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-13C] cobalamin and titanium citrate, both [methyl-13C] 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.

Consecutive radical S -adenosylmethionine methylations form the ethyl side chain in thienamycin biosynthesis

“…The relative stoichiometry of iron and sulfide bound by the as-isolated protein was determined to be 7.4 ± 1.4 and 3.7 ± 0.8 per polypeptide, respectively, consistent with the presence of a [4Fe-4S] cluster. Apparent excess iron has been observed with other RS enzymes (11,14), and was similarly seen in an alanine variant control.…”

“…The (3S,5S)-carbapenam (2) was synthesized as described previously (32). Compounds 8,9,13,14,16 (33), 10-12 (34, 35), and 15 (36, 37) were derived from previous synthetic strategies as indicated. Details are presented in SI Appendix.…”

Vitamin B 12 in the spotlight again

“…Indeed, as predicted based on sequence, several of these enzymes have been shown to require Cbl for chemistry [36–43] and one member, TsrM, has been shown spectroscopically to bind Cbl in a “base-off”, “His-off” conformation [44]. Despite the large family size, only a small number of these enzymes have been isolated and characterized; in addition to the arduous oxygen and light sensitive cofactors, several have been reported to be insoluble [36,39–41]. …”

“…Of particular interest is the reaction catalyzed by PhpK, which is responsible for the formation of a C-P bond linkage in a bialaphos precursor. This challenging methylation as well as other methylation reactions catalyzed by members of this enzyme class have been hypothesized to require both Cbl and AdoMet as shown in Figure 4a [36,37,39–41,48,49,51,52]. Unlike the traditional Cbl-dependent methyltransferase reactions, these enzymes are hypothesized to catalyze methylation of a substrate following homolytic Co-C bond cleavage of MeCbl; precedent for transfer of a methyl radical species comes from Cbl model chemistry [53].…”

The Birth and Demise of Hypotheses on Evolution of S-Adenosyl-Lmethionine and Adenosylcobalamin

“…Certain radical SAM enzymes are methyltransferases that methylate non-nucleophilic centers, such as phosphorus in phosphonates and trigonal carbons in amino acids and nucleic acid bases [24]. Moreover, certain methyltransferases require both SAM and a cobalamin, generally methylcobalamin [24,25]. The biochemical and mechanistic relations between SAM and cobalamins might be indicative of special relationships that eventually led to adenosylcobalamin-dependent enzymes.…”