Abstract
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.