Cloning and characterization of a gene (msdA) encoding methylmalonic acid semialdehyde dehydrogenase from Streptomyces coelicolor.
A homolog of the mmsA gene of Pseudomonas aeruginosa, which encodes methylmalonic acid semialdehyde dehydrogenase (MSDH) and is involved in valine catabolism in pseudomonads and mammals, was cloned and sequenced from Streptomyces coelicolor. Of the two open reading frames (ORFs) found, which are convergently transcribed and separated by a 62-nucleotide noncoding region, the deduced amino acid sequence of the msdA ORF (homologous to mmsA) is similar to a variety of prokaryotic and eukaryotic aldehyde dehydrogenases that utilize NAD+, particularly to the MmsA protein from P. aeruginosa. No significant similarity was found between the deduced product of ORF1 and known proteins in the databases. An S. coelicolor msdA mutant, constructed by insertion of a hygromycin resistance gene (hyg) into the msdA coding region, lost the MSDH activity and the ability to grow in a minimal medium with valine or isobutyrate as the sole carbon source but grew on propionate. The msdA::hyg mutation was complemented by introduction of the msdA gene on a plasmid. When the S. coelicolor msdA gene was overexpressed in Escherichia coli under the control of the T7 promoter, a protein of 51-kDa, corresponding to the approximate mass of the predicted S. coelicolor msdA product (52.6 kDa), and specific MSDH activity were detected. These results strongly suggest that msdA indeed encodes the MSDH that is involved in valine catabolism in S. coelicolor.
School of Pharmacy, University of Wisconsin, Madison 53706, USA.
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Zhang
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Tang
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Hutchinson
D000445:Aldehyde Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D050646:Methylmalonate-Semialdehyde Dehydrogenase (Acylating); D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D014633:Valine / Q000378:metabolism
D000445:Aldehyde Oxidoreductases; D050646:Methylmalonate-Semialdehyde Dehydrogenase (Acylating); D014633:Valine