[Show abstract][Hide abstract] ABSTRACT: [Fe]-Hydrogenase catalyses the reversible hydrogenation of a methenyltetrahydromethanopterin substrate, which is an intermediate step during the methanogenesis from CO2 and H2. The active site contains an iron-guanylylpyridinol cofactor, in which Fe(2+) is coordinated by two CO ligands, as well as an acyl carbon atom and a pyridinyl nitrogen atom from a 3,4,5,6-substituted 2-pyridinol ligand. However, the mechanism of H2 activation by [Fe]-hydrogenase is unclear. Here we report the reconstitution of [Fe]-hydrogenase from an apoenzyme using two FeGP cofactor mimics to create semisynthetic enzymes. The small-molecule mimics reproduce the ligand environment of the active site, but are inactive towards H2 binding and activation on their own. We show that reconstituting the enzyme using a mimic that contains a 2-hydroxypyridine group restores activity, whereas an analogous enzyme with a 2-methoxypyridine complex was essentially inactive. These findings, together with density functional theory computations, support a mechanism in which the 2-hydroxy group is deprotonated before it serves as an internal base for heterolytic H2 cleavage.
[Show abstract][Hide abstract] ABSTRACT: [Fe]-hydrogenase (Hmd), an enzyme of the methanogenic energy metabolism, harbors an iron-guanylylpyridinol (FeGP) cofactor used for H2 cleavage. The generated hydride is transferred to methenyl-tetrahydromethanopterin (methenyl-H4 MPT(+) ). Most hydrogenotrophic methanogens contain the hmd related genes hmdII and hmdIII. Their function is still elusive. We were able to reconstitute HmdII holoenzyme of Methanocaldococcus jannaschii with recombinantly produced apoenzyme and the FeGP cofactor, which is a prerequisite for an in vitro functional analysis. Infrared spectroscopic and X-ray structural data clearly indicated binding of the FeGP cofactor. Methylene-H4 MPT binding was detectable in the significantly altered infrared spectra of the HmdII holoenzyme and in the HmdII apoenzyme-methylene-H4 MPT complex structure. The related binding mode of the FeGP cofactor and methenyl-H4 MPT(+) compared to Hmd and their multiple contacts to the polypeptide highly suggest a biological role in HmdII. However, holo-HmdII did not catalyze the Hmd reaction, not even in a single turn-over process, as demonstrated by kinetic measurements. The found inactivity can be rationalized by an increased contact area between the C- and N-terminal folding units in HmdII compared to in Hmd that impairs the catalytically necessary open-to-close transition and by an exchange of a crucial histidine to a tyrosine. Mainly based on the presented data, a function of HmdII as Hmd isoenzyme, H2 sensor, FeGP-cofactor storage protein and scaffold protein for FeGP-cofactor biosynthesis could be excluded. Inspired by the recently found binding of HmdII to aminoacyl-tRNA synthases and tRNA, we tentatively consider HmdII as a regulatory protein for protein synthesis that senses the intracellular methylene-H4 MPT concentration. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: The iron-guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase contains a prominent iron centre with an acyl-Fe bond and is the only acyl-organometallic iron compound found in nature. Here, we identify the functions of HcgE and HcgF, involved in the biosynthesis of the FeGP cofactor using structure-to-function strategy. Analysis of the HcgE and HcgF crystal structures with and without bound substrates suggest that HcgE catalyses the adenylylation of the carboxy group of guanylylpyridinol (GP) to afford AMP-GP, and subsequently HcgF catalyses the transesterification of AMP-GP to afford a Cys (HcgF)-S-GP thioester. Both enzymatic reactions are confirmed by in vitro assays. The structural data also offer plausible catalytic mechanisms. This strategy of thioester activation corresponds to that used for ubiquitin activation, a key event in the regulation of multiple cellular processes. It further implicates a nucleophilic attack onto the acyl carbon presumably via an electron-rich Fe(0)- or Fe(I)-carbonyl complex in the Fe-acyl formation.
No preview · Article · Apr 2015 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: HcgD, a homolog of the ubiquitous Nif3-like protein family, is found in a gene cluster involved in the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase. The presented crystal structure and biochemical analyses indicated that HcgD has a dinuclear iron-center, which provides a pronounced binding site for anionic ligands. HcgD contains a stronger and a weaker bound iron; the latter being removable by chelating reagents preferentially in the oxidized state. Therefore, we propose HcgD as an iron chaperon in FeGP cofactor biosynthesis, which might also stimulate investigations on the functionally unknown but physiologically important eukaryotic Nif3-like protein family members.
[Show abstract][Hide abstract] ABSTRACT: The reversible redox reaction between coenzyme F420 and H2 to F420H2 is catalyzed by a F420-reducing [NiFe]-hydrogenase (FrhABG) which is an enzyme of the energy metabolism of methanogenic archaea. FrhABG is a group 3 [NiFe]-hydrogenase with a dodecameric quaternary structure of 1.25 MDa as recently revealed by high resolution cryo electron microscopy. We report on the crystal structure of FrhABG from Methanothermobacter marburgensis at 1.7Å resolution and compare it with the structures of group 1 [NiFe]-hydrogenases, the only group structurally characterized yet. FrhA is similar to the large subunit of group 1 [NiFe]-hydrogenases regarding its core structure and the embedded [NiFe]-center but different because of the truncation of ca. 160 residues which results in similar but modified H2- and proton- transport pathways and in suitable interfaces for oligomerization. The small subunit FrhG is composed of a N-terminal domain related to group 1 enzymes and a new C-terminal ferredoxin-like domain carrying the distal and medial [4Fe-4S] clusters. FrhB adopts a novel fold, binds one [4Fe-4S] cluster as well as one FAD in a U-shaped conformation and provides the F420-binding site at the Si-face of the isoalloxazine ring. Similar electrochemical potentials of both catalytic reactions and the electron-transferring [4Fe-4S] clusters, determined to be E°' ≈ -400mV, are in full agreement with the equalized forward and backward rates of the FrhABG reaction. The protein might contribute to balanced redox potentials by the aspartate coordination of the proximal [4Fe-4S] cluster, the new ferredoxin module and a rather negatively charged FAD surrounding.
No preview · Article · May 2014 · Journal of Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Consortia of anaerobic methanotrophic (ANME) archaea and delta-proteobacteria anaerobically oxidize methane coupled to sulfate reduction to sulfide. The metagenome of ANME-1 archaea contains genes homologous to genes otherwise only found in methanogenic archaea, and transcription of some of these genes in ANME-1 cells has been shown. We now have heterologously expressed three of these genes in Escherichia coli, namely those homologous to genes for formylmethanofuran:tetrahydromethanopterin formyltransferase (Ftr), methenyltetrahydromethanopterin cyclohydrolase (Mch), and coenzyme F420-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd), and have characterized the overproduced enzymes with respect to their coenzyme specificity and other catalytic properties. The three enzymes from ANME-1 were found to catalyze the same reactions and with similar specific activities using identical coenzymes as the respective enzymes in methanogenic archaea, the apparent Km for their substrates being in the same concentration range. The results support the proposal that anaerobic oxidation of methane to CO2 in ANME involves the same enzymes and coenzymes as CO2 reduction to methane in methanogenic archaea. Interestingly, the activity of Mch and the stability of Mtd from ANME-1were found to be dependent on the presence of 0.5-1.0 M potassium phosphate, which suggested that ANME-1 archaea contain high concentrations of lyotropic salts, presumably as compatible solutes.
No preview · Article · Apr 2014 · Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: Coenzyme F430 is a nickel hydrocorphinoid, and is the prosthetic group of methyl-coenzyme M reductase that catalyzes the last step of the methanogenic reaction sequence and its reversed reaction for anaerobic methane oxidation by ANME. As such, function-specific compound analysis has the potential to reveal the microbial distribution and activity associated with methane production and consumption in natural environments and, in particular, in deep subsurface sediments where microbiological and geochemical techniques are restricted. Herein, we report the development of a technique for high-sensitivity analysis of F430 in environmental samples, including paddy soils, marine sediments, microbial mats, and an anaerobic groundwater. The lower detection limit of F430 analysis by liquid chromatography / mass spectrometry is 0.1 fmol, which corresponds to 6 × 102 to 1 × 104 cells of methanogens. F430 concentrations in these natural environmental samples range from 6 to 44 nmol g-1 and are consistent with the methanogenic archaeal biomass estimated by microbiological analyses.
Full-text · Article · Mar 2014 · Analytical Chemistry
[Show abstract][Hide abstract] ABSTRACT: One reaction step of the biosynthesis of [Fe]‐hydrogenase‐cofactor is elucidated by S. Shima et al. in their Communication on page 12555 ff. A structural genomics approach, in combination with model reactions and thorough product analysis by X‐ray crystallography of the protein–product complexes, revealed that HcgB is the enzyme that catalyzes guanylylpyridinol formation from a 2,4‐dihydroxypyridine derivative and guanosine triphosphate.
No preview · Article · Nov 2013 · Angewandte Chemie International Edition
[Show abstract][Hide abstract] ABSTRACT: [Fe]-Hydrogenase requires the iron guanylylpyridinol (FeGP) cofactor for activity. The function of HcgB, an enzyme in the biosynthesis of the FeGP cofactor, was predicted by structural genomics and confirmed by model reactions and various analytical methods: HcgB catalyzes the terminal guanylyltransferase reaction for the formation of guanylylpyridinol. GMP=guanosine monophosphate.
No preview · Article · Nov 2013 · Angewandte Chemie International Edition
[Show abstract][Hide abstract] ABSTRACT: Ein Reaktionsschritt der Biosynthese des [Fe]‐Hydrogenase‐Cofaktors wurde von S. Shima et al. in ihrer Zuschrift auf S. 12787 ff. aufgeklärt. Mithilfe struktureller Genomik in Kombination mit Modellreaktionen und gründlicher Produktanalyse durch Röntgenkristallographie von Protein‐Produkt‐Komplexen wurde gefunden, dass HcgB das Enzym ist, das die Guanylylpyridinolbildung aus einem 2,4‐Dihydroxypyridinderivat und Guanosintriphosphat katalysiert.
No preview · Article · Nov 2013 · Angewandte Chemie
[Show abstract][Hide abstract] ABSTRACT: Inhibition mechanism: Isocyanides strongly inhibit [Fe]-hydrogenase. X-ray crystallography and X-ray absorption spectroscopy revealed that the isocyanide binds to the trans position, versus the acyl carbon of the Fe center, and is covalently bound to the pyridinol hydroxy oxygen. These results also indicated that the hydroxy group is essential for H2 activation.
No preview · Article · Sep 2013 · Angewandte Chemie International Edition
[Show abstract][Hide abstract] ABSTRACT: We report the presence of coenzyme factor 430 (F430), a prosthetic group of methyl coenzyme M reductase for archaeal methanogenesis, in the deep sub-seafloor biosphere. At 106.7 m depth in sediment collected off Shimokita Peninsula, northwestern Pacific, its concentration was estimated to be at least 40 fmol g sediment−1 (i.e. 36 pg g−1 wet sediment). This is about three orders of magnitude lower than typical concentrations of archaeal intact polar lipids in similar sub-seafloor sediments. On the basis of the concentration of F430 in methanogens and conversion to biomass composed of typical sub-seafloor microbial cells, we estimated that ca. 2 × 106 cells g−1 could be methanogens in the deeply buried marine sediment.
Full-text · Article · May 2013 · Organic Geochemistry
[Show abstract][Hide abstract] ABSTRACT: Methanogenic archaea use a [NiFe]-hydrogenase, Frh, for oxidation/reduction of F420, an important hydride carrier in the methanogenesis pathway from H2 and CO2. Frh accounts for about 1% of the cytoplasmic protein and forms a huge complex consisting of FrhABG heterotrimers with each a [NiFe] center, four Fe-S clusters and an FAD. Here, we report the structure determined by near-atomic resolution cryo-EM of Frh with and without bound substrate F420. The polypeptide chains of FrhB, for which there was no homolog, was traced de novo from the EM map. The 1.2-MDa complex contains 12 copies of the heterotrimer, which unexpectedly form a spherical protein shell with a hollow core. The cryo-EM map reveals strong electron density of the chains of metal clusters running parallel to the protein shell, and the F420-binding site is located at the end of the chain near the outside of the spherical structure. DOI: http://dx.doi.org/10.7554/eLife.00218.001
[Show abstract][Hide abstract] ABSTRACT: Anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR) at marine gas seeps is performed by archaeal-bacterial consortia that have so far not been cultivated in axenic binary or pure cultures. Knowledge about possible biochemical reactions in AOM consortia is based on metagenomic retrieval of genes related to those in archaeal methanogenesis and bacterial sulfate reduction, and identification of a few catabolic enzymes in protein extracts. Whereas the possible enzyme for methane activation (a variant of methyl-coenzyme M reductase, Mcr) was shown to be harboured by the archaea, enzymes for sulfate activation and reduction have not been localized so far. We adopted a novel approach of fluorescent immunolabelling on semi-thin (0.3-0.5 μm) cryosections to localize two enzymes of the SR pathway, adenylyl : sulfate transferase (Sat; ATP sulfurylase) and dissimilatory sulfite reductase (Dsr) in microbial consortia from Black Sea methane seeps. Both Sat and Dsr were exclusively found in an abundant microbial morphotype (c. 50% of all cells), which was tentatively identified as Desulfosarcina/Desulfococcus-related bacteria. These results show that ANME-2 archaea in the Black Sea AOM consortia did not express bacterial enzymes of the canonical sulfate reduction pathway and thus, in contrast to previous suggestions, most likely cannot perform canonical sulfate reduction. Moreover, our results show that fluorescent immunolabelling on semi-thin cryosections which to our knowledge has been so far only applied on cell tissues, is a powerful tool for intracellular protein detection in natural microbial associations.
No preview · Article · Sep 2012 · Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: Methenyltetrahydromethanopterin (methenyl-H(4)MPT(+)) cyclohydrolase (Mch) catalyzes the interconversion of methenyl-H(4)MPT(+) and formyl-H(4)MPT in the one-carbon energy metabolism of methanogenic, methanotrophic, and sulfate-reducing archaea and of methylotrophic bacteria. To understand the catalytic mechanism of this reaction, we kinetically characterized site-specific variants of Mch from Archaeoglobus fulgidus (aMch) and determined the X-ray structures of the substrate-free aMch(E186Q), the aMch:H(4)MPT complex, and the aMch(E186Q):formyl-H(4)MPT complex. (Formyl-)H(4)MPT is embedded inside a largely preformed, interdomain pocket of the homotrimeric enzyme with the pterin and benzyl rings being oriented nearly perpendicular to each other. The active site is primarily built up by the segment 93:95, Arg183 and Glu186 that either interact with the catalytic water attacking methenyl-H(4)MPT(+) or with the formyl oxygen of formyl-H(4)MPT. The catalytic function of the strictly conserved Arg183 and Glu186 was substantiated by the low enzymatic activities of the E186A, E186D, E186N, E186Q, R183A, R183Q, R183E, R183K, and R183E-E186Q variants. Glu186 most likely acts as a general base. Arg183 decisively influences the pK(a) value of Glu186 and the proposed catalytic water mainly by its positive charge. In addition, Glu186 appears to be also responsible for product specificity by donating a proton to the directly neighbored N(10) tertiary amine of H(4)MPT. Thus, N(10) becomes a better leaving group than N(5) which implies the generation of N(5)-formyl-H(4)MPT. For comparison, methenyltetrahydrofolate (H(4)F) cyclohydrolase produces N(10)-formyl-H(4)F in an analogous reaction. An enzymatic mechanism of Mch is postulated and compared with that of other cyclohydrolases.
[Show abstract][Hide abstract] ABSTRACT: [Fe]-hydrogenase catalyzes the reversible hydride transfer from H(2) to methenyltetrahydromethanoptherin, which is an intermediate in methane formation from H(2) and CO(2) in methanogenic archaea. The enzyme harbors a unique active site iron-guanylylpyridinol (FeGP) cofactor, in which a low-spin Fe(II) is coordinated by a pyridinol-N, an acyl group, two carbon monoxide, and the sulfur of the enzyme's cysteine. Here, we studied the biosynthesis of the FeGP cofactor by following the incorporation of (13)C and (2)H from labeled precursors into the cofactor in growing methanogenic archaea and by subsequent NMR, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) and IR analysis of the isolated cofactor and reference compounds. The pyridinol moiety of the cofactor was found to be synthesized from three C-1 of acetate, two C-2 of acetate, two C-1 of pyruvate, one carbon from the methyl group of l-methionine, and one carbon directly from CO(2). The metabolic origin of the two CO-ligands was CO(2) rather than C-1 or C-2 of acetate or pyruvate excluding that the two CO are derived from dehydroglycine as has previously been shown for the CO-ligands in [FeFe]-hydrogenases. A formation of CO from CO(2) via direct reduction catalyzed by a nickel-dependent CO dehydrogenase or from formate could also be excluded. When the cells were grown in the presence of (13)CO, the two CO-ligands and the acyl group became (13)C-labeled, indicating either that free CO is an intermediate in their synthesis or that free CO can exchange with these iron-bound ligands. Based on these findings, we propose pathways for how the FeGP cofactor might be synthesized.
Full-text · Article · Feb 2012 · Journal of the American Chemical Society
[Show abstract][Hide abstract] ABSTRACT: [Fe]-hydrogenase is one of the three types of hydrogenases. This enzyme is found in many hydrogenotrophic methanogenic archaea and catalyzes the reversible hydride transfer from H(2) to methenyl-H(4)MPT(+) in methanogenesis from H(2) and CO(2). The enzyme harbors a unique iron-guanylyl pyridinol (FeGP) cofactor as a prosthetic group. Here, we describe the purification of [Fe]-hydrogenase from Methanothermobacter marburgensis, the isolation of the FeGP cofactor from the native holoenzyme, and the reconstitution of [Fe]-hydrogenase from the isolated FeGP cofactor and the heterologously produced apoenzyme.
No preview · Article · Dec 2011 · Methods in enzymology