Open AccessOverall Retention of Methyl Stereochemistry during B12-Dependent Radical SAM Methyl Transfer in Fosfomycin Biosynthesis
Author(s) -
Martin I. McLaughlin,
Katharina Pallitsch,
Gabriele Wallner,
Wilfred A. van der Donk,
Friedrich Hammerschmidt
Publication year - 2021
Publication title -
biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.43
H-Index - 253
eISSN - 1520-4995
pISSN - 0006-2960
DOI - 10.1021/acs.biochem.1c00113
Subject(s) - methylcobalamin , chemistry , sn2 reaction , methyl group , biosynthesis , stereochemistry , fosfomycin , methyltransferase , walden inversion , methyl radical , enzyme , biochemistry , organic chemistry , methylation , alkyl , dna , radical , vitamin b12 , antibiotics
Methylcobalamin-dependent radical S -adenosylmethionine (SAM) enzymes methylate non-nucleophilic atoms in a range of substrates. The mechanism of the methyl transfer from cobalt to the receiving atom is still mostly unresolved. Here we determine the stereochemical course of this process at the methyl group during the biosynthesis of the clinically used antibiotic fosfomycin. In vitro reaction of the methyltransferase Fom3 using SAM labeled with 1 H, 2 H, and 3 H in a stereochemically defined manner, followed by chemoenzymatic conversion of the Fom3 product to acetate and subsequent stereochemical analysis, shows that the overall reaction occurs with retention of configuration. This outcome is consistent with a double-inversion process, first in the S N 2 reaction of cob(I)alamin with SAM to form methylcobalamin and again in a radical transfer of the methyl group from methylcobalamin to the substrate. The methods developed during this study allow high-yield in situ generation of labeled SAM and recombinant expression and purification of the malate synthase needed for chiral methyl analysis. These methods facilitate the broader use of in vitro chiral methyl analysis techniques to investigate the mechanisms of other novel enzymes.