S50: Hygromycin A: Activity, biosynthesis, export, and resistance

Hygromycin A (HA) is an aminocyclitol antibiotic produced and excreted by Streptomyces hygroscopicus. The antibacterial activity of hygromycin A (HA) arises from protein synthesis inhibition and has previously been shown to be dependent upon a methylenedioxy bridged-aminocyclitol moiety. Selective gene deletions and chemical complementation in the HA- producer Streptomyces hygroscopicus NRRL 2388 have shown which gene products are responsible for generation of this unusual moiety.   A metallo-dependant hydrolase homolog gene is shown to be required for the last step, an oxidative cyclization step of methylenedioxy bridge formation.  The methyl/methylene group is not required for in vitro protein synthesis inhibition but is essential for activity against Escherichia coli.  Recent work on this pathway has also shown that the final step in the biosynthetic pathway is NAD(H)-dependent reversible interconversion of HA and 5”-dihydrohygromycin A (DHHA) and catalyzed by Hyg26.  The equilibrium for the Hyg26-catalyzed reaction heavily favors the DHHA intermediate. High titer production of HA product by S. hygroscopicus must be dependant upon a subsequent energetically favorable enzyme-catalyzed process such as selective and efficient export of HA. The hyg19 encodes a putative proton gradient-dependent transporter, and a mutant lacking this gene was observed to produce less HA and produce the DHHA intermediate. The DHHA produced by either the Δhyg19 or the Δhyg26 mutant had slightly reduced antibacterial activity against E. coli and reduced in vitro protein synthesis inhibitory activity. The data indicate that Hyg26 and Hyg19 have evolved to produce and export the final potent HA product in a coordinated fashion.