K-079. 2-Phosphonomethylmalate Synthase Is a Novel Driving Reaction for C-P Bond Formation in the Biosynthesis of the Antimalarial Compound FR900098
The phosphonic acid FR900098 (N-acetyl-N-hydroxy-3-aminopropylphosphonate) is a potent antimalarial compound that inhibits 1-deoxy-D-xylulose 5-phosphate reductoisomerase in the nonmevalonate pathway of isoprenoid biosynthesis. We recently cloned and sequenced the FR900098 biosynthetic pathway from Streptomyces rubellomurinus. Unlike other characterized phosphonate biosynthetic pathways (e.g. fosfomycin, phosphinothricin tripeptide, and aminoethylphosphonate), C-P bond formation in the FR900098 pathway is not initiated by the sequential action of phosphoenolpyruvate (PEP) mutase and 3-phosphonopyruvate (PnPy) decarboxylase. Instead, the thermodynamically unfavorable PEP mutase reaction is driven by removal of PnPy through condensation with acetyl-CoA to form phosphonomethylmalate (PnMM). This new reaction is catalyzed by a homolog of homocitrate synthase (HCS), PnMM synthase (acetyl-coenzyme A:3-phosphonopyruvate C-transferase) encoded by the frbC gene in S. rubellomurinus. We have cloned and expressed soluble, recombinant PnMM synthase and PEP mutase in E. coli. Using purified PnMM synthase or PEP mutase plus PnMM synthase, we have shown PnMM formation from acetyl-CoA and PnPy or PEP, respectively. We have developed colorimetric and HPLC assays that are being used for characterization of the kinetic mechanism of PnMM synthase. Sequence alignment of PnMM synthase with HCS and isopropylmalate synthase (IPMS), shows PnMM synthase has the three conserved residues (Asp14, His194, and His196) proposed as metal ligands in HCS and IPMS. By analogy, this suggests the α-carboxylate and α-keto of PnPy may coordinate to a similar metal in PnMM synthase, as opposed to active site residues as in the case of citrate synthase. Further characterization of the enzymes involved in the biosynthesis of FR900098 will aid in the development of low cost biosynthetic production approaches for this important antimalarial phosphonic acid.