Christoph H. Hagemeier

The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features

Clostridium kluyveri is unique among the clostridia; it grows anaerobically on ethanol and acetate as sole energy sources. Fermentation products are butyrate, caproate, and H2. We report here the genome sequence of C. kluyveri, which revealed new insights into the metabolic capabilities of this well studied organism. A membrane-bound energy-converting NADH:ferredoxin oxidoreductase (RnfCDGEAB) and a cytoplasmic butyryl-CoA dehydrogenase complex (Bcd/EtfAB) coupling the reduction of crotonyl-CoA to butyryl-CoA with the reduction of ferredoxin represent a new energy-conserving module in anaerobes. The genes for NAD-dependent ethanol dehydrogenase and NAD(P)-dependent acetaldehyde dehydrogenase are located next to genes for microcompartment proteins, suggesting that the two enzymes, which are isolated together in a macromolecular complex, form a carboxysome-like structure. Unique for a strict anaerobe, C. kluyveri harbors three sets of genes predicted to encode for polyketide/nonribosomal peptide synthetase hybrides and one set for a nonribosomal peptide synthetase. The latter is predicted to catalyze the synthesis of a new siderophore, which is formed under iron-deficient growth conditions.

Insight into the mechanism of biological methanol activation based on the crystal structure of the methanol-cobalamin methyltransferase complex

Some methanogenic and acetogenic microorganisms have the catalytic capability to cleave heterolytically the CO bond of methanol. To obtain insight into the elusive enzymatic mechanism of this challenging chemical reaction we have investigated the methanol-activating MtaBC complex from Methanosarcina barkeri composed of the zinc-containing MtaB and the 5-hydroxybenzimidazolylcobamide-carrying MtaC subunits. Here we report the 2.5-Å crystal structure of this complex organized as a (MtaBC)2 heterotetramer. MtaB folds as a TIM barrel and contains a novel zinc-binding motif. Zinc(II) lies at the bottom of a funnel formed at the C-terminal β-barrel end and ligates to two cysteinyl sulfurs (Cys-220 and Cys-269) and one carboxylate oxygen (Glu-164). MtaC is structurally related to the cobalamin-binding domain of methionine synthase. Its corrinoid cofactor at the top of the Rossmann domain reaches deeply into the funnel of MtaB, defining a region between zinc(II) and the corrinoid cobalt that must be the binding site for methanol. The active site geometry supports a SN2 reaction mechanism, in which the CO bond in methanol is activated by the strong electrophile zinc(II) and cleaved because of an attack of the supernucleophile cob(I)amide. The environment of zinc(II) is characterized by an acidic cluster that increases the charge density on the zinc(II), polarizes methanol, and disfavors deprotonation of the methanol hydroxyl group. Implications of the MtaBC structure for the second step of the reaction, in which the methyl group is transferred to coenzyme M, are discussed.

Re-Citrate Synthase from Clostridium kluyveri Is Phylogenetically Related to Homocitrate Synthase and Isopropylmalate Synthase Rather Than to Si-Citrate Synthase

The synthesis of citrate from acetyl-coenzyme A and oxaloacetate is catalyzed in most organisms by a Si-citrate synthase, which is Si-face stereospecific with respect to C-2 of oxaloacetate. However, in Clostridium kluyveri and some other strictly anaerobic bacteria, the reaction is catalyzed by a Re-citrate synthase, whose primary structure has remained elusive. We report here that Re-citrate synthase from C. kluyveri is the product of a gene predicted to encode isopropylmalate synthase. C. kluyveri is also shown to contain a gene for Si-citrate synthase, which explains why cell extracts of the organism always exhibit some Si-citrate synthase activity.

Structure of coenzyme F420H2 oxidase (FprA), a di‐iron flavoprotein from methanogenic Archaea catalyzing the reduction of O2 to H2O

The di‐iron flavoprotein F420H2 oxidase found in methanogenic Archaea catalyzes the four‐electron reduction of O2 to 2H2O with 2 mol of reduced coenzyme F420(7,8‐dimethyl‐8‐hydroxy‐5‐deazariboflavin). We report here on crystal structures of the homotetrameric F420H2 oxidase from Methanothermobacter marburgensis at resolutions of 2.25 Å, 2.25 Å and 1.7 Å, respectively, from which an active reduced state, an inactive oxidized state and an active oxidized state could be extracted. As found in structurally related A‐type flavoproteins, the active site is formed at the dimer interface, where the di‐iron center of one monomer is juxtaposed to FMN of the other. In the active reduced state [Fe(II)Fe(II)FMNH2], the two irons are surrounded by four histidines, one aspartate, one glutamate and one bridging aspartate. The so‐called switch loop is in a closed conformation, thus preventing F420 binding. In the inactive oxidized state [Fe(III)FMN], the iron nearest to FMN has moved to two remote binding sites, and the switch loop is changed to an open conformation. In the active oxidized state [Fe(III)Fe(III)FMN], both irons are positioned as in the reduced state but the switch loop is found in the open conformation as in the inactive oxidized state. It is proposed that the redox‐dependent conformational change of the switch loop ensures alternate complete four‐electron O2 reduction and redox center re‐reduction. On the basis of the known Si–Si stereospecific hydride transfer, F420H2 was modeled into the solvent‐accessible pocket in front of FMN. The inactive oxidized state might provide the molecular basis for enzyme inactivation by long‐term O2 exposure observed in some members of the FprA family.

Crystal structure of methylenetetrahydromethanopterin reductase (Mer) in complex with coenzyme F420: Architecture of the F420/FMN binding site of enzymes within the nonprolyl cis‐peptide containing bacterial luciferase family

Methylenetetratetrahydromethanopterin reductase (Mer) is involved in CO2 reduction to methane in methanogenic archaea and catalyses the reversible reduction of methylenetetrahydromethanopterin (methylene‐H4MPT) to methyl‐H4MPT with coenzyme F420H2, which is a reduced 5′‐deazaflavin. Mer was recently established as a TIM barrel structure containing a nonprolyl cis‐peptide bond but the binding site of the substrates remained elusive. We report here on the crystal structure of Mer in complex with F420 at 2.6 Å resolution. The isoalloxazine ring is present in a pronounced butterfly conformation, being induced from the Re‐face of F420 by a bulge that contains the non‐prolyl cis‐peptide bond. The bindingmode of F420 is very similar to that in F420‐dependent alcohol dehydrogenase Adf despite the low sequence identity of 21%. Moreover, binding of F420 to the apoenzyme was only associated with minor conformational changes of the polypeptide chain. These findings allowed us to build an improved model of FMN into its binding site in bacterial luciferase, which belongs to the same structural family as Mer and Adf and also contains a nonprolyl cis‐peptide bond in an equivalent position.

Coenzyme F420-dependent Methylenetetrahydromethanopterin Dehydrogenase (Mtd) from Methanopyrus kandleri: A Methanogenic Enzyme with an Unusual Quarternary Structure

The fourth reaction step of CO(2)-reduction to methane in methanogenic archaea is catalyzed by coenzyme F(420)-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd). We have structurally characterized this enzyme in the selenomethionine-labelled form from the hyperthermophilic methanogenic archaeon Methanopyrus kandleri at 1.54A resolution using the single wavelength anomalous dispersion method for phase determination. Mtd was found to be a homohexameric protein complex that is organized as a trimer of dimers. The fold of the individual subunits is composed of two domains: a larger alpha,beta domain and a smaller helix bundle domain with a short C-terminal beta-sheet segment. In the homohexamer the alpha,beta domains are positioned at the outside of the enzyme, whereas, the helix bundle domains assemble towards the inside to form an unusual quarternary structure with a 12-helix bundle around a 3-fold axis. No structural similarities are detectable to other enzymes with F(420) and/or substituted tetrahydropterins as substrates. The substrate binding sites of F(420) and methylenetetrahydromethanopterin are most likely embedded into a crevice between the domains of one subunit, their isoalloxazine and tetrahydropterin rings being placed inside a pocket formed by this crevice and a loop segment of the adjacent monomer of the dimer. Mtd revealed the highest stability at low salt concentrations of all structurally characterized enzymes from M.kandleri. This finding might be due to the compact quaternary structure that buries 36% of the monomer surface and to the large number of ion pairs.

How an Enzyme Binds the C1 Carrier Tetrahydromethanopterin STRUCTURE OF THE TETRAHYDROMETHANOPTERIN-DEPENDENT FORMALDEHYDE-ACTIVATING ENZYME (Fae) FROM METHYLOBACTERIUM EXTORQUENS AM1

Tetrahydromethanopterin (H4 MPT) is a tetrahydrofolate analogue involved as a C1 carrier in the metabolism of various groups of microorganisms. How H4MPT is bound to the respective C1 unit converting enzymes remained elusive. We describe here the structure of the homopentameric formaldehyde-activating enzyme (Fae) from Methylobacterium extorquens AM1 established at 2.0 Å without and at 1.9 Å with methylene-H4MPT bound. Methylene-H4MPT is bound in an “S”-shaped conformation into the cleft formed between two adjacent subunits. Coenzyme binding is accompanied by side chain rearrangements up to 5 Å and leads to a rigidification of the C-terminal arm, a formation of a new hydrophobic cluster, and an inversion of the amide side chain of Gln88. Methylene-H4MPT in Fae shows a characteristic kink between the tetrahydropyrazine and the imidazolidine rings of 70° that is more pronounced than that reported for free methylene-H4MPT in solution (50°). Fae is an essential enzyme for energy metabolism and formaldehyde detoxification of this bacterium and catalyzes the formation of methylene-H4MPT from H4MPT and formaldehyde. The molecular mechanism ofthis reaction involving His22 as acid catalyst is discussed.

MtdC, a Novel Class of Methylene Tetrahydromethanopterin Dehydrogenases

Novel methylene tetrahydromethanopterin (H4MPT) dehydrogenase enzymes, named MtdC, were purified after expressing in Escherichia coli genes from, respectively, Gemmata sp. strain Wa1-1 and environmental DNA originating from unidentified microbial species. The MtdC enzymes were shown to possess high affinities for methylene-H4MPT and NADP but low affinities for methylene tetrahydrofolate or NAD. The substrate range and the kinetic properties revealed by MtdC enzymes distinguish them from the previously characterized bacterial methylene-H4MPT dehydrogenases, MtdA and MtdB. While revealing higher sequence similarity to MtdA enzymes, MtdC enzymes appear to fulfill a function homologous to the function of MtdB, as part of the H4MPT-linked pathway for formaldehyde oxidation/detoxification.

Structure of methylene-tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1

NADP-dependent methylene-H(4)MPT dehydrogenase, MtdA, from Methylobacterium extorquens AM1 catalyzes the dehydrogenation of methylene-tetrahydromethanopterin and methylene-tetrahydrofolate with NADP(+) as cosubstrate. The X-ray structure of MtdA with and without NADP bound was established at 1.9 A resolution. The enzyme is present as a homotrimer. The alpha,beta fold of the monomer is related to that of methylene-H(4)F dehydrogenases, suggesting a common evolutionary origin. The position of the active site is located within a large crevice built up by the two domains of one subunit and one domain of a second subunit. Methylene-H(4)MPT could be modeled into the cleft, and crucial active site residues such as Phe18, Lys256, His260, and Thr102 were identified. The molecular basis of the different substrate specificities and different catalytic demands of MtdA compared to methylene-H(4)F dehydrogenases are discussed.

Re-face stereospecificity of NADP dependent methylenetetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1 as determined by NMR spectroscopy

MtdA catalyzes the dehydrogenation of N 5,N 10‐methylenetetrahydromethanopterin (methylene‐H4MPT) with NADP+ as electron acceptor. In the reaction two prochiral centers are involved, C14a of methylene‐H4MPT and C4 of NADP+, between which a hydride is transferred. The two diastereotopic protons at C14a of methylene‐H4MPT and at C4 of NADPH can be seen separately in 1H‐NMR spectra. This fact was used to determine the stereospecificity of the enzyme. With (14aR)‐[14a‐2H1]‐[14a‐13C]methylene‐H4MPT as the substrate, it was found that the pro‐R hydrogen of methylene‐H4MPT is transferred by MtdA into the pro‐R position of NADPH.