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

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.