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Identification and characterization of novel broad-spectrum amino acid racemases from Escherichia coli and Bacillus subtilis

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Abstract

The peptidoglycan layer of the bacterial cell wall typically contains d-alanine (d-Ala) and d-glutamic acid (d-Glu), and also various non-canonical d-amino acids that have been linked to peptidoglycan remodeling, inhibition of biofilm formation, and triggering of biofilm disassembly. Bacteria produce d-amino acids when adapting to environmental changes as a common survival strategy. In our previous study, we detected non-canonical d-amino acids in Escherichia coli grown in minimal medium. However, the biosynthetic pathways of non-canonical d-amino acids remain poorly understood. In the present study, we identified amino acid racemases in E. coli MG1655 (YgeA) and Bacillus subtilis (RacX) that produce non-canonical d-amino acids other than d-Ala and d-Glu. We characterized their enzymatic properties, and both displayed broad substrate specificity but low catalytic activity. YgeA preferentially catalyzes the racemization of homoserine, while RacX preferentially racemizes arginine, lysine, and ornithine. RacX is dimeric, and appears not to require pyridoxal 5′-phosphate (PLP) as a coenzyme as is the case with YgeA. To our knowledge, this is the first report on PLP-independent amino acid racemases possessing broad substrate specificity in E. coli and B. subtilis.

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Abbreviations

PLP:

Pyridoxal 5′-phosphate

BN-PAGE:

Blue native polyacrylamide gel electrophoresis

OPA:

o-Phthalaldehyde

NAC:

N-Acetyl-l-cysteine

Boc-l-Cys:

N-tert-Butoxycarbonyl-l-cysteine

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Authors and Affiliations

  1. Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan

    Tetsuya Miyamoto, Masumi Katane, Yasuaki Saitoh, Masae Sekine & Hiroshi Homma

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  1. Tetsuya Miyamoto
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  2. Masumi Katane
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  3. Yasuaki Saitoh
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  4. Masae Sekine
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  5. Hiroshi Homma
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Correspondence to Hiroshi Homma.

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Miyamoto, T., Katane, M., Saitoh, Y. et al. Identification and characterization of novel broad-spectrum amino acid racemases from Escherichia coli and Bacillus subtilis . Amino Acids 49, 1885–1894 (2017). https://doi.org/10.1007/s00726-017-2486-2

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