Identification of a Unique Radical S -Adenosylmethionine 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 the 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 CX 3 CX 2 C 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 the loss of C-7 methylation, suggesting that the first motif could be 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 that methylation does not require cobalamin. When E. coli cells expressing MJ0619 were grown 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.

A Pterin-Dependent Signaling Pathway Regulates a Dual-Function Diguanylate Cyclase-Phosphodiesterase Controlling Surface Attachment in Agrobacterium tumefaciens

“…Whole-cell extracts prepared from late-exponential-phase cultures of A. tumefaciens were prepared, and pterins were enriched using cation exchange chromatography. These preparations were analyzed using HPLC on a reverse-phase C 18 column with fluorescence detection as previously described ( 79 ) and provided in Text S1 in the supplemental material in more detail.…”

Mechanistic Diversity of Radical S-Adenosylmethionine (SAM)-dependent Methylation

“…Finally, a new class of RS methylases, designated Class D, has recently been described (22). The prototype, MJ0619 from Methanocaldococcus jannaschii, is hypothesized to be involved in the biosynthesis of methanopterin (MPT), a coenzyme used in C1 metabolism during methanogenesis and methylotrophy in certain archaea and bacteria.…”

“…Lastly, Class C enzymes exhibit sequence homology to the RS protein HemN, and may bind two molecules of SAM simultaneously to effect methylation of sp 2 -hybridized carbon centers (11). Herein, we recapitulate some of the most salient features of that review, update current understanding and theory about RS methylases, and describe a new class (Class D) of RS methylases that, unlike Classes A, B, and C, are proposed to use methylenetetrahydrofolate rather than SAM as the source of the appended methyl carbon atom (22).…”

TsrM as a Model for Purifying and Characterizing Cobalamin-Dependent Radical S -Adenosylmethionine Methylases

“…Class C RS methylases are predicted to use two simultaneously bound SAM molecules to methylate sp 2 -hybridized carbon centers during the biosynthesis of several complex natural products. Class D methylases also methylate sp 2 -hybridized carbon centers, but have been suggested to use methylenetetrahydrofolate as the methyl source (Allen, Xu, & White, 2014). The recently annotated Class E RS methylases, by contrast, are involved in the maturation of the M cluster of nitrogenase, and appear to methylate a sulfide ion as part of a metallocluster (Hu & Ribbe, 2016).…”