title: An O-phosphotransferase catalyzes phosphorylation of hygromycin A in the antibiotic-producing organism Streptomyces hygroscopicus.
abstract: The antibiotic hygromycin A (HA) binds to the 50S ribosomal subunit and inhibits protein synthesis in gram-positive and gram-negative bacteria. The HA biosynthetic gene cluster in Streptomyces hygroscopicus NRRL 2388 contains 29 open reading frames, which have been assigned putative roles in biosynthesis, pathway regulation, and self-resistance. The hyg21 gene encodes an O-phosphotransferase with a proposed role in self-resistance. We observed that insertional inactivation of hyg21 in S. hygroscopicus leads to a greater than 90% decrease in HA production. The wild type and the hyg21 mutant were comparably resistant to HA. Using Escherichia coli as a heterologous host, we expressed and purified Hyg21. Kinetic analyses revealed that the recombinant protein catalyzes phosphorylation of HA (K(m) = 30 +/- 4 microM) at the C-2''' position of the fucofuranose ring in the presence of ATP (K(m) = 200 +/- 20 microM) or GTP (K(m) = 350 +/- 60 microM) with a k(cat) of 2.2 +/- 0.1 min(-1). The phosphorylated HA is inactive against HA-sensitive Delta tolC E. coli and Streptomyces lividans. Hyg21 also phosphorylates methoxyhygromycin A and desmethylenehygromycin A with k(cat) and K(m) values similar to those observed with HA. Phosphorylation of the naturally occurring isomers of 5'''-dihydrohygromycin A and 5'''-dihydromethoxyhygromycin A was about 12 times slower than for the corresponding non-natural isomers. These studies demonstrate that Hyg21 is an O-phosphotransferase with broad substrate specificity, tolerating changes in the aminocyclitol moiety more than in the fucofuranose moiety, and that phosphorylation by Hyg21 is one of several possible mechanisms of self-resistance in S. hygroscopicus NRRL 2388.
authors.affiliation: Department of Chemistry, Portland State University, Portland, OR 97207-0751, USA.
authors.forename: Vidya
authors.initials: V
authors.lastname: Dhote
authors.forename: Shuchi
authors.initials: S
authors.lastname: Gupta
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Reynolds
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002934:Cinnamates / Q000737:chemistry; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics / Q000502:physiology; D005798:Genes, Bacterial; D006921:Hygromycin B / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry; D007700:Kinetics; D009154:Mutation; D010766:Phosphorylation; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002934:Cinnamates; D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D006921:Hygromycin B; C026273:hygromycin A; D010770:Phosphotransferases