Journal
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
Volume 1844, Issue 12, Pages 2135-2144Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbapap.2014.09.009
Keywords
Radical S-adenosyl-L-methionine; Methyltransferase; Cobalamin; Phosphinate
Categories
Funding
- NIH [RR0631401, RR12948]
- NSF [CHE-9115282, DBI-9604689]
- MJ Murdock Charitable Trust
- NIH training grant [T32GM008336]
- Bank of America Poncin Trust Fellowship
- Washington State University
- Faculty Early Career Development Award (CAREER) from the NSF [CHE-0953721]
- Division Of Chemistry
- Direct For Mathematical & Physical Scien [0953721] Funding Source: National Science Foundation
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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-C-13] cobalamin and titanium citrate, both [methyl-C-13] 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. denitnficans produces a phosphinate natural product. (C) 2014 Elsevier B.V. All rights reserved.
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