The Function of Two Radical‐SAM Enzymes, HcgA and HcgG, in the Biosynthesis of the [Fe]‐Hydrogenase Cofactor

Abstract

In the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor, 6-carboxymethyl-5-methyl-4hydroxy-2-pyridinol ( 1) is 3-methylated to form 2, then 4-guanylylated to form 3, and converted into the full cofactor. HcgA-G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S-adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis ΔhcgA strain was complemented with HcgA or precursors 1, 2 or 3. The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1. We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the ΔhcgG system was recovered by HcgG but not by 3, which indicated that HcgG catalyzes the reactions after the biosynthesis of 3. The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.

Acyl and CO Ligands in the [Fe]‐Hydrogenase Cofactor Scramble upon Photolysis

“…[2b] By photolysis, the acyl ligand of the isolated FeGP cofactor is hydrolyzed to form the 6-carboxymethyl group of 6-carboxymethyl-3,5-dimethyl-4-guanylyl-2-pyridone (GP) (Figure 1b). [2a,5b] GP is also known as a precursor of biosynthesis of the FeGP cofactor [8,10] The FeGP cofactor is biosynthesized by the reactions catalyzed by HcgAÀ G proteins. GP is produced by the reactions by HcgA, HcgB and HcgC.…”

“…GP is produced by the reactions by HcgA, HcgB and HcgC. [10][11] The CO ligand is biosynthesized from CO gas or an unknown organic substance by HcgG. [8,10] The carboxy group of GP is proposed to be converted to the acyl ligand by the consecutive biosynthetic reactions of HcgE, HcgF and HcgG.…”

“…[10][11] The CO ligand is biosynthesized from CO gas or an unknown organic substance by HcgG. [8,10] The carboxy group of GP is proposed to be converted to the acyl ligand by the consecutive biosynthetic reactions of HcgE, HcgF and HcgG. [8,10,11c,12] The hypothesis of acyl-ligand biosynthesis was supported by the observation that 6-decarboxylated GP did not produce active [Fe]-hydrogenase in in vitro biosyn-thesis and up to two 13 C were incorporated into the FeGP cofactor from [ 13 C]-CO (Figure 2a).…”