- 10:20 AM

Deciphering the Late Steps In FR900098 Biosynthesis

Matthew A. DeSieno, Tyler W. Johannes, and Huimin Zhao. Chemical & Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave., 221 RAL, Box C-3, MC-712, Urbana, IL 61801

Antibiotics containing C-P bonds, phosphonates and phosphinates, are a potent group of bioactive compounds.  The phosphonic acid FR-900098 is a novel chemotherapeutic agent for the treatment of malaria and has been shown to be effective in humans and other animals.  This compound inhibits 1-deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase, the first committed enzyme in the nonmevalonate pathway for isoprenoid biosynthesis in many bacteria as well as Plasmodium falciparum, the parasite responsible for the most virulent form of the disease. The biosynthetic cluster for FR-900098 has recently been cloned from Streptomyces rubellomurinus and heterologously expressed in Streptomyces lividans.  The gene cluster was also successfully reconstituted in E. coli, resulting in 6 mg/mL FR-900098.

The initial step in FR-900098 biosynthesis is similar to almost all other known phosphonate biosyntheses, a phosphoenolpyruvate (PEP) mutase catalyzed reaction of PEP to phosphonopyruvate.  The subsequent steps parallel the tricarboxylic (TCA) cycle, resulting in the formation of 2-oxo-4-phosphonobutyrate, an analog of α-ketoglutarate.  The final steps were proposed based only on sequence homology of the remaining genes, without any definitive assignment of the order or function.  Here we report the deciphering of the last steps in FR-900098 biosynthesis, verified through whole cell feeding experiments and in vitro assays with purified enzymes.  Two novel enzymes were revealed in the process; one a bifunctional enzyme capable of performing a nucleotide transfer and decarboxylation and the other an amide hydroxylase.  A complete understanding of the FR-900098 biosynthetic pathway now opens the possibilities for metabolic engineering in the native or heterologous Streptomyces strain or in E. coli to increase production of the antibiotic and combinatorial biosynthesis to generate novel derivatives of FR-900098.