Rifamycin Biosynthesis

The rifamycins (e.g., rifamycin B) are a family of antibacterial antibiotics produced by the soil bacterium, Amycolatopsis mediterranei. In chemically modified form, to make them orally active, they have found widespread clinical use in the treatment of tuberculosis, leoprosy and aids-associated mycobacterial infections. Rifamycin B is produced by fermentation, and its biosynthesis involves the assembly of a polyketide by addition of acetate and propionate chain extension units to a unique starter unit, 3-amino-5-hydroxybenzoic acid (AHBA) (Figure 1). Further downstream processing of the initially generated polyketide, proansamycin X, leads to the various rifamycin structures isolated from the fermentation.

Our group has established that AHBA is formed by a novel branch of the shikimate pathway, the aminoshikimate pathway, which parallels the first three steps of shikimate biosynthesis. We purified the enzyme AHBA synthase which aromatizes the 5-amino analog of dehydroshikimic acid to AHBA and cloned the gene encoding it. Overexpression of the gene in E. coli allowed us to study the enzyme mechanism (AHBA synthase is a dimeric PLP enzyme which catalyzes an alpha, beta elimination of water and stereospecific abstraction of a proton from C-6 of its substrate), and allowed Dr. Janina Eads, Department of Biochemistry, University of Birmingham, UK, to solve the crystal structure of the holoenzyme at 2.0 angstroms and of an enzyme-inhibitor complex at 2.2 angstroms (Figure 2).

Using the AHBA synthase gene as a probe we then cloned the gene cluster encoding rifamycin biosynthesis. The 90+ kb cluster, which includes a 50 kb type I polyketide synthase was sequenced in collaboration with the group of C. R. Hutchinson, University of Wisconsin, Madison (Figure 3). Work in both laboratories is now underway to analyze the functions of the individual genes in this cluster by gene inactivation/deletion/replacement and gene expression experiments. In this work our group is focusing mostly on the formation of AHBA and on the post-PKS downstream processing, whereas the Hutchinson group is primarily studying the rifamycin polyketide synthase.