Bioorganic Chemical Studies on the Formation Mechanisms of Bioactic C-P Compounds

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This reseach has been made in order to reveal the formation mechanism of the C-P bond found in some microbial metabolites. As a target of this research we have selected a herbicide named bialaphos which contains a unique C-P-C bond. This metabolite is produced by Streptomyces hygroscopicus SF1293.At first, the transformation mechanism of phosphimopyruvic acid (an analog of oxalacetic acid) to demethylphosphinothricin (an analog of glutamic acid) resulting in the increase of one carbor unit of the skeleton was studied by using enzyme inhibitors of TCA cycle. Addition of monofluoro acetic acid caused the accumulation of a new phosphinic acid named 2-phosphinomethylmalic acid (PMM) which was isolated by ion exchange resin cellulose chromatograpgy and gel filtration. Its structure was determined to be an analog of citric acid by ^+H- and ^<13>C-NMR spectral analysis. The absolute stereochemistry of PMM was established to be (R) by comparing with the (S)-form isomer prepared by the use of porcine heart citrate synthase.The enzyme catalyzing this reaction was named PMM synthase and was purified to a pure protein by several chromatographic procedures. It is a homodimer consisting of 48K monomers. The N-terminal amino acid sequence up to 28 amino acids was determined. The corresponding open reading frame was found in the already cloned DNA which codes for the biosynthetic genes of bialaphos. The properties of this enzyme clearly shows that PMM synthase is very closely related to (R)-citrate synthase obtained from some anaerobic bacteria.PMM was converted to demethylphosphinothricin (<alpha>-ketoglutaric acid analog) by aconitase and isocitrate dehydrogenase; this finding suggests strongly that TCA cycle enzymes are involved in the biosynthesis of bialaphos. Thus, the detailed mechanism of bialaphos operating in the later stage has been revealed by this research work.