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Sulfate-reducing bacteria and archaea are important players in the biogeochemical sulfur cycle. ATP sulfurylase, adenosine 5'-phosphosulfate reductase and dissimilatory sulfite reductase are the key enzymes in the energy conserving process of SO4(2-) → H2S reduction. This review summarizes recent advances in our understanding of the activation of sulfate to adenosine 5'-phosphosulfate, the following reductive cleavage to SO3(2-) and AMP, and the final six-electron reduction of SO3(2-) to H2S in the hyperthermophilic archaeon Archaeoglobus fulgidus. Structure based mechanisms will be discussed for these three enzymes which host unique metal centers at their catalytic sites.

Isoprenoid precursor biosynthesis occurs through the mevalonate or the methylerythritol phosphate (MEP) pathway, used i.e., by humans and by many human pathogens, respectively. In the MEP pathway, 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate (MEcPP) is converted to (E)-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) by the iron-sulfur cluster enzyme HMBPP synthase (GcpE). The presented X-ray structure of the GcpE-MEcPP complex from Thermus thermophilus at 1.55Å resolution provides valuable information about the catalytic mechanism and for rational inhibitor design. MEcPP binding inside the TIM-barrel funnel induces a 60° rotation of the [4Fe-4S] cluster containing domain onto the TIM-barrel entrance. The apical iron of the [4Fe-4S] cluster ligates with the C3 oxygen atom of MEcPP., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Isoprenoids are biosynthesized via the mevalonate or the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways the latter being used by most pathogenic bacteria, some parasitic protozoa, plant plastids, but not by animals. We determined the X-ray structure of the homodimeric [4Fe-4S] cluster carrying E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate synthase (GcpE) of Thermus thermophilus which catalyzes the penultimate reaction of the MEP pathway and is therefore an attractive target for drug development. The [4Fe-4S] cluster ligated to three cysteines and one glutamate is encapsulated at the intersubunit interface. The substrate binding site lies in front of an (αβ)(8) barrel. The great [4Fe-4S] cluster-substrate distance implicates large-scale domain rearrangements during the reaction cycle., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Most methanogenic archaea use the rudimentary hydrogenotrophic pathway—from CO2 and H2 to methane—as the terminal step of microbial biomass degradation in anoxic habitats. The barely exergonic process that just conserves sufficient energy for a modest lifestyle involves chemically challenging reactions catalyzed by complex enzyme machineries with unique metal-containing cofactors. The basic strategy of the methanogenic energy metabolism is to covalently bind C1 species to the C1 carriers methanofuran, tetrahydromethanopterin, and coenzyme M at different oxidation states. The four reduction reactions from CO2 to methane involve one molybdopterin-based two-electron reduction, two coenzyme F420–based hydride transfers, and one coenzyme F430–based radical process. For energy conservation, one ion-gradient-forming methyl transfer reaction is sufficient, albeit supported by a sophisticated energy-coupling process termed flavin-based electron bifurcation for driving the endergonic CO2 reduction and fixation. Here, we review the knowledge about the structure-based catalytic mechanism of each enzyme of hydrogenotrophic methanogenesis. [ABSTRACT FROM AUTHOR]

[Fe]‐hydrogenase (Hmd) catalyzes the reversible hydrogenation of methenyltetrahydromethanopterin (methenyl‐H4MPT+) with H2. Hmd contains the iron–guanylylpyridinol (FeGP) cofactor, which is sensitive to light and oxidative stress. A natural protection mechanism is reported for Hmd based on structural and biophysical data. Hmd from Methanothermobacter marburgensis (mHmd) was found in a hexameric state, where an expanded oligomerization loop is detached from the dimer core and intrudes into the active site of a neighboring dimer. An aspartic acid residue from the loop ligates to FeII of the FeGP cofactor and thus blocks the postulated H2‐binding site. In solution, this enzyme is in a hexamer‐to‐dimer equilibrium. Lower enzyme concentrations, and the presence of methenyl‐H4MPT+, shift the equilibrium toward the active dimer side. At higher enzyme concentrations—as present in the cell—the enzyme is predominantly in the inactive hexameric state and is thereby protected against light and oxidative stress. [Fe]‐hydrogenase (Hmd) contains the iron–guanylylpyridinol (FeGP) cofactor, which is sensitive to UV‐A/blue light and H2O2. In hexameric Hmd from Methanothermobacter marburgensis (mHmd), an expanded loop is detached from the dimer core and intrudes into the active site of a neighboring dimer. An aspartic acid (Asp) residue from the loop ligates to the Fe center. In the hexameric form, mHmd is protected against light and oxidative stress. [ABSTRACT FROM AUTHOR]

Mono‐iron hydrogenase ([Fe]‐hydrogenase) reversibly catalyzes the transfer of a hydride ion from H2 to methenyltetrahydromethanopterin (methenyl‐H4MPT+) to form methylene‐H4MPT. Its iron guanylylpyridinol (FeGP) cofactor plays a key role in H2 activation. Evidence is presented for O2 sensitivity of [Fe]‐hydrogenase under turnover conditions in the presence of reducing substrates, methylene‐H4MPT or methenyl‐H4MPT+/H2. Only then, H2O2 is generated, which decomposes the FeGP cofactor; as demonstrated by spectroscopic analyses and the crystal structure of the deactivated enzyme. O2 reduction to H2O2 requires a reductant, which can be a catalytic intermediate transiently formed during the [Fe]‐hydrogenase reaction. The most probable candidate is an iron hydride species; its presence has already been predicted by theoretical studies of the catalytic reaction. The findings support predictions because the same type of reduction reaction is described for ruthenium hydride complexes that hydrogenate polar compounds. [ABSTRACT FROM AUTHOR]

8-demethyl-8-aminoriboflavin-5′-phosphate (AFP) synthase (RosB) catalyzes the key reaction of roseoflavin biosynthesis by forming AFP from riboflavin-5′-phosphate (RP) and glutamate via the intermediates 8-demethyl-8-formylriboflavin-5′-phosphate (OHC-RP) and 8-demethyl-8-carboxylriboflavin-5′-phosphate (HO2C-RP). To understand this reaction in which a methyl substituent of an aromatic ring is replaced by an amine we structurally characterized RosB in complex with OHC-RP (2.0 Å) and AFP (1.7 Å). RosB is composed of four flavodoxin-like subunits which have been upgraded with specific extensions and a unique C-terminal arm. It appears that RosB has evolved from an electron- or hydride-transferring flavoprotein to a sophisticated multi-step enzyme which uses RP as a substrate (and not as a cofactor). Structure-based active site analysis was complemented by mutational and isotope-based mass-spectrometric data to propose an enzymatic mechanism on an atomic basis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Hydrocarbons are abundant in anoxic environments and pose biochemical challenges to their anaerobic degradation by microorganisms. Within the framework of the Priority Program 1319, investigations funded by the Deutsche Forschungsgemeinschaft on the anaerobic microbial degradation of hydrocarbons ranged from isolation and enrichment of hitherto unknown hydrocarbon-degrading anaerobic microorganisms, discovery of novel reactions, detailed studies of enzyme mechanisms and structures to process-oriented in situ studies. Selected highlights from this program are collected in this synopsis, with more detailed information provided by theme-focused reviews of the special topic issue on 'Anaerobic biodegradation of hydrocarbons' [this issue, pp. 1-244]. The interdisciplinary character of the program, involving microbiologists, biochemists, organic chemists and environmental scientists, is best exemplified by the studies on alkyl-/arylalkylsuccinate synthases. Here, research topics ranged from in-depth mechanistic studies of archetypical toluene-activating benzylsuccinate synthase, substrate-specific phylogenetic clustering of alkyl-/arylalkylsuccinate synthases (toluene plus xylenes, p -cymene, p -cresol, 2-methylnaphthalene, n -alkanes), stereochemical and co-metabolic insights into n -alkane-activating (methylalkyl)succinate synthases to the discovery of bacterial groups previously unknown to possess alkyl-/arylalkylsuccinate synthases by means of functional gene markers and in situ field studies enabled by state-of-the-art stable isotope probing and fractionation approaches. Other topics are Mo-cofactor-dependent dehydrogenases performing O 2 -independent hydroxylation of hydrocarbons and alkyl side chains (ethylbenzene, p -cymene, cholesterol, n -hexadecane), degradation of p -alkylated benzoates and toluenes, glycyl radical-bearing 4-hydroxyphenylacetate decarboxylase, novel types of carboxylation reactions (for acetophenone, acetone, and potentially also benzene and naphthalene), W-cofactor-containing enzymes for reductive dearomatization of benzoyl-CoA (class II benzoyl-CoA reductase) in obligate anaerobes and addition of water to acetylene, fermentative formation of cyclohexanecarboxylate from benzoate, and methanogenic degradation of hydrocarbons. [ABSTRACT FROM AUTHOR]

[Fe] ‐ Hydrogenase benötigt für seine Aktivität einen Eisen ‐ Guanylylpyridinol(FeGP) ‐ Cofaktor. Die Funktion von HcgB, einem Enzym in der Biosynthese des Cofaktors FeGP, wurde durch strukturelle Genomik prognostiziert und durch Modellreaktionen und verschiedene analytische Methoden bestätigt: HcgB katalysiert die terminale Guanylyltransferase ‐ Reaktion zur Bildung von Guanylylpyridinol. GMP=Guanosinmonophosphat. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Investigates the crystallization of 4-hydroxybenzoyl-CoA reductase and the structure of its electron donor ferredoxin. Structure of the ferrodoxin solved at 2.9 A resolution by the molecular-replacement method using the highly related structure of the ferredoxin from Allochromatium vinosum as a model.

Formyltransferase from Methanopyrus kandleri is composed of only one type of subunits of molecular mass 32 kDa. The enzyme requires the presence of lyotropic salts for activity and thermostability. We report here that the enzyme is in a monomer/dimer/tetramer association equilibrium, the association constant being affected by lyotropic salts. At 0.01 M K2HPO4/KH2PO4, pH 7.2, the enzyme (0.4 mg/ml) was mainly present in a monomeric form. Upon increase of the phosphate concentration, the concentration of the dimer increased up to a phosphate concentration of 0.6 M, then decrease at the expense of tetramer formation up to a phosphate concentration of 1.0 M. The specific activity at 4 +C increased from < 0.1 U/mg at 0.01 M, over 1.5 U/mg at 0.6 M to 3.6 U/mg at 1.0 M. Similar results were obtained with ammonium sulfate as lyotropic salt. The findings indicate that both oligomerization and activity increase with increasing salt concentrations, suggesting that there is a causal connection. To determine this, we exploited the observation that oligomer formation was not induced by the weak lyotropic salt NaCl up to a concentration of 1.5 M and that the dissociation of the dimer into the monomer at 4 +C proceeded very slowly (50 % in approximately 6 h). This allowed us to study the effect of NaCl on the activity of the oligomers at NaCl concentrations not sufficient to induce oligomerization. At 4 +C, the activity of the oligomers increased from 0.3 U/mg at 0.25 M NaCl to 3.4 U/mg at 1.0 M NaCl. At these NaCl concentrations, the monomers were inactive. The findings indicate that oligomerization is a prerequisite for enzyme activity in the presence of NaCl. The salt-dependent induction of oligomerization was parallelled by an increase in thermostability; strong lyotropic salts conferred thermostability at much lower concentrations than the weak lyotropic NaCl. [ABSTRACT FROM AUTHOR]

Coenzyme F[SUB420]-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd ) is an enzyme involved in methanogenic energy metabolism which reversibly catalyzes the reduction of methenyl-tetrahydromethanopterin (methenyl-H[SUB4]MPT[SUP+] to methylenetera-hydromethanopterin (methylene-H[SUB4]MPT). The enzyme from the hyperthermophilic methanoarchacon Methanopyrus kandleri could be crystallized: the non-labelled enzyme had unit-cell parameters a = 119.1, b = 151.0, c = 219.4Å and also belonged to space group C222[SUB1], indicating a surprising bisection of the c axis. The crystals grown from the non-labelled and labeled enzyme contained six and three monomers in the asymmetric unit and diffracted to about 1.9 and 1.5Å, respectively. The crystal packing of the two crystal forms seems to be similar. In particular, the crystals of the selenomethionine-labelled enzyme are highly suitable for X-ray structure determination. [ABSTRACT FROM AUTHOR]

In methanogenic archaea, the carbon dioxide (CO2) fixation and methane-forming steps are linked through the heterodisulfide reductase (HdrABC)–[NiFe]-hydrogenase (MvhAGD) complex that uses flavin-based electron bifurcation to reduce ferredoxin and the heterodisulfide of coenzymes M and B. Here, we present the structure of the native heterododecameric HdrABC-MvhAGD complex at 2.15-angstrom resolution. HdrB contains two noncubane [4Fe-4S] clusters composed of fused [3Fe-4S]-[2Fe-2S] units sharing 1 iron (Fe) and 1 sulfur (S), which were coordinated at the CCG motifs. Soaking experiments showed that the heterodisulfide is clamped between the two noncubane [4Fe-4S] clusters and homolytically cleaved, forming coenzyme M and B bound to each iron. Coenzymes are consecutively released upon one-by-one electron transfer. The HdrABC-MvhAGD atomic model serves as a structural template for numerous HdrABC homologs involved in diverse microbial metabolic pathways. [ABSTRACT FROM AUTHOR]

Formylmethanofuran:tetrahydromethanopterin formyltransferase is an essential enzyme in the one-carbon metabolism of methanogenic and sulfate-reducing archaea and of methylotrophic bacteria. The enzyme, which is devoid of a prosthetic group, catalyzes the reversible formyl transfer between the two substrates coenzyme methanofuran and coenzyme tetrahydromethanopterin (H4MPT) in a ternary complex catalytic mechanism. The structure of the formyltransferase without its coenzymes has been determined earlier. We report here the structure of the enzyme in complex with both coenzymes at a resolution of 2.0Å. Methanofuran, characterized for the first time in an enzyme structure, is embedded in an elongated cleft at the homodimer interface and fixed by multiple hydrophobic interactions. In contrast, tetrahydromethanopterin is only weakly bound in a shallow and wide cleft that provides two binding sites. It is assumed that the binding of the bulky coenzymes induces conformational changes of the polypeptide in the range of 3Å that close the H4MPT binding cleft and position the reactive groups of both substrates optimally for the reaction. The key residue for substrate binding and catalysis is the strictly conserved Glu245. Glu245, embedded in a hydrophobic region and completely buried upon tetrahydromethanopterin binding, is presumably protonated prior to the reaction and is thus able to stabilize the tetrahedral oxyanion intermediate generated by the nucleophilic attack of the N5 atom of tetrahydromethanopterin onto the formyl carbon atom of formylmethanofuran. [Copyright &y& Elsevier]

The coenzyme A (CoA)-dependent aerobic benzoate metabolic pathway uses an unprecedented chemical strategy to overcome the high aromatic resonance energy by forming the non-aromatic 2,3-epoxybenzoyl-CoA. The crucial dearomatizing reaction is catalyzed by three enzymes, BoxABC, where BoxA is an NADPH-dependent reductase, BoxB is a benzoyl-CoA 2,3-epoxidase, and BoxC is an epoxide ring hydrolase. We characterized the key enzyme BoxB from Azoarcus evansii by structural and Mössbauer spectroscopic methods as a new member of class I diiron enzymes. Several family members were structurally studied with respect to the diiron center architecture, but no structure of an intact diiron enzyme with its natural substrate has been reported. X-ray structures between 1.9 and 2.5 Å resolution were determined for BoxB in the diferric state and with bound substrate benzoyl-CoA in the reduced state. The substrate-bound reduced state is distinguished from the diferric state by increased iron-ligand distances and the absence of directly bridging groups between them. The position of benzoyl-CoA inside a 20 Å long channel and the position of the phenyl ring relative to the diiron center are accurately defined. The C2 and C3 atoms of the phenyl ring are closer to one of the irons. Therefore, one oxygen of activated O2 must be ligated predominantly to this proximate iron to be in a geometrically suitable position to attack the phenyl ring. Consistent with the observed iron/phenyl geometry, BoxB stereoselectively should form the 2S,3R-epoxide. We postulate a reaction cycle that allows a charge delocalization because of the phenyl ring and the electron-withdrawing CoA thioester. [ABSTRACT FROM AUTHOR]

The iron-sulfur flavoenzyme adenosine-5′-phosphosulfate (APS) reductase catalyzes a key reaction of the global sulfur cycle by reversibly transforming APS to sulfite and AMP. The structures of the dissimilatory enzyme from Archaeoglobus fulgidus in the reduced state (FADred) and in the sulfite adduct state (FAD-sulfite-AMP) have been recently elucidated at 1.6 and 2.5 Å resolution, respectively. Here we present new structural features of the enzyme trapped in four different catalytically relevant states that provide us with a detailed picture of its reaction cycle. In the oxidized state (FADox), the isoalloxazine moiety of the FAD cofactor exhibits a similarly bent conformation as observed in the structure of the reduced enzyme. In the APS-bound state (FADox—APS), the substrate APS is embedded into a 17 Å long substrate channel in such a way that the isoalloxazine ring is pushed toward the channel bottom, thereby producing a compressed enzyme—substrate complex. A clamp formed by residues ArgA317 and LeuA278 to fix the adenine ring and the curved APS conformation appear to be key factors to hold APS in a strained conformation. This energy-rich state is relaxed during the attack of APS on the reduced FAD. A relaxed FAD—sulfite adduct is observed in the structure of the FAD—sulfite state. Finally, a FAD—sulfite—AMP1 state with AMP within van der Waals distance of the sulfite adduct could be characterized. This structure documents how adjacent negative charges are stabilized by the protein matrix which is crucial for forming APS from AMP and sulfite in the reverse reaction. [ABSTRACT FROM AUTHOR]

Tetrahydromethanopterin (H4MPT) 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 AMI 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 Gin88. 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 of this reaction involving His22 as acid catalyst is discussed. [ABSTRACT FROM AUTHOR]

The fourth reaction step of CO2-reduction to methane in methanogenic archaea is catalyzed by coenzyme F420-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd). We have structurally characterized this enzyme in the selenomethionine-labelled form from the hyperthermophilic methanogenic archaeon Methanopyrus kandleri at 1.54 A˚ 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 α,β domain and a smaller helix bundle domain with a short C-terminal β-sheet segment. In the homohexamer the α,β 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 F420 and/or substituted tetrahydropterins as substrates. The substrate binding sites of F420 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. [Copyright &y& Elsevier]

Methane is an end product of anaerobic degradation of organic compounds in fresh water environments such as lake sediments and the intestinal tract of animals. Methanogenic archaea produce methane from carbon dioxide and molecular hydrogen, acetate and C1 compounds such as methanol in an energy gaining process. The methanogenic pathway utilizing carbon dioxide and molecular hydrogen involves ten methanogen specific enzymes, which catalyze unique reactions using novel coenzymes. These enzymes have been purified and biochemically characterized. The genes encoding the enzymes have been cloned and sequenced. Recently, crystal structures of five methanogenic enzymes: formylmethanofuran : tetrahydromethanopterin formyltransferase, methenyltetrahydromethanopterin cyclohydrolase, methylenetetrahydromethanopterin reductase, F420H2: NADP oxidoreductase and methyl-coenzyme M reductase were reported. In this review, we describe the pathway utilizing carbon dioxide and molecular hydrogen and the catalytic mechanisms of the enzymes based on their crystal structures. [Copyright &y& Elsevier]