Multiple drug resistance is becoming a major threat to public health. We are facing a need for both new biologically active compounds and for new methods of making such compounds. Streptomyces produce the majority of antibiotics that are made by bacteria and study of such antibiotic biosynthesis pathways is a basic prerequisite to developing new methods of antibiotic production. The unusual polyketide, nonactin, produced by Streptomyces griseus, is an inhibitor of the P 170-glycoprotein efflux pump found in multiple drug resistant cancer cells. Nonactin is also an ionophore active against Gram positive bacteria, mycobacteria, and fungi. Nonactin is an achiral compound; a tetramer made up from both enantiomers of a precursor nonactic acid. The organism has two mirror image biosynthesis pathways, one for each of the precursor enantiomers. Of particular interest are the late stages of nonactin biosynthesis wherein the complete macrocycle is assembled l as these steps can be manipulated to generate natural product- synthetic compound hybrid analogs of nonactin. The fundamental and applied biochemistry of the highly unusual nonactin biosynthesis system will be studied by: (1) Analysis of the (+)- and (-)-nonactate synthase enzymes, two enzymes that catalyze the same chemical reaction, yet upon different enantiomers of the substrate. (2) Analysis of the ATP-dependent formation of nonactin from dimeric nonactate precursors catalyzed by the enzyme NonL. The process is a unique example of a biological Coupe du Roi synthesis where an achiral molecule is made by the condensation of two homochiral precursors. (3) Analysis of NonR, the enzyme product of the nonactin resistance gene. NonR is likely a serine esterase that renders nonactin inactive. This is a study of a fundamental antibiotic resistance mechanism. We hypothesize that NonR, and a second likely esterase NonD, act together to recycle nonactin and thereby control nonactin synthesis levels. (4) Study of in vivo and in vitro systems derived from the basic biochemistry of the late steps of nonactin biosynthesis to produce a number of natural product-synthetic compound hybrid antibiotics. The biological and chemical properties of the new compounds will be evaluated.
