Investigation of enzymatic C–P bond formation using multiple quantum HCP nuclear magnetic resonance spectroscopy

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 13C-31P multiple quantum 1H-13C-31P (HCP) experiment in 1H-31P two-dimensional mode directly on a PhpK-catalyzed reaction mixture using 13CH3-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 13CH3 and 31P. Related nuclear magnetic resonance experiments based upon these principles may be designed for the study of enzymatic and/or synthetic chemical reaction mechanisms.

Mechanistic Studies on CysS – A Vitamin B12-Dependent Radical SAM Methyltransferase Involved in the Biosynthesis of the tert-Butyl Group of Cystobactamid

“…PhpK catalyzes the conversion of 5 to 6 and occurs on the biosynthesis of the broadspectrum herbicide phosphinothricin. 15 GenK catalyzes the Calkylation of an alcohol (7 to 8) during the biosynthesis of the antibiotic gentamicin, and Fom3 catalyzes a similar reaction in the biosynthesis of the antibiotic fosfomycin. [11][12][13][14]18 Nosiheptide biosynthesis uses TsrM to catalyze an indole methylation (11 to 12).…”

“…The CysS catalyzed iterative methylation of the methoxy group of 1 is the topic of this paper. PhpK catalyzes the conversion of 5 to 6 and occurs on the biosynthesis of the broad-spectrum herbicide phosphinothricin . GenK catalyzes the C-alkylation of an alcohol ( 7 to 8 ) during the biosynthesis of the antibiotic gentamicin, and Fom3 catalyzes a similar reaction in the biosynthesis of the antibiotic fosfomycin. − , Nosiheptide biosynthesis uses TsrM to catalyze an indole methylation ( 11 to 12 ). − ThnK catalyzes an iterative C-methylation to assemble an ethyl group during the biosynthesis of the antibiotic thienamycin ( 13 to 14 ). , PoyC catalyzes valine Cβ-methylation during polytheonamide biosynthesis ( 15 to 16 ) …”

“…While sequence analysis indicates that Cbl-dependent radical SAM methyltransferases are widely used in biosynthesis, it was not until recently that enzymes in this class were reconstituted in defined biochemical systems. − Detailed mechanistic studies are still at an early stage because of substrate complexity and the low yield of pure active enzyme available from most overexpression systems. − No Cbl-dependent radical SAM methyltransferase has yet been structurally characterized.…”

Biosynthesis of Oxetanocin-A Includes a B12-Dependent Radical SAM Enzyme That Can Catalyze both Oxidative Ring Contraction and the Demethylation of SAM

“…Determination of the OxsB crystal structure revealed juxtaposition of the [4Fe-4S]-SAM and the Cbl cofactors in the active site, suggesting that both may participate directly in the catalytic cycle of OxsB . Thus far, a total of 12 B 12 -dependent radical SAM enzymes (TsrM, − Fom3, − PhpK, − ThnK, TokK, CysS, , PoyC, GenK, , GenD1, MaMmp10, orf29 in bacterial MeACC synthase, and OxsB 3 ) have been studied in vitro. Unlike other members of this group, which are all involved in the methylation of unactivated carbons or phosphinate centers, OxsB is the only enzyme in this class known to catalyze an oxidative ring contraction .…”

Reaction Catalyzed by GenK, a Cobalamin-Dependent Radical S-Adenosyl-l-methionine Methyltransferase in the Biosynthetic Pathway of Gentamicin, Proceeds with Retention of Configuration

“…In summary, the Cbl-dependent RSMTs represent the largest and most diverse collection of RSMTs presently known. Based on prior in vitro investigations of eight of these enzymes (TsrM, − Fom3, − GenD1, ThnK, PhpK, − PoyC, CysS and GenK), a model of substrate binding wherein the substrate is sandwiched between the Cbl cofactor and the radical SAM machinery has been suggested. However, the present results with GenK suggest that this may not necessarily be a general property of these enzymes.…”