Identification of HcgC as a SAM-Dependent Pyridinol Methyltransferase in [Fe]-Hydrogenase Cofactor Biosynthesis

Lichen Bai, Xile Hu, Tao Xu
Wiley-Blackwell, 2016
Journal paper

Previous retrosynthetic and isotope-labeling studies have indicated that biosynthesis of the iron guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase requires a methyltransferase. This hypothetical enzyme covalently attaches the methyl group at the 3-position of the pyridinol ring. We describe the identification of HcgC, a gene product of the hcgA-G cluster responsible for FeGP cofactor biosynthesis. It acts as an S-adenosylmethionine (SAM)-dependent methyltransferase, based on the crystal structures of HcgC and the HcgC/SAM and HcgC/S-adenosylhomocysteine (SAH) complexes. The pyridinol substrate, 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol, was predicted based on properties of the conserved binding pocket and substrate docking simulations. For verification, the assumed substrate was synthesized and used in a kinetic assay. Mass spectrometry and NMR analysis revealed 6-carboxymethyl-3,5-dimethyl-4-hydroxy-2-pyridinol as the reaction product, which confirmed the function of HcgC.

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Lichen Bai, Xile Hu, Tao Xu
Wiley-Blackwell, 2016
Journal paper

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https://infoscience.epfl.ch/record/222237?ln=en

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Chemical reaction

A chemical reaction is a process that leads to the chemical transformation of one set of chemical substances to another. Classically, chemical reactions encompass changes that only involve the posit

Methyl group

In organic chemistry, a methyl group is an alkyl derived from methane, containing one carbon atom bonded to three hydrogen atoms, having chemical formula . In formulas, the group is often abbreviate

Hydrogenase

A hydrogenase is an enzyme that catalyses the reversible oxidation of molecular hydrogen (H2), as shown below: Hydrogen uptake () is coupled to the reduction of electron acceptors such as oxygen,

Towards artificial methanogenesis: biosynthesis of the [Fe]-hydrogenase cofactor and characterization of the semisynthetic hydrogenase

Lichen Bai, Xile Hu, Tao Xu

The greenhouse gas and energy carrier methane is produced on Earth mainly by methanogenic archaea. In the hydrogenotrophic methanogenic pathway the reduction of one CO2 to one methane molecule requires four molecules of H-2 containing eight electrons. Four of the electrons from two H-2 are supplied for reduction of an electron carrier F-420, which is catalyzed by F-420-reducing [NiFe]-hydrogenase under nickel-sufficient conditions. The same reaction is catalysed under nickel-limiting conditions by [Fe]-hydrogenase coupled with a reaction catalyzed by F-420-dependent methylene tetrahydromethanopterin dehydrogenase. [Fe]-hydrogenase contains an iron-guanylylpyridinol (FeGP) cofactor for H-2 activation at the active site. FeII of FeGP is coordinated to a pyridinol-nitrogen, an acyl-carbon, two CO and a cysteine-thiolate. We report here on comparative genomic analyses of biosynthetic genes of the FeGP cofactor, which are primarily located in a hmd-co-occurring (hcg) gene cluster. One of the gene products is HcgB which transfers the guanosine monophosphate (GMP) moiety from guanosine triphosphate (GTP) to a pyridinol precursor. Crystal structure analysis of HcgB from Methanococcus maripaludis and its complex with 6-carboxymethyl- 3,5-dimethyl-4-hydroxy-2-pyridinol confirmed the physiological guanylyltransferase reaction. Furthermore, we tested the properties of semi-synthetic [Fe]-hydrogenases using the [Fe]-hydrogenase apoenzyme from several methanogenic archaea and a mimic of the FeGP cofactor. On the basis of the enzymatic reactions involved in the methanogenic pathway, we came up with an idea how the methanogenic pathway could be simplified to develop an artificial methanogenesis system.

Royal Soc Chemistry2017

A Water-Bridged H-Bonding Network Contributes to the Catalysis of the SAM-Dependent C-Methyltransferase HcgC

Lichen Bai, Xile Hu, Tao Xu

[Fe]-hydrogenase hosts an iron-guanylylpyridinol (FeGP) cofactor. The FeGP cofactor contains a pyridinol ring substituted with GMP, two methyl groups, and an acylmethyl group. HcgC, an enzyme involved in FeGP biosynthesis, catalyzes methyl transfer from S-adenosylmethionine (SAM) to C3 of 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol (2). We report on the ternary structure of HcgC/S-adenosylhomocysteine (SAH, the demethylated product of SAM) and 2 at 1.7 angstrom resolution. The proximity of C3 of substrate 2 and the S atom of SAH indicates a catalytically productive geometry. The hydroxy and carboxy groups of substrate 2 are hydrogen-bonded with I115 and T179, as well as through a series of water molecules linked with polar and a few protonatable groups. These interactions stabilize the deprotonated state of the hydroxy groups and a keto form of substrate 2, through which the nucleophilicity of C3 is increased by resonance effects. Complemented by mutational analysis, a structure-based catalytic mechanism was proposed.

2017