Allen KD, Xu H, White RH

Identification of a unique radical SAM methylase likely involved in methanopterin biosynthesis in Methanocaldococcus jannaschii

▸ Abstract

Methanopterin (MPT) and its analogs are coenzymes required for methanogenesis and methylotrophy in specialized microorganisms. The methyl groups at C-7 and C-9 of the pterin ring distinguish MPT from all other pterin-containing natural products. However, the enzyme(s) responsible for addition of these methyl groups has yet to be identified. Here we demonstrate that a putative radical S-adenosyl-L-methionine (SAM) enzyme superfamily member encoded by the mj0619 gene in the methanogen Methanocaldococcus jannaschii is likely this missing methylase. When MJ0619 was heterologously expressed in Escherichia coli, various methylated pterins were detected, consistent with MJ0619 catalyzing methylation at C-7 and C-9 of 7,8-dihydro-6-hydroxymethylpterin, a common intermediate in both folate and MPT biosynthesis. Site-directed mutagenesis of Cys77 present in the first of two canonical radical SAM CX3CX2C motifs present in MJ0619 did not inhibit C-7 methylation while mutation of Cys102, found in the other radical SAM amino acid motif, resulted in loss of C-7 methylation, suggesting that the first motif is likely involved in C-9 methylation while the second motif is required for C-7 methylation. Further experiments demonstrated that the C-7 methyl group is not derived from methionine and methylation does not require cobalamin. When the E. coli expressing MJ0619 was grown in minimal media with deuterium-labeled acetate as the sole carbon source, the resulting methyl group on the pterin was predominantly labeled with three deuteriums. Based on these results, we propose that this archaeal radical SAM methylase employs a previously uncharacterized mechanism for methylation, using methylenetetrahydrofolate as a methyl group donor.

J Bacteriol 2014;None(None):None-None | PubMed ID: 25002541