An O -Phosphotransferase Catalyzes Phosphorylation of Hygromycin A in the Antibiotic-Producing Organism Streptomyces hygroscopicus

Abstract

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 μM) at the C-2‴ position of the fucofuranose ring in the presence of ATP ( K m = 200 ± 20 μM) or GTP ( K m = 350 ± 60 μM) with a k cat of 2.2 ± 0.1 min −1 . The phosphorylated HA is inactive against HA-sensitive Δ 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.