Open AccessThe Final Step of Hygromycin A Biosynthesis, Oxidation of C-5″-Dihydrohygromycin A, Is Linked to a Putative Proton Gradient-Dependent Efflux
Author(s) -
Vidya Dhote,
Agata L. Starosta,
Daniel N. Wilson,
Kevin A. Reynolds
Publication year - 2009
Publication title -
antimicrobial agents and chemotherapy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.07
H-Index - 259
eISSN - 1070-6283
pISSN - 0066-4804
DOI - 10.1128/aac.01069-09
Subject(s) - biosynthesis , efflux , chemistry , biochemistry , electrochemical gradient , proton , biology , stereochemistry , enzyme , physics , quantum mechanics , membrane
Hygromycin A (HA) is an aminocyclitol antibiotic produced and excreted byStreptomyces hygroscopicus . Deletion ofhyg26 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 inE scherichia 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 byS. 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 Δhyg19 or the Δhyg26 mutant had slightly reduced activity againstE. 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.