Isolation and characterization of 27-O-demethylrifamycin SV methyltransferase provides new insights into the post-PKS modification steps during the biosynthesis of the antitubercular drug rifamycin B by Amycolatopsis mediterranei S699.
The gene rif orf14 in the rifamycin biosynthetic gene cluster of Amycolatopsis mediterranei S699, producer of the antitubercular drug rifamycin B, encodes a protein of 272 amino acids identified as an AdoMet: 27-O-demethylrifamycin SV methyltransferase. Frameshift inactivation of rif orf14 generated a mutant of A. mediterranei S699 that produces no rifamycin B, but accumulates 27-O-demethylrifamycin SV (DMRSV) as the major new metabolite, together with a small quantity of 27-O-demethyl-25-O-desacetylrifamycin SV (DMDARSV). Heterologous expression of rif orf14 in Escherichia coli yielded a 33.8-kDa polyhistidine-tagged polypeptide, which efficiently catalyzes the methylation of DMRSV to rifamycin SV, but not that of DMDARSV or rifamycin W. 27-O-Demethylrifamycin S was methylated poorly, if at all, by the enzyme to produce rifamycin S. The purified enzyme does not require a divalent cation for catalytic activity. While Ca(2+) or Mg(2+) inhibits the enzyme activity slightly, Zn(2+), Ni(2+), and Co(2+) are strongly inhibitory. The K(m) values for DMRSV and S-adenosyl-L-methionine (AdoMet) are 18.0 and 19.3 microM, respectively, and the K(cat) is 87s(-1). The results indicate that DMRSV is a direct precursor of rifamycin SV and that acetylation of the C-25 hydroxyl group must precede the methylation reaction. They also suggest that rifamycin S is not the precursor of rifamycin SV in rifamycin B biosynthesis, but rather an oxidative shunt-product.
Xu
Jun
J
Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195-1700, USA.
Mahmud
Taifo
T
Floss
Heinz G
HG
D000192:Actinomycetales / Q000378:metabolism*; D000595:Amino Acid Sequence; D000904:Antibiotics, Antitubercular / Q000096:biosynthesis*; D005786:Gene Expression Regulation; D020868:Gene Silencing; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012294:Rifamycins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
C473196:27-O-demethylrifamycin S; C473197:27-O-demethylrifamycin SV; D000904:Antibiotics, Antitubercular; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D012294:Rifamycins; D012436:S-Adenosylmethionine; C033974:rifamycin B; C473198:27-O-demethylrifamycin SV methyltransferase; D008780:Methyltransferases