Crystal structure of methyl-coenzyme M reductase: the key enzyme of biological methane formation.

Methyl-coenzyme M reductase (MCR), the enzyme responsible for the microbial formation of methane, is a 300-kilodalton protein organized as a hexamer in an alpha2beta2gamma2 arrangement. The crystal structure of the enzyme from Methanobacterium thermoautotrophicum, determined at 1.45 angstrom resolution for the inactive enzyme state MCRox1-silent, reveals that two molecules of the nickel porphinoid coenzyme F430 are embedded between the subunits alpha, alpha', beta, and gamma and alpha', alpha, beta', and gamma', forming two identical active sites. Each site is accessible for the substrate methyl-coenzyme M through a narrow channel locked after binding of the second substrate coenzyme B. Together with a second structurally characterized enzyme state (MCRsilent) containing the heterodisulfide of coenzymes M and B, a reaction mechanism is proposed that uses a radical intermediate and a nickel organic compound.

Active sites of transition-metal enzymes with a focus on nickel.

Since 1995, crystal structures have been determined for many transition-metal enzymes, in particular those containing the rarely used transition metals vanadium, molybdenum, tungsten, manganese, cobalt and nickel. Accordingly, our understanding of how an enzyme uses the unique properties of a specific transition metal has been substantially increased in the past few years. The different functions of nickel in catalysis are highlighted by describing the active sites of six nickel enzymes - methyl-coenyzme M reductase, urease, hydrogenase, superoxide dismutase, carbon monoxide dehydrogenase and acetyl-coenzyme A synthase.

Formylmethanofuran: tetrahydromethanopterin formyltransferase from Methanopyrus kandleri - new insights into salt-dependence and thermostability.

BACKGROUND: Formylmethanofuran: tetrahydromethanopterin formyltransferase (Ftr) from the methanogenic Archaeon Methanopyrus kandleri (optimum growth temperature 98 degrees C) is a hyperthermophilic enzyme that is absolutely dependent on the presence of lyotropic salts for activity and thermostability. The enzyme is involved in the pathway of carbon dioxide reduction to methane and catalyzes the transfer of formyl from formylmethanofuran to tetrahydromethanopterin. RESULTS: The crystal structure of Ftr, determined to a resolution of 1:73 AE reveals a homotetramer composed essentially of two dimers. Each subunit is subdivided into two tightly associated lobes both consisting of a predominantly antiparallel beta sheet flanked by alpha helices forming an alpha/beta sandwich structure. The approximate location of the active site was detected in a region close to the dimer interface. CONCLUSIONS: The adaptation of Ftr against high lyotropic salt concentrations is structurally reflected by a large number of negatively charged residues and their high local concentration on the surface of the protein. The salt-dependent thermostability of Ftr might be explained on a molecular basis by ionic interactions at the protein surface, involving both protein and inorganic salt ions, and the mainly hydrophobic interactions between the subunits and within the core.

Structure of the photosynthetic reaction centre from Rhodobacter sphaeroides at 2.65 A resolution: cofactors and protein-cofactor interactions.

BACKGROUND: Photosynthetic reaction centres (RCs) catalyze light-driven electron, transport across photosynthetic membranes. The photosynthetic bacterium Rhodobacter, sphaeroides is often used for studies of RCs, and three groups have determined the structure of its reaction centre. There are discrepancies between these structures, however, and to resolve these we have determined the structure to higher resolution than before, using a new crystal form. RESULTS: The new structure provides a more detailed description of the Rb. sphaeroides RC, and allows us to compare it with the structure of the RC from Rhodopseudomonas viridis. We find no evidence to support most of the published differences in cofactor binding between the RCs from Rps. viridis and Rb. sphaeroides. Generally, the mode of cofactor binding is conserved, particularly along the electron transfer pathway. Substantial differences are only found at ring V of one bacteriochlorophyll of the 'special pair' and for the secondary quinone, QB. A water chain with a length of about 23 A including 14 water molecules extends from the QB to the cytoplasmic side of the RC. CONCLUSIONS: The cofactor arrangement and the mode of binding to the protein seem to be very similar among the non-sulphur bacterial photosynthetic RCs. The functional role of the displaced QB molecule, which might be present as quinol, rather than quinone, is not yet clear. The newly discovered water chain to the QB binding site suggests a pathway for the protonation of the secondary quinone QB.

The crystal structure of methenyltetrahydromethanopterin cyclohydrolase from the hyperthermophilic archaeon Methanopyrus kandleri.

BACKGROUND: The reduction of carbon dioxide to methane in methanogenic archaea involves the tetrahydrofolate analogue tetrahydromethanopterin (H(4)MPT) as a C(1) unit carrier. In the third step of this reaction sequence, N(5)-formyl-H(4)MPT is converted to methenyl-H(4)MPT(+) by the enzyme methenyltetrahydromethanopterin cyclohydrolase. The cyclohydrolase from the hyperthermophilic archaeon Methanopyrus kandleri (Mch) is extremely thermostable and adapted to a high intracellular concentration of lyotropic salts. RESULTS: Mch was crystallized and its structure solved at 2.0 A resolution using a combination of the single isomorphous replacement (SIR) and multiple anomalous dispersion (MAD) techniques. The structure of the homotrimeric enzyme reveals a new alpha/beta fold that is composed of two domains forming a large sequence-conserved pocket between them. Two phosphate ions were found in and adjacent to this pocket, respectively; the latter is displaced by the phosphate moiety of the substrate formyl-H(4)MPT according to a hypothetical model of the substrate binding. CONCLUSIONS: Although the exact position of the substrate is not yet known, the residues lining the active site of Mch could be tentatively assigned. Comparison of Mch with the tetrahydrofolate-specific cyclohydrolase/dehydrogenase reveals similarities in domain arrangement and in some active-site residues, whereas the fold appears to be different. The adaptation of Mch to high salt concentrations and high temperatures is reflected by the excess of acidic residues at the trimer surface and by the higher oligomerization state of Mch compared with its mesophtic counterparts.

Crystal structure of cancer chemopreventive Bowman-Birk inhibitor in ternary complex with bovine trypsin at 2.3 A resolution. Structural basis of Janus-faced serine protease inhibitor specificity.

Understanding molecular recognition on a structural basis is an objective with broad academic and applied significance. In the complexes of serine proteases and their proteinaceous inhibitors, recognition is governed mainly by residue P1 in accord with primary serine protease specificity. The bifunctional soybean Bowman-Birk inhibitor (sBBI) should, therefore, interact at LysI16 (subdomain 1) with trypsin and at LeuI43 (subdomain 2) with chymotrypsin. In contrast with this prediction, a 2:1 assembly with trypsin was observed in solution and in the crystal structure of sBBI in complex with trypsin, determined at 2.3 A resolution by molecular replacement. Strikingly, P1LeuI43 of sBBI was fully embedded into the S(1) pocket of trypsin in contrast to primary specificity. The triple-stranded beta-hairpin unique to the BBI-family and the surface loops surrounding the active site of the enzyme formed a protein-protein-interface far extended beyond the primary contact region. Polar residues, hydrophilic bridges and weak hydrophobic contacts were predominant in subdomain 1, interacting specifically with trypsin. However, close hydrophobic contacts across the interface were characteristic of subdomain 2 reacting with both trypsin and chymotrypsin. A Met27Ile replacement shifted the ratio with trypsin to the predicted 1:1 ratio. Thus, the buried salt-bridge responsible for trypsin specificity was stabilised in a polar, and destabilized in a hydrophobic, environment. This may be used for adjusting the specificity of protease inhibitors for applications such as insecticides and cancer chemopreventive agents.

Comparison of three methyl-coenzyme M reductases from phylogenetically distant organisms: unusual amino acid modification, conservation and adaptation.

The nickel enzyme methyl-coenzyme M reductase (MCR) catalyzes the terminal step of methane formation in the energy metabolism of all methanogenic archaea. In this reaction methyl-coenzyme M and coenzyme B are converted to methane and the heterodisulfide of coenzyme M and coenzyme B. The crystal structures of methyl-coenzyme M reductase from Methanosarcina barkeri (growth temperature optimum, 37 degrees C) and Methanopyrus kandleri (growth temperature optimum, 98 degrees C) were determined and compared with the known structure of MCR from Methanobacterium thermoautotrophicum (growth temperature optimum, 65 degrees C). The active sites of MCR from M. barkeri and M. kandleri were almost identical to that of M. thermoautotrophicum and predominantly occupied by coenzyme M and coenzyme B. The electron density at 1.6 A resolution of the M. barkeri enzyme revealed that four of the five modified amino acid residues of MCR from M. thermoautotrophicum, namely a thiopeptide, an S-methylcysteine, a 1-N-methylhistidine and a 5-methylarginine were also present. Analysis of the environment of the unusual amino acid residues near the active site indicates that some of the modifications may be required for the enzyme to be catalytically effective. In M. thermoautotrophicum and M. kandleri high temperature adaptation is coupled with increasing intracellular concentrations of lyotropic salts. This was reflected in a higher fraction of glutamate residues at the protein surface of the thermophilic enzymes adapted to high intracellular salt concentrations.

New crystal form of the photosynthetic reaction centre from Rhodobacter sphaeroides of improved diffraction quality.

Trigonal crystals of photosynthetic reaction centres from the wild-type purple bacterium, Rhodobacter sphaeroides (ATCC 17023), have been grown from potassium phosphate solutions at 18 degrees C. They belong to the space group P3(1/2)21 and have unit cell dimensions of a = b = 141.4 A and c = 187.2 A. The crystals diffract to at least 2.65 A resolution and are suitable for detailed structural studies.

Structure of coenzyme F(420) dependent methylenetetrahydromethanopterin reductase from two methanogenic archaea.

Coenzyme F(420)-dependent methylenetetrahydromethanopterin reductase (Mer) is an enzyme of the Cl metabolism in methanogenic and sulfate reducing archaea. It is composed of identical 35-40 kDa subunits and lacks a prosthetic group. The crystal structure of Mer from Methanopyrus kandleri (kMer) revealed in one crystal form a dimeric and in another a tetrameric oligomerisation state and that from Methanobacterium thermoautotrophicum (tMer) a dimeric state. Each monomer is primarily composed of a TIM-barrel fold enlarged by three insertion regions. Insertion regions 1 and 2 contribute to intersubunit interactions. Insertion regions 2 and 3 together with the C-terminal end of the TIM-barrel core form a cleft where the binding sites of coenzyme F(420) and methylene-tetrahydromethanopterin are postulated. Close to the coenzyme F(420)-binding site lies a rarely observed non-prolyl cis-peptide bond. It is surprising that Mer is structurally most similar to a bacterial FMN-dependent luciferase which contains a non-prolyl cis-peptide bond at the equivalent position. The structure of Mer is also related to that of NADP-dependent FAD-harbouring methylenetetrahydrofolate reductase (MetF). However, Mer and MetF do not show sequence similarities although they bind related substrates and catalyze an analogous reaction.

On the mechanism of biological methane formation: structural evidence for conformational changes in methyl-coenzyme M reductase upon substrate binding.

Methyl-coenzyme M reductase (MCR) catalyzes the final reaction of the energy conserving pathway of methanogenic archaea in which methylcoenzyme M and coenzyme B are converted to methane and the heterodisulfide CoM-S-S-CoB. It operates under strictly anaerobic conditions and contains the nickel porphinoid F430 which is present in the nickel (I) oxidation state in the active enzyme. The known crystal structures of the inactive nickel (II) enzyme in complex with coenzyme M and coenzyme B (MCR-ox1-silent) and in complex with the heterodisulfide CoM-S-S-CoB (MCR-silent) were now refined at 1.16 A and 1.8 A resolution, respectively. The atomic resolution structure of MCR-ox1-silent describes the exact geometry of the cofactor F430, of the active site residues and of the modified amino acid residues. Moreover, the observation of 18 Mg2+ and 9 Na+ ions at the protein surface of the 300 kDa enzyme specifies typical constituents of binding sites for either ion. The MCR-silent and MCR-ox1-silent structures differed in the occupancy of bound water molecules near the active site indicating that a water chain is involved in the replenishment of the active site with water molecules. The structure of the novel enzyme state MCR-red1-silent at 1.8 A resolution revealed an active site only partially occupied by coenzyme M and coenzyme B. Increased flexibility and distinct alternate conformations were observed near the active site and the substrate channel. The electron density of the MCR-red1-silent state aerobically co-crystallized with coenzyme M displayed a fully occupied coenzyme M-binding site with no alternate conformations. Therefore, the structure was very similar to the MCR-ox1-silent state. As a consequence, the binding of coenzyme M induced specific conformational changes that postulate a molecular mechanism by which the enzyme ensures that methylcoenzyme M enters the substrate channel prior to coenzyme B as required by the active-site geometry. The three different enzymatically inactive enzyme states are discussed with respect to their enzymatically active precursors and with respect to the catalytic mechanism.

Crystallization and preliminary X-ray analysis of glucose-fructose oxidoreductase from Zymomonas mobilis.

Glucose-fructose oxidoreductase (E.C. 1.1.99.-) from the ethanol-producing Gram-negative bacterium Zymomonas mobilis is a periplasmic, soluble enzyme that forms a homotetramer of 160 kDa with one NADP(H) cofactor per subunit that is tightly, but noncovalently, bound. The enzyme was crystallized by the hanging drop vapor diffusion method using sodium citrate as precipitant. The obtained crystals belong to the space group P2(1)2(1)2, with unit cell constants of 84.6 A, 94.1 A, and 117.0 A, consistent with two monomers in the asymmetric unit. They diffract to a resolution of about 2 A and are suitable for X-ray structure determination.

Crystallization and X-ray analysis of the reaction center from the thermophilic green bacterium Chloroflexus aurantiacus.

The photochemical reaction center of Chloroflexus (Cf.) aurantiacus, a membrane bound pigment-protein complex, has been crystallized in the presence of monodisperse polyoxyethylene detergents. The crystals possessed a pronounced polymorphism. Three different crystal forms belonging to triclinic, monoclinic and orthorhombic space groups have been characterized by X-ray analysis. The triclinic crystal form, with unit cell dimensions of a = 88 A, b = 115 A and c = 151 A, diffracts up to 3.2 A in two directions and to 4.0 A in the third direction.

Crystallization and preliminary X-ray diffraction studies of methyl-coenzyme M reductase from methanobacterium thermoautotrophicum.

Methyl-coenzyme M reductase isoenzyme I from the methanogenic Archaeon, Methanobacterium thermoautotrophicum (strain Marburg), was crystallized by vapor diffusion methods. Crystal form M obtained with 2-methyl-2,4-pentanediol as the precipitant displayed space group P2(1), with unit cell parameters of a=83.2 A, b=117.4 A, c=125.1 A, and beta= 92.6 degrees, and diffracted at better than 2.8 A resolution. Crystal form P grown from polyethylene glycol 400 belonged to space group P2(1), and had unit cell parameters of a=83.1 A, b=120.2 A, c=123.1 A, and beta=91.7 degrees, diffracting at least to 1.7 A resolution. Both crystal forms have one molecule per asymmetric unit and are suitable for X-ray structure analysis.

The three-dimensional structure of glutathione reductase from Escherichia coli at 3.0 A resolution.

The structure of glutathione reductase from Escherichia coli has been solved at 3 A resolution using multiple isomorphous replacement, solvent flattening, and molecular replacement on the basis of the homologous (53% identical residues) and structurally well-established human enzyme. The structures of both enzyme species agree with each other in a global way; there is no domain rearrangement. In detail, clear structural differences can be observed. The structure analysis of the E. coli enzyme was tackled in order to understand site-directed mutants, the most spectacular of which changed the cofactor specificity of this enzyme from NADP to NAD (Scrutton et al., 1990, Nature 343:38-43).

Structure and function of the photosynthetic reaction center from Rhodobacter sphaeroides.

The three-dimensional structure of the photosynthetic reaction center from Rhodobacter sphaeroides is described. The reaction center is a transmembrane protein that converts light into chemical energy. The protein has three subunits: L, M, and H. The mostly helical L and M subunits provide the scaffolding and the finely tuned environment in which the chromophores carry out electron transfer. The details of the protein-chromophore interactions are from studies of a trigonal crystal form that diffracted to 2.65-A resolution. Functional studies of the multi-subunit complex by site-specific replacement of key amino acid residues are summarized in the context of the molecular structure.

Structure at 2.7 A resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody FV fragment.

The aa3 type cytochrome c oxidase consisting of the core subunits I and II only was isolated from the soil bacterium Paracoccus denitrificans and crystallized as complex with a monoclonal antibody Fv fragment. Crystals could be grown in the presence of a number of different nonionic detergents. However, only undecyl-beta-D-maltoside and cyclohexyl-hexyl-beta-D-maltoside yielded well-ordered crystals suitable for high resolution x-ray crystallographic studies. The crystals belong to space group P212121 and diffract x-rays to at least 2.5 A (1 A = 0.1 nm) resolution using synchrotron radiation. The structure was determined to a resolution of 2.7 A using molecular replacement and refined to a crystallographic R-factor of 20.5% (Rfree = 25.9%). The refined model includes subunits I and II and the 2 chains of the Fv fragment, 2 heme A molecules, 3 copper atoms, and 1 Mg/Mn atom, a new metal (Ca) binding site, 52 tentatively identified water molecules, and 9 detergent molecules. Only four of the water molecules are located in the cytoplasmic half of cytochrome c oxidase. Most of them are near the interface of subunits I and II. Several waters form a hydrogen-bonded cluster, including the heme propionates and the Mg/Mn binding site. The Fv fragment binds to the periplasmic polar domain of subunit II and is critically involved in the formation of the crystal lattice. The crystallization procedure is well reproducible and will allow for the analysis of the structures of mechanistically interesting mutant cytochrome c oxidases.

Three-dimensional structure of transketolase, a thiamine diphosphate dependent enzyme, at 2.5 A resolution.

The crystal structure of Saccharomyces cerevisiae transketolase, a thiamine diphosphate dependent enzyme, has been determined to 2.5 A resolution. The enzyme is a dimer with the active sites located at the interface between the two identical subunits. The cofactor, vitamin B1 derived thiamine diphosphate, is bound at the interface between the two subunits. The enzyme subunit is built up of three domains of the alpha/beta type. The diphosphate moiety of thiamine diphosphate is bound to the enzyme at the carboxyl end of the parallel beta-sheet of the N-terminal domain and interacts with the protein through a Ca2+ ion. The thiazolium ring interacts with residues from both subunits, whereas the pyrimidine ring is buried in a hydrophobic pocket of the enzyme, formed by the loops at the carboxyl end of the beta-sheet in the middle domain in the second subunit. The structure analysis identifies amino acids critical for cofactor binding and provides mechanistic insights into thiamine catalysis.

Hyperthermophilic and salt-dependent formyltransferase from Methanopyrus kandleri.

Methanopyrus kandleri is a hyperthermophilic methanogenic archaeon, which grows on H(2) and CO(2) as its sole energy source. Its growth temperature optimum is 98 degrees C. One of the interesting characteristics of this archaeon is its high intracellular salt content. The organism has been reported to contain the trianionic cDPG (cyclic 2,3-diphosphoglycerate) and K+ at concentrations of 1.1 and 3 M, respectively. Reflecting the high cellular salt concentration, the enzymes in this organism are adapted not only to high temperature but also to high salt concentrations. The formyltransferase from M. kandleri was characterized extensively with respect to thermo- and halophilicity. The crystal structure of the formyltransferase at 1.73 A shows the enzyme to be composed of four identical subunits of molecular mass 32 kDa. The formyltransferase is thermostable and active only at relatively high concentrations of potassium phosphate (1 M) or other salts with strongly hydrated anions (strong salting-out salts). Potassium phosphate and potassium cDPG were found to be equivalent in activating and stabilizing the enzyme. At low concentrations of these salts, the enzyme is inactive and thermolabile. It was shown by equilibrium sedimentation analysis that the enzyme is in a monomer/dimer/tetramer equilibrium, the equilibrium constant being dependent on the concentration of salts: the higher oligomeric species increase with increasing salt concentrations. Evidence was provided that the monomer is both inactive and thermolabile. Experiments using a mutation which is directed to break surface ion pairs between two dimers indicated that dimerization is required for activity and tetramerization leads to thermostability.

Structural, spectroscopic and catalytic activity studies on glutathione reductase reconstituted with FAD analogues.

FAD-modified human glutathione reductases were reconstituted from apoenzyme using the FAD analogues 6-SH-FAD, 6-SCN-FAD, 6-OH-FAD, 6-NH2-FAD and 8-OH-FAD. The catalytic activities of the modified enzymes were substantially lower than for the native enzyme. All five species could be crystallized, but only those containing 6-SH-FAD, 6-OH-FAD and 6-NH2-FAD yielded crystals that could be analyzed. X-ray analyses and structural refinements were performed at 0.27 nm and 0.30 nm resolution resulting in R factors around 13.5%. The crystal structures showed the additional non-hydrogen atoms and small conformational changes of the polypeptide that were obviously induced by the substituents of the FAD analogues. The observed changes together with spectroscopic and activity data permit some conclusions about the chemical nature of the substituents.

Crystallization and preliminary X-ray diffraction studies of formylmethanofuran: tetrahydromethanopterin formyltransferase from Methanopyrus kandleri.

Formylmethanofuran:tetrahydromethanopterin formyltransferase from the hyperthermophilic methanogenic Archaeon Methanopyrus kandleri (growth temperature optimum 98 degrees C) was crystallized by vapor diffusion methods. Crystal form M obtained with 2-methyl-2,4-pentanediol as precipitant displayed the space group P2(1) with unit cell parameters of a = 87.0 A, b = 75.4 A, c = 104.7 A, and beta = 113.9 degrees and diffracted better than 2 A resolution. Crystal form P grown from polyethylene glycol 8000 belonged to the space group I4(1)22 and had unit cell parameters of 157.5 A and 242.1 A. Diffraction data to 1.73 A were recorded. Crystal form S which was crystallized from (NH4)2SO4 in the space group I4(1)22 with unit cell parameters of 151.3 A and 249.5 A diffracted at least to 2.2 A resolution. All crystal forms probably have four molecules per asymmetric unit and are suitable for X-ray structure analysis.