Nonstereospecific Transamination Catalyzed by Pyridoxal Phosphate-dependent Amino Acid Racemases of Broad Substrate Specificity

Lim, Young Hee; Yoshimura, Teizo; Kurokawa, Yoichi; Esaki, Nobuyoshi; Soda, Kenji

doi:10.1074/jbc.273.7.4001

Cited by 14 publications

(9 citation statements)

References 38 publications

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“…The utilization of both L-and D-alanine as substrates by the M. tuberculosis KAPA synthase demonstrates its broad substrate stereospecificity. PLP-dependent enzymes exhibit stereospecificity at the level of the exchange of ␣-protons of their amino acid substrates by catalyzing this reaction either on the si-(sinister) or the re-(rectus) face of the planar external aldimine intermediate (29,32). The ability of the M. tuberculosis KAPA synthase to exchange ␣-protons of both L-and D-enantiomers on both the si-and the re-faces as reported in case of the PLPdependent amino acid racemases (32) may give rise to its broad stereospecificity.…”

Section: Discussionmentioning

confidence: 99%

Broad Substrate Stereospecificity of the Mycobacterium tuberculosis 7-Keto-8-aminopelargonic Acid Synthase

Bhor

Dev

Vasanthakumar

et al. 2006

Journal of Biological Chemistry

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Biotin is an essential enzyme cofactor required for carboxylation and transcarboxylation reactions. The absence of the biotin biosynthesis pathway in humans suggests that it can be an attractive target for the development of novel drugs against a number of pathogens. 7-Keto-8-aminopelargonic acid (KAPA) synthase (EC 2.3.1.47), the enzyme catalyzing the first committed step in the biotin biosynthesis pathway, is believed to exhibit high substrate stereospecificity. Biotin (vitamin H) is an essential cofactor required for a number of carboxylation, transcarboxylation, and decarboxylation reactions in all living organisms. Its participation in gluconeogenesis, metabolism of amino acids, and initiation of fatty acid biosynthesis makes it an indispensable entity in cellular metabolism. However, only plants and microorganisms are capable of synthesizing biotin, whereas mammals obtain it either from the diet or from the intestinal microflora (1). The biotin biosynthesis pathway has been studied in detail in microbes like Escherichia coli, Bacillus subtilis, Bacillus sphaericus, and Saccharomyces cerevisiae and in plants like Arabidopsis thaliana and Lavandula vera. With the exception of the first step, which is the synthesis of pimeloyl-CoA, the remaining four steps of the pathway (Scheme 1) are invariant in all biotin-synthesizing organisms and are carried out by four committed enzymes (2). The first of these four steps is the decarboxylative condensation of pimeloyl-CoA and L-alanine to 7-keto-8-aminopelargonic acid (KAPA) 3 catalyzed by KAPA synthase, followed by the DAPA synthase-catalyzed transfer of an amino group from S-adenosylmethionine to KAPA, forming DAPA. DAPA is then converted to dethiobiotin by the addition of CO 2 in an ATP-dependent reaction catalyzed by dethiobiotin synthetase, and finally the biotin synthase-catalyzed insertion of a sulfur atom between the reactive methyl and methylene carbon atoms adjacent to the ureido ring of dethiobiotin to generate biotin (1-3).KAPA synthase is a PLP-dependent enzyme belonging to subclass II of the aminotransferase family. Along with 5-aminolevulinate synthase, serine palmitoyltransferase, and 2-amino-3-oxobutyrate-CoA ligase, which typically catalyze the Claisen condensations between amino acids and acyl-CoA thioesters, it forms the ␣-oxoamine synthase subfamily. The similarities in the sequences of these enzymes as well as the reactions catalyzed by them indicate that they share a common catalytic mechanism (4 -6). Availability of the three-dimensional structures of these enzymes has provided a structural basis for understanding the reactions catalyzed by them (4, 7-9). The knowledge of the reaction mechanism of KAPA synthase in particular is based on studies on the Bacillus sphaericus and E. coli enzymes (7, 10 -11). It involves the displacement of lysine from the internal aldimine complex with PLP resulting in the formation of the external aldimine between PLP and L-alanine. This is followed by the abstraction of the C␣-H proton of the L-alanine external ald...

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Section: Discussionmentioning

confidence: 99%

Broad Substrate Stereospecificity of the Mycobacterium tuberculosis 7-Keto-8-aminopelargonic Acid Synthase

Bhor

Dev

Vasanthakumar

et al. 2006

Journal of Biological Chemistry

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“…Certain amino acid racemases, particularly those with a relaxed substrate specificity, also facilitate transamination as a side reaction (52)(53)(54). This transamination half reaction eventually results in accumulation of PMP in vitro, as opposed to the epimerization reaction, which freely regenerates the PLP cofactor.…”

Section: Analysis Of Nocj-mentioning

confidence: 99%

Mutational Analysis and Characterization of Nocardicin C-9′ Epimerase

Kelly

Townsend

2004

Journal of Biological Chemistry

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The biosynthetic gene cluster for the nocardicin A producer Nocardia uniformis subsp. tsuyamanensis ATCC 21806 was recently identified. Nocardicin A is the most potent of a series of monocyclic ␤-lactam antibiotics produced by this organism. Its activity has been attributed to a syn-configured oxime moiety and a D-homoseryl side chain attached through an unusual ether linkage to the core nocardicin framework. Notably present in the nocardicin biosynthetic gene cluster is nocJ, encoding a protein with sequence similarity to the pyridoxal 5 -phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid deaminases. Insertional mutagenesis of nocJ abolished nocardicin A production, while the L-homoseryl isomer, isonocardicin A, was still observed. Expression of the disrupted nocJ gene in trans was sufficient to restore production of nocardicin A in the disruption mutant. Heterologous expression, purification, and in vitro characterization of NocJ by UV spectroscopy, cofactor reduction, chiral HPLC analysis of the products and their exchange behavior in deuterium oxide led to confirmation of its role as the PLP-dependent nocardicin C-9 epimerase responsible for interconversion of the nocardicin homoseryl side chain in both nocardicin A with isonocardicin A, and nocardicin C with isonocardicin C. NocJ is the first member of a new class of ␤-lactam aminoacyl side chain epimerases, the first two classes being the evolutionarily distinct prokaryotic PLP-dependent isopenicillin N epimerase and the fungal isopenicillin N epimerase two protein system.

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“…5(A′)] are generated by the subsequent transaldimination between the aldimine complex and active‐site lysyl residue. Various amino acid racemases, such as arginine racemase,26 amino acid racemase with low substrate specificity,27 and alanine racemase,28 also catalyze the transamination as a side reaction. Transamination is characterized by hydrogen transfer between the C‐2 of the substrate and the C‐4′ of the cofactor.…”

Section: Reaction Mechanism Of Plp‐dependent Amino Acid Racemasesmentioning

confidence: 99%

“…We studied the stereochemistry of a C‐4′ hydrogen transfer in a transamination catalyzed by PLP‐dependent amino acid racemases from various sources 27. The hydrogen transfer in side‐reaction transaminations by amino acid racemases was expected to be nonstereospecific, if the reaction proceeded through the two‐base mechanism.…”

Section: Stereospecificity For the Hydrogen Transfer In Transaminatiomentioning

confidence: 99%

Stereospecificity for the hydrogen transfer of pyridoxal enzyme reactions

Soda

Yoshimura

Esaki

2001

The Chemical Record

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We have studied the stereospecificities of various pyridoxal 5'-phosphate dependent enzymes for the hydrogen transfer between the C-4' of a bound coenzyme and the C-2 of a substrate in the transamination catalyzed by the enzymes. Prior to our studies, pyridoxal enzymes so far studied were reported to catalyze the hydrogen transfer only on the si-face of the planar imine intermediate formed from substrate and coenzyme. This finding had been considered as the evidence that pyridoxal enzymes have evolved divergently from a common ancestral protein, because identity in the stereospecificity reflects the similarity in the active-site structure, in particular in the geometrical relationship between the coenzyme and the active site base participating in the hydrogen transfer. However, we found that D-amino acid aminotransferase, branched-chain L-amino acid aminotransferase, and 4-amino-4-deoxychorismate lyase catalyze the re-face specific hydrogen transfer, and that amino acid racemases catalyze the nonstereospecific hydrogen transfer. These findings suggest the convergent evolution of pyridoxal enzymes. Crystallographical studies have shown that the stereospecificity reflects the active-site structure of the enzymes, and that the enzymes with the same fold exhibit the same stereospecificity. The active site structure with the catalytic base being situated on the specific face of the cofactor has been conserved during the evolution among the pyridoxal enzymes of the same family.

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Nonstereospecific Transamination Catalyzed by Pyridoxal Phosphate-dependent Amino Acid Racemases of Broad Substrate Specificity

Cited by 14 publications

References 38 publications

Broad Substrate Stereospecificity of the Mycobacterium tuberculosis 7-Keto-8-aminopelargonic Acid Synthase

Broad Substrate Stereospecificity of the Mycobacterium tuberculosis 7-Keto-8-aminopelargonic Acid Synthase

Mutational Analysis and Characterization of Nocardicin C-9′ Epimerase

Stereospecificity for the hydrogen transfer of pyridoxal enzyme reactions

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