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.

Keywords: Cobalamin; Methyltransferase; Phosphinate; Radical S-adenosyl-l-methionine.

Figure 1

P-methylation and C-methylation reactions of interest.

Figure 2

Radical SAM cleavage.

Figure 3

Comparison of putative S. denitrificans OS217 and Kitasatospora NRRL F-6133 C-P homologous genes [23]. Genes in color are homologous. Red indicates Cbl-dependent radical SAM methyltransferases. Genes in gray are not homologous. Not all genes from each putative pathway are shown. PepM: phosphoenolpyruvate mutase, Ppd: phosphonopyruvate decarboxylase.

Figure 4

UV-Vis spectra of ~30 μM SD_1168. Black: reconstituted SD_1168; orange: SD_1168 + 4 mM sodium dithionite; blue: SD_1168 + 10 mM Ti citrate.

Figure 5

EPR spectra of SD_1168. Spectrum A: Refolded and reconstituted SD_1168, Spectrum B: SD_1168 + dithionite, Spectrum C: SD_1168 refolded with HO-Cbl + dithionite, Spectrum D: SD_1168 refolded with HO-Cbl + dithionite + SAM, Spectrum E: SD_1168 (−SAM) + dithionite.

Figure 6

H-P gHSQC NMR spectra of partially purified, Ti citrate-reduced SD_1168-catalyzed reactions of interest. A. NAcDMPT + CH₃Cbl reaction. The cross-peaks at (1.66, 35) ppm and (1.95, 35) ppm correspond to the C-4 methylene protons and the C-3 methylene protons, respectively, of NAcDMPT. The cross-peak at (1.35, 56) ppm corresponds to the methyl protons of NAcPT. B. DMPT + CH₃Cbl reaction. The cross-peaks at (1.45, 26) ppm and (1.90, 26) ppm correspond to the C-4 methylene protons and the C-3 methylene protons, respectively, of DMPT. The cross-peak at (1.12, 41) ppm corresponds to the methyl protons of PT. C. NAcDMPT + [methyl-¹³C]Cbl reaction. The two cross-peaks indicated by the double daggers (‡) at (1.30, 56) ppm and (1.09, 56) ppm correspond to the methyl protons of [methyl-¹³C]NAcPT, which are split by the NMR-active ¹³C and ³¹P nuclei. The cross-peak indicated by the asterisk (*) at (1.20, 56) ppm corresponds to the methyl protons of unlabeled NAcPT. D. NAcDMPT + HOCbl reaction. The major cross-peak at (1.68, 35) ppm corresponds to the C-4 methylene protons of NAcDMPT, and the cross-peak at (1.29, 56.5) ppm corresponds to the methyl protons of NAcPT.

Figure 7

Proposed radical SAM P-methylation mechanism.