In Vitro Biosynthesis of the [Fe]‐Hydrogenase Cofactor Verifies the Proposed Biosynthetic Precursors

Abstract

In the FeGP cofactor of [Fe]‐hydrogenase, low‐spin FeII is in complex with two CO ligands and a pyridinol derivative; the latter ligates the iron with a 6‐acylmethyl substituent and the pyridinol nitrogen. A guanylylpyridinol derivative, 6‐carboxymethyl‐3,5‐dimethyl‐4‐guanylyl‐2‐pyridinol (3), is produced by the decomposition of the FeGP cofactor under irradiation with UV‐A/blue light and is also postulated to be a precursor of FeGP cofactor biosynthesis. HcgC and HcgB catalyze consecutive biosynthesis steps leading to 3. Here, we report an in vitro biosynthesis assay of the FeGP cofactor using the cell extract of the ΔhcgBΔhcgC strain of Methanococcus maripaludis, which does not biosynthesize 3. We chemically synthesized pyridinol precursors 1 and 2, and detected the production of the FeGP cofactor from 1, 2 and 3. These results indicated that 1, 2 and 3 are the precursors of the FeGP cofactor, and the carboxy group of 3 is converted to the acyl ligand.

On the evolution of coenzyme biosynthesis

“…5 This homodimeric enzyme contains one non-redox active iron per subunit linked to a guanylylpyridinol cofactor 27 in short the FeGP cofactor. 90 The biosynthesis of FeGP has not yet been fully elucidated. So far, it is known that 2-(4,6-dihydroxy-3,5-dimethylpyridin-2yl)acetic acid 75 is the biosynthetic precursor for FeGP 27, while so far only a few preliminary feeding experiments with 13 C-labeled building blocks such as acetic acid and pyruvate could give some clues about the origin of the pyridine ligand.…”

“…So far, it is known that 2-(4,6-dihydroxy-3,5-dimethylpyridin-2yl)acetic acid 75 is the biosynthetic precursor for FeGP 27, while so far only a few preliminary feeding experiments with 13 C-labeled building blocks such as acetic acid and pyruvate could give some clues about the origin of the pyridine ligand. 90 3. Can the analysis provide insight into coenzyme evolution?…”

“…5 This homodimeric enzyme contains one non-redox active iron per subunit linked to a guanylylpyridinol cofactor 27 in short the FeGP cofactor. 90…”

Current State of [Fe]‐Hydrogenase and Its Biomimetic Models

“…Proposed biosynthesis pathway of the FeGP cofactor. [11,[24][25][26][27][28][29] Chemistry-A European Journal which the distance could be shortened to ~3.8 Å. [33] Thus, open/closed conformation changes were proposed as the required step for H À transfer.…”

“…HcgA and HcgG may be SAM‐dependent enzymes, but their real functions are unknown [28] . A recent report showed that the FeGP cofactor can be synthesized in vitro using synthetic ligand precursors, Fe, CO, H 2 and cell extracts from Methanococcus maripaludis ΔhcgBΔhcgC , HcgB and HcgC enzymes [29] …”