Overall Retention of Methyl Stereochemistry during B12-Dependent Radical SAM Methyl Transfer in Fosfomycin Biosynthesis.

Martin I Mclaughlin,Katharina Pallitsch,Wilfred A Van Der Donk,Friedrich Hammerschmidt,Gabriele Wallner

doi:10.1021/acs.biochem.1c00113

Martin I Mclaughlin, Katharina Pallitsch + Show 3 more

Open Access

https://doi.org/10.1021/acs.biochem.1c00113

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Abstract

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 1H, 2H, and 3H 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 SN2 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.

Highlights

The mechanism of the methyl transfer from cobalt to the receiving atom is still mostly unresolved
Our chiral methyl analysis of the Fom3-catalyzed methyl transfer from SAM to 2-HEP-CMP demonstrates that this transformation occurs with net retention of stereochemistry at the methyl center under in vitro reconstituted reaction conditions
This finding is consistent with previous studies on S. f radiae cultures that were fed methionine with a stereodefined methyl group as a precursor to fosfomycin.[41]

Summary

Introduction

The mechanism of the methyl transfer from cobalt to the receiving atom is still mostly unresolved. In vitro reaction of the methyltransferase Fom[3] using SAM labeled with 1H, 2H, and 3H in a stereochemically defined manner, followed by chemoenzymatic conversion of the Fom[3] product to acetate and subsequent stereochemical analysis, shows that the overall reaction occurs with retention of configuration This outcome is consistent with a doubleinversion process, first in the SN2 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. HPP-CMP leaves cob(II)alamin in the enzyme active site; MeCbl is regenerated by one-electron reduction of cob(II)alamin followed by standard SN2-type methyl transfer from a second molecule of SAM

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