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.

FIG. 1.

Structures of HA and related compounds.

FIG. 2.

Reversed-phase HPLC-MS analyses of phosphorylation of HA by Hyg21. (A) HPLC chromatogram of a control reaction of HA and ATP without Hyg21. (B) HPLC chromatogram of a reaction of HA and ATP after incubation with Hyg21. The resulting HA-P product has a shorter retention time than HA under the tested HPLC conditions (see Materials and Methods). (C) MS analysis (in positive mode) of the HA-P.

FIG. 3.

Disk diffusion assay demonstrating that HA but not HA-P has bioactivity against the efflux pump-deficient ΔtolC E. coli strain. Filter paper disks with 30 or 60 μg of both HA and HA-P were placed on LB agar plate seeded with ΔtolC E. coli and incubated overnight at 37°C.

FIG. 4.

Hyg21-catalyzed phosphorylation of a diastereomeric mixture of 5‴-dihydrohygromycin A (compounds 2a and 2b). (A) HPLC chromatogram of a control reaction of dihydrohygromycin A and ATP without Hyg21. (B) HPLC chromatogram of a reaction of dihydrohygromycin A and ATP after incubation with Hyg21. Compound 2a is the naturally occurring isomer also detected in fermentation broths of the wild type and the hyg26 blocked mutant. Compound 2b is the non-natural isomer observed only in the diastereomeric mixture after chemical reduction of HA. The phosphorylated products of compounds 2a and 2b are labeled as compounds 2a-P and 2b-P, respectively.

FIG. 5.

Schematic representation of the hyg20-22 region in the genome of S. hygroscopicus wild type (A) and Δhyg21 mutant (B). The hyg20 and hyg22 gene products are homologous to transglucosylases and acyltransferases, respectively, and have been proposed to be involved in HA biosynthesis. The hyg21 gene is replaced with the apramycin resistance gene, aac3(IV), in the Δhyg21 strain.

FIG. 6.

Reversed-phase HPLC analyses and radiolabel detection of wild-type and Δhyg21 culture broths grown in the presence of [2-³H]myo-inositol. The production of HA was significantly decreased in the mutant. Radiolabeled analogs of HA (see Fig. 1 for structures) are present in fermentation broths of both the wild-type and the Δhyg21 strains.