The final step of hygromycin A biosynthesis, oxidation of C-5''-dihydrohygromycin A, is linked to a putative proton gradient-dependent efflux

Abstract

Hygromycin A (HA) is an aminocyclitol antibiotic produced and excreted by Streptomyces hygroscopicus. Deletion of hyg26 from the hygromycin A biosynthetic gene cluster has previously been shown to result in a mutant that produces 5''-dihydrohygromycin A (DHHA). We report herein on the purification and characterization of Hyg26 expressed in Escherichia coli. The enzyme catalyzes an NAD(H)-dependent reversible interconversion of HA and DHHA, supporting the role of the reduced HA as the penultimate biosynthetic pathway intermediate and not a shunt product. The equilibrium for the Hyg26-catalyzed reaction heavily favors the DHHA intermediate. The high-titer production of the HA product by S. hygroscopicus must be dependent upon a subsequent energetically favorable enzyme-catalyzed process, such as the selective and efficient export of HA. hyg19 encodes a putative proton gradient-dependent transporter, and a mutant lacking this gene was observed to produce less HA and to produce the DHHA intermediate. The DHHA produced by either the Deltahyg19 or the Deltahyg26 mutant had slightly reduced activity against E. coli and reduced protein synthesis-inhibitory activity in vitro. The data indicate that Hyg26 and Hyg19 have evolved to produce and export the final potent HA product in a coordinated fashion.

FIG. 1.

Structures of HA and related compounds.

FIG. 2.

Reverse-phase HPLC analysis of DHHA (compound 2) dehydrogenation by Hyg26. (A) Control reaction of DHHA and NAD⁺ without Hyg26; (B) reaction of DHHA and NAD⁺ after incubation with Hyg26 showing the formation of HA. mAU, milli-absorbance units.

FIG. 3.

Reverse-phase HPLC analysis of hygromycin A (HA) reduction by Hyg26. (A) Control reaction of HA and NADH without Hyg26; (B) reaction of HA and NADH after incubation with Hyg26, showing the formation of DHHA. An additional peak (11.1 min) eluting earlier than HA is the C-4″ epimer of HA and not a substrate for Hyg26. mAU, milli-absorbance units.

FIG. 4.

Reverse-phase HPLC analysis of fermentation broth of the wild-type (A) and Δhyg19 (B) strains. Reduced HA production and accumulation of DHHA were observed in the Δhyg19 strain. mAU, milli-absorbance units.

FIG. 5.

Schematic representation of replacement of the hyg19, hyg20, and hyg21 genes with the apramycin resistance cassette. WT, wild type.

FIG. 6.

Effects of HA and derivatives on in vitro transcription-translation. (A) Detection of template-dependent synthesis of GFP by using fluorescence and native polyacrylamide gel electrophoresis in the presence of template with increasing concentrations (μM) of the antibiotic HA, DHHA, and compound 7 on GFP synthesis; (B) quantitation of effects of HA, DHHA, and compound 7 on GFP synthesis; (C) quantitation of effects of HA and compounds 3, 4, 5, and 6 on GFP synthesis. The experiments whose results are shown in panels B and C were performed in triplicate. GFP fluorescence is given as a percentage, where 100% is defined as the fluorescence detected in the absence of the antibiotic. Rel., relative.

FIG. 7.

Working hypothesis for the roles of Hyg19 and Hyg28 in antibiotic efflux. The proton gradient-dependent transporter, Hyg19, is the primary HA transporter in the wild type. Efflux in the Δhyg19 strain is hypothesized to be mediated by the ABC transporter, Hyg28, which is less selective for HA.