solvent tolerant P. putida strain demonstrated several advantages in the applied biphasic reaction system. The optimized P. putida whole-cell system produced ∼16 mM (S)-2-octanol with 87% ee from octane, which is more than sevenfold higher than the previously described system with isolated enzymes. The achieved enantiopurity of the product could further be increased up to 99% ee by adding an alcohol dehydrogenase (ADH) to the alkane-oxidizing P. putida whole-cell systems. By using this setup for the individual conversions of heptane, octane or nonane, 2.6 mM (S)-2-heptanol with 91% ee, 5.4 mM (S)-2-octanol with 97% ee, or 5.5 mM (S)-2-nonanol with 97% ee were produced, respectively. The achieved concentrations of chiral 2-alkanols are the highest reported for a P450-based whole-cell system so far. Biotechnol. Bioeng. 2016;113: 1845-1852. © 2016 Wiley Periodicals, Inc.

Lysine ε-aminotransferases: kinetic constants, substrate specificities, and the variation in active site residues.

L-Lysine ε-aminotransferase (lysAT) is an important enzyme in tailoring the terminal amino group of L-lysine or L-ornithine and can be directed to the synthesis of various value-added chemicals such as adipic acid. Three lysATs, lysAT from Saccharopolyspora erythraea NRRL 2338 (lysAT_Sery), lysAT from Nocardia farcinica IFM 10152, and lysAT from Rhodococcus jostii RHA1, were cloned, and their kinetic values and substrate specificities were investigated. In the reaction using 5mM L-lysine and 10mM α-ketoglutarate, lysAT_Sery from S. erythraea NRRL 2338 showed 72% higher specific activity than lysAT from Nocardia farcinica IFM 10152 and 42% higher specific activity than lysAT from R. jostii RHA1. More interesting result was that lysAT Sery, exhibiting the highest activity among three lysATs, did not show any activity to L-ornithine. The alignment of 146 lysAT sequences from RefSeq database was searched by the EC number of lysAT to compare the active site residues among the lysAT sequences. The sequence alignment showed that only two residues, corresponding to Ala129 and Asn328 of lysAT from Mycobacterium tuberculosis H37Rv (lysAT_Mtub), showed variations among the active site residues. All the active site residues except those two residues were completely conserved throughout 145 lysAT sequences. lysAT from S. erythraea NRRL 2338 has A129T and N328S variations (residue numbers are those of the crystal structure of lysAT_Mtub). The structural analysis by the homology model indicate that Thr126 by A129T variation in lysAT_Sery is appeared to interact more tightly with the phosphate group of PLP than alanine (the distance between Thr126 and the phosphate group of PLP was 2.92Å). In addition, Ser328 is located at the substrate recognition site of active site and, therefore, N328S variation may be connected to the substrate specificity of lysAT.