Identification and Purification of an L-Alanine Dehydrogenase Homolog that Catalyzes the Formation of L-Ornithine Lactam from Sinefungin-Producing Streptomyces incarnatus NRRL8089
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概要
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Sinefungin is a nucleoside antibiotic in which <SMALL>L</SMALL>-ornithine is covalently bound to adenosine. <SUP>14</SUP>C-Incorporation studies indicate that <SMALL>L</SMALL>-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 <I>Streptomyces incarnatus</I> 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 <I>N</I>-terminal sequence MKVGIRP was identical to those of <SMALL>L</SMALL>-alanine dehydrogenases (40 kDa) encoded in the genomes of <I>Streptomyces griseus</I>, <I>Streptomyces avermitilis</I>, <I>Streptomyces coelicolor</I>, and other Gram-positive bacteria. The purified enzyme catalyzed the NAD-dependent oxidation of <SMALL>L</SMALL>-Ala, as well as the formation of OrnLcm from <SMALL>L</SMALL>-Arg. It did not show any activity towards <SMALL>L</SMALL>-Orn or <SMALL>D</SMALL>-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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