Biosynthetic Origin of Hygromycin A

Abstract

ABSTRACT Hygromycin A, an antibiotic produced by Streptomyces hygroscopicus , is an inhibitor of bacterial ribosomal peptidyl transferase. The antibiotic binds to the ribosome in a distinct but overlapping manner with other antibiotics and offers a different template for generation of new agents effective against multidrug-resistant pathogens. Reported herein are the results from a series of stable-isotope-incorporation studies demonstrating the biosynthetic origins of the three distinct structural moieties which comprise hygromycin A. Incorporation of [1- 13 C]mannose and intact incorporation of d -[1,2- 13 C 2 ]glucose into the 6-deoxy-5-keto- d -arabino-hexofuranose moiety are consistent with a pathway in which mannose is converted to an activated l -fucose, via a 4-keto-6-deoxy- d -mannose intermediate, with a subsequent unusual mutation of the pyranose to the corresponding furanose. The aminocyclitol moiety was labeled by d -[1,2- 13 C 2 ]glucose in a manner consistent with formation of myo -inositol and a subsequent unprecedented oxidation and transamination of the C-2 hydroxyl group to generate neo -inosamine-2. Incorporation of [ carboxy- 13 C]-4-hydroxybenzoic acid and intact incorporation of [2,3- 13 C 2 ]propionate are consistent with a polyketide synthase-type decarboxylation condensation to generate the 3,4-dihydroxy-α-methylcinnamic acid moiety of hygromycin A. No labeling of hygromycin A was observed when [3- 13 C]tyrosine, [3- 13 C]phenylalanine, or [ carboxy- 13 C]benzoic acid was used, suggesting that the 4-hydroxybenzoic acid is derived directly from chorismic acid. Consistent with this hypothesis was the observation that hygromycin A titers could be reduced by addition of N -(phosphonomethyl)-glycine (an inhibitor of chorismic acid biosynthesis) and restored by coaddition of 4-hydroxybenzoic acid. The convergent biosynthetic pathway established for hygromycin A offers significant versatility for applying the techniques of combinatorial and directed biosynthesis to production of new antibiotics which target the ribosomal peptidyl transferase activity.