title: Significance of the 20-kDa subunit of heterodimeric 2-deoxy-scyllo-inosose synthase for the biosynthesis of butirosin antibiotics in Bacillus circulans.
abstract: A gene (btrC2) encoding the 20-kDa subunit of 2-deoxy-scyllo-inosose (DOI) synthase, a key enzyme in the biosynthesis of 2-deoxystreptamine, was identified from the butirosin-producer Bacillus circulans by reverse genetics. The deduced amino acid sequence of BtrC2 closely resembled that of YaaE of B. subtilis, but the function of the latter has not been known to date. Instead, BtrC2 appeared to show sequence similarity to a certain extent with HisH of B. subtilis, an amidotransferase subunit of imidazole glycerol phosphate synthase. Disruption of btrC2 reduced the growth rate compared with the wild type, and simultaneously antibiotic producing activity was lost. Addition of NH4Cl to the medium complemented only the growth rate of the disruptant, and both the growth rate and antibiotic production were restored by addition of yeast extract. In addition, a heterologous co-expression system of btrC2 with btrC was constructed in Escherichia coli. The simultaneously over-expressed BtrC2 and BtrC constituted a heterodimer, the biochemical features of which resembled those of DOI synthase from B. circulans more than those of the recombinant homodimeric BtrC. Despite the similarity of BtrC2 to HisH the heterodimer showed neither aminotransfer nor amidotransfer activity for 2-deoxy-scyllo-inosose as a substrate. All the observations suggest that BtrC2 is involved not only in the secondary metabolism, but also in the primary metabolism in B. circulans. The function of BtrC2 in the butirosin biosynthesis appears to be indirect, and may be involved in stabilization of DOI synthase and in regulation of its enzyme activity.
authors.affiliation: Department of Chemistry, Tokyo Institute of Technology, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Tamegai
authors.forename: Eriko
authors.initials: E
authors.lastname: Nango
authors.forename: Ayumi
authors.initials: A
authors.lastname: Koike-Takeshita
authors.forename: Fumitaka
authors.initials: F
authors.lastname: Kudo
authors.forename: Katsumi
authors.initials: K
authors.lastname: Kakinuma
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000494:pharmacology; D001407:Bacillus / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D002076:Butirosin Sulfate / Q000096:biosynthesis* / Q000494:pharmacology; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D007700:Kinetics; D008190:Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002076:Butirosin Sulfate; D008190:Lyases; C085631:2-deoxy-scyllo-inosose synthase