title: Spectroscopic characterization and mechanistic investigation of P-methyl transfer by a radical SAM enzyme from the marine bacterium Shewanella denitrificans OS217.
abstract: 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.
authors.affiliation: School of Molecular Biosciences, College of Veterinary Medicine, PO Box 647520, Washington State University, Pullman, WA 99164-7520, USA. Electronic address: kdallen@vt.edu.
authors.forename: Kylie D
authors.initials: KD
authors.lastname: Allen
authors.affiliation: School of Molecular Biosciences, College of Veterinary Medicine, PO Box 647520, Washington State University, Pullman, WA 99164-7520, USA. Electronic address: susan_wang@wsu.edu.
authors.forename: Susan C
authors.initials: SC
authors.lastname: Wang
keywords: Cobalamin; Methyltransferase; Phosphinate; Radical S-adenosyl-l-methionine