Characterization of A Novel S‐adenosylmethionine‐dependent Methylase by Electron Paramagnetic Resonance and Mössbauer Spectroscopies

Abstract

Radical S‐adenosyl‐L‐methionine dependent methylases are divided into four classes (A–D) with only A through C being extensively characterized in vitro. Class D methylases separate themselves by purportedly utilizing methylenetetrahydrofolate in their catalysis‐. The gene product from Methanocaldococcus jannaschii, MJ0619, is the pioneer member of this class and is hypothesized to install methyl groups into C7 and C9 of methanopterin, a key coenzyme for C1 metabolism in methanogeneic and methylotrophic bacteria. By extracting folates from E. coli transformed with a plasmid containing the MJ0619 gene, Allen, et.al. were able to detect and characterize methylated substrates and determine that 7‐methylpterin and 6‐ethyl‐7‐methylpterin were produced in vivo. Feeding studies in which E. coli were grown in media containing C2H3‐ methionine or C2H3‐acetate suggested that MJ0619, unlike members of classes A‐C, does not use SAM, but rather methylenetetrahydrofolate as the methyl donor. Excitingly, MJ0619 harbors a second CX3CX2C motif hypothesized to ligate an additional [4Fe‐4S] cluster and when the middle cysteine of this motif is changed to an alanine only 7‐methylpterin is detected. Although it is suggested that the N‐terminal Fe/S cluster is involved in C9 methylation there is little in vitro evidence to support this function and no spectroscopic characterization to identify it as a [4Fe‐4S] cluster. Herein, we report the heterologous expression and isolation of MJ0619, a member of a class D methylases, and employ Mössbauer and electron paramagnetic resonance spectroscopies to characterize its Fe/S cluster(s) of MJ0619.Support or Funding InformationHoward Hughes Medical InstituteThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.