title: Towards the Extension of the Substrate Spectrum of the [Fe]-Hydrogenase Hmd
title: Zur Erweiterung des Substratspektrums der [Fe]-Hydrogenase Hmd
titleType: AlternativeTitle
Creators: name: Gehl, Manuel
Publisher: name: Philipps-Universität Marburg
Year: 2023
Subjects: crystal structure
katalytischer Mechanismus
Methylen-Tetrahydromethanopterin-Reduktase
methylene-tetrahydrofolate reductase
methylene-tetrahydromethanopterin reductase
Methylen-Tetrahydrofolat-Reduktase
evolution
catalytic mechanism
[Fe]-Hydrogena
Kristallstruktur
[Fe]-hydrogenase
[Fe]-hydrogenas
Life sciences
Biowissenschaften, Biologie
Evolution

description: In hydrogenotrophic methanogenesis, methenyl-tetrahydromethanopterin (methenyl H4MPT+) is sequentially reduced to methylene-H4MPT and methyl H4MPT. The H2-forming methylene H4MPT dehydrogenase Hmd catalyzes the reduction of methenyl H4MPT+ to methylene H4MPT using H2 as electron donor. The reduction of methylene H4MPT to methyl H4MPT is catalyzed by the F420-dependent methylene H4MPT reductase Mer using reduced F420 (F420H2) as electron donor. Despite the very similar structure of the H4MPT derivatives and the fact that Hmd can bind methylene H4MPT and generate H2 by oxidation of the methylene group, Hmd is unable to reduce methylene H4MPT. The question that arises is what are the structural features that allow Hmd to reduce only methenyl H4MPT+ but not methylene-H4MPT. To this end, the catalytic mechanism of Hmd was first investigated to determine whether the H2 activation reaction requires the presence of methenyl-H4MPT+. In collaboration with researchers at the MPI Göttingen, an NMR method based on the parahydrogen induced polarization (PHIP) effect was applied to study the trajectories of H2, hydrides and protons during the catalytic cycle of Hmd. For the first time, significant PHIP-NMR signals of H2 and hydride binding to Hmd were obtained, supporting the proposed catalytic mechanism that does not involve the methenyl group in hydride formation. Following this finding, the catalytic mechanism of Mer and the flavin-independent methylene-tetrahydrofolate reductase Mfr, which catalyzes the analogous reduction of methylene-tetrahydrofolate (methylene-H4F) to methyl-H4F, was investigated. Mer from Methanocaldococcus jannaschii (jMer) and Mfr from Mycolicibacterium hassiacum (hMfr) were heterologously produced in Escherichia coli and the crystal structures of the apoenzymes of jMer and hMfr as well as the binary complex of jMer with F420 were solved. Since no ternary complex of jMer or hMfr including the C1 carrier and reducing agent could be obtained, a functional alignment approach was used to derive information on the geometry of the ternary complex. The structure of jMer complexed with F420 was aligned with the published ternary complex structure of the FAD-dependent methylene-H4F reductase from E. coli (eMTHFR) in such a way that the proteins were first manually aligned using the hydride-carrying atoms of the electron carriers as a fixed point. In a second step, the apoenzyme structure of hMfr was incorporated into the model by manually aligning it with the structure of eMTHFR. Amino acids found at equivalent positions in all three reductases were mutated to investigate their putative function. The mutational analysis indicated that although eMTHFR, hMfr and jMer share a limited degree of sequence identity, the active site amino acid residues and their geometries are very similar and may serve the same function. Furthermore, a glutamate was found as the key catalytic residue at the equivalent position in all three enzymes, suggesting that they share a common catalytic mechanism, which involves the formation of a 5-iminium cation intermediate. This knowledge was used to construct a docking model of hMfr in complex with NADH and methylene-H4F. A phylogenetic analysis indicated that the three reductases do not share a common ancestor and the conserved active site structures of the three reductases may be the result of divergent evolution. Through this series of studies, it was suggested that if the active site of Hmd could be modified to carry out the desired protonation of methylene-H4MPT, it would be possible to construct an Hmd mutant capable of reducing methylene-H4MPT to methyl-H4MPT using H2. Some possible mutations to form such a methylene-H4MPT-reducing Hmd are suggested.