Actinomycetologica
Online ISSN : 1881-6371
Print ISSN : 0914-5818
ISSN-L : 0914-5818
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Identification and Purification of an L-Alanine Dehydrogenase Homolog that Catalyzes the Formation of L-Ornithine Lactam from Sinefungin-Producing Streptomyces incarnatus NRRL8089
Koji FukudaTakashi TamuraKumi SatoKenji Inagaki
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2010 Volume 24 Issue 2 Pages 63-65

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Abstract

Sinefungin is a nucleoside antibiotic in which L-ornithine is covalently bound to adenosine. 14C-Incorporation studies indicate that L-Arg and ATP are precursor substrates. To identify the biosynthetic intermediates in the sinefungin biosynthetic pathway, a shunt metabolite accumulated in a resting cell system was isolated from the cell suspension broth, and identified as ornithine-lactam (OrnLcm). The enzyme that catalyzes the formation of OrnLcm was purified from the lysed cell extract of Streptomyces incarnatus NRRL8089 by four-steps of column chromatography using DEAE-Toyopearl, Sephacryl S-200 HR gel filtration, Butyl-Toyopearl and hydroxy-apatite. The molecular mass of the purified protein was determined to be 43 kDa by SDS-PAGE. The N-terminal sequence MKVGIRP was identical to those of L-alanine dehydrogenases (40 kDa) encoded in the genomes of Streptomyces griseus, Streptomyces avermitilis, Streptomyces coelicolor, and other Gram-positive bacteria. The purified enzyme catalyzed the NAD-dependent oxidation of L-Ala, as well as the formation of OrnLcm from L-Arg. It did not show any activity towards L-Orn or D-Arg. Although it has not yet been clarified whether OrnLcm serves as a direct substrate for enzymatic C-C bond formation intracellularly, the lactam intermediate has the advantage of facile transport across the cell membrane as well as the enhanced nucleophilicity at the C5 carbon.

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© 2010 The Society for Actinomycetes Japan
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