Publications

• Studies on the biosynthesis of bialaphos (SF-1293) 12. C-P bond formation mechanism of bialaphos: discovery of a P-methylation enzyme.

  Kamigiri K., Hidaka T., Imai S., Murakami T., Seto H.

  An enzymatic activity catalyzing P-methylation of N-acetyldemethylphosphinothricin, a biosynthetic intermediate of the herbicide bialaphos, was detected in a cell extract of Streptomyces hygroscopicus SF-1293, a bialaphos producing organism. The gene coding for this P-methylation enzyme in the bia ... >> More

  An enzymatic activity catalyzing P-methylation of N-acetyldemethylphosphinothricin, a biosynthetic intermediate of the herbicide bialaphos, was detected in a cell extract of Streptomyces hygroscopicus SF-1293, a bialaphos producing organism. The gene coding for this P-methylation enzyme in the bialaphos biosynthetic gene cluster was also expressed in Streptomyces lividans. The methyl donor of the reaction was determined to be methylcobalamin. The P-methylation enzyme utilized both N-acetyldemethylbialaphos and N-acetyldemethylphosphinothricin as substrates. << Less

  J Antibiot (Tokyo) 45:781-787(1992) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Spectroscopic characterization and mechanistic investigation of P-methyl transfer by a radical SAM enzyme from the marine bacterium Shewanella denitrificans OS217.

  Allen K.D., Wang S.C.

  Natural products containing carbon-phosphorus bonds elicit important bioactivity in many organisms. l-Phosphinothricin contains the only known naturally-occurring carbon-phosphorus-carbon bond linkage. In actinomycetes, the cobalamin-dependent radical S-adenosyl-l-methionine (SAM) methyltransferas ... >> More

  Natural products containing carbon-phosphorus bonds elicit important bioactivity in many organisms. l-Phosphinothricin contains the only known naturally-occurring carbon-phosphorus-carbon bond linkage. In actinomycetes, the cobalamin-dependent radical S-adenosyl-l-methionine (SAM) methyltransferase PhpK catalyzes the formation of the second C-P bond to generate the complete C-P-C linkage in phosphinothricin. Here we use electron paramagnetic resonance and nuclear magnetic resonance spectroscopies to characterize and demonstrate the activity of a cobalamin-dependent radical SAM methyltransferase denoted SD_1168 from Shewanella denitrificans OS217, a marine bacterium that has not been reported to synthesize phosphinothricin. Recombinant, refolded, and reconstituted SD_1168 binds a four-iron, four-sulfur cluster that interacts with SAM and cobalamin. In the presence of SAM, a reductant, and methylcobalamin, SD_1168 surprisingly catalyzes the P-methylation of N-acetyl-demethylphosphinothricin and demethylphosphinothricin to produce N-acetyl-phosphinothricin and phosphinothricin, respectively. In addition, this enzyme is active in the absence of methylcobalamin if the strong reductant titanium (III) citrate and hydroxocobalamin are provided. When incubated with [methyl-(13)C] cobalamin and titanium citrate, both [methyl-(13)C] and unlabeled N-acetylphosphinothricin are produced. Our results suggest that SD_1168 catalyzes P-methylation using radical SAM-dependent chemistry with cobalamin as a coenzyme. In light of recent genomic information, the discovery of this P-methyltransferase suggests that S. denitrificans produces a phosphinate natural product. << Less

  Biochim Biophys Acta 1844:2135-2144(2014) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• In vitro phosphinate methylation by PhpK from Kitasatospora phosalacinea.

  Werner W.J., Allen K.D., Hu K., Helms G.L., Chen B.S., Wang S.C.

  Radical S-adenosyl-L-methionine, cobalamin-dependent methyltransferases have been proposed to catalyze the methylations of unreactive carbon or phosphorus atoms in antibiotic biosynthetic pathways. To date, none of these enzymes has been purified or shown to be active in vitro. Here we demonstrate ... >> More

  Radical S-adenosyl-L-methionine, cobalamin-dependent methyltransferases have been proposed to catalyze the methylations of unreactive carbon or phosphorus atoms in antibiotic biosynthetic pathways. To date, none of these enzymes has been purified or shown to be active in vitro. Here we demonstrate the activity of the P-methyltransferase enzyme, PhpK, from the phosalacine producer Kitasatospora phosalacinea. PhpK catalyzes the transfer of a methyl group from methylcobalamin to 2-acetylamino-4-hydroxyphosphinylbutanoate (N-acetyldemethylphosphinothricin) to form 2-acetylamino-4-hydroxymethylphosphinylbutanoate (N-acetylphosphinothricin). This transformation gives rise to the only carbon-phosphorus-carbon linkage known to occur in nature. << Less

  Biochemistry 50:8986-8988(2011) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Sequence of a P-methyltransferase-encoding gene isolated from a bialaphos-producing Streptomyces hygroscopicus.

  Hidaka T., Hidaka M., Kuzuyama T., Seto H.

  The nucleotide sequence of the Streptomyces hygroscopicus gene encoding P-methyltransferase, catalyzing the formation of a carbon-phosphorus bond, involved in bialaphos biosynthesis, has been determined. The amino-acid sequence deduced from the nt sequence, shows homology with those of magnesium-p ... >> More

  The nucleotide sequence of the Streptomyces hygroscopicus gene encoding P-methyltransferase, catalyzing the formation of a carbon-phosphorus bond, involved in bialaphos biosynthesis, has been determined. The amino-acid sequence deduced from the nt sequence, shows homology with those of magnesium-protoporphyrin IX monomethyl ester oxidative cyclase (Mg-ProtoMe cyclase) of Rhodobacter capsulatus and the enzyme catalyzing the methylation of the aldehyde carbon of phosphonoacetaldehyde in fosfomycin biosynthesis. << Less

  Gene 158:149-150(1995) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Investigation of enzymatic C-P bond formation using multiple quantum HCP nuclear magnetic resonance spectroscopy.

  Hu K., Werner W.J., Allen K.D., Wang S.C.

  The biochemical mechanism for the formation of the C-P-C bond sequence found in l-phosphinothricin, a natural product with antibiotic and herbicidal activity, remains unclear. To obtain further insight into the catalytic mechanism of PhpK, the P-methyltransferase responsible for the formation of t ... >> More

  The biochemical mechanism for the formation of the C-P-C bond sequence found in l-phosphinothricin, a natural product with antibiotic and herbicidal activity, remains unclear. To obtain further insight into the catalytic mechanism of PhpK, the P-methyltransferase responsible for the formation of the second C-P bond in l-phosphinothricin, we utilized a combination of stable isotopes and two-dimensional nuclear magnetic resonance spectroscopy. Exploiting the newly emerged Bruker QCI probe (Bruker Corp.), we specifically designed and ran a (13) C-(31) P multiple quantum (1) H-(13) C-(31) P (HCP) experiment in (1) H-(31) P two-dimensional mode directly on a PhpK-catalyzed reaction mixture using (13) CH3 -labeled methylcobalamin as the methyl group donor. This method is particularly advantageous because minimal sample purification is needed to maximize product visualization. The observed 3:1:1:3 multiplet specifically and unequivocally illustrates direct bond formation between (13) CH3 and (31) P. Related nuclear magnetic resonance experiments based upon these principles may be designed for the study of enzymatic and/or synthetic chemical reaction mechanisms. << Less

  Magn Reson Chem 53:267-272(2015) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.