Publications

• Site-directed mutagenesis and X-ray crystallography of the PQQ-containing quinoprotein methanol dehydrogenase and its electron acceptor, cytochrome c(L).

  Afolabi P.R., Mohammed F., Amaratunga K., Majekodunmi O., Dales S.L., Gill R., Thompson D., Cooper J.B., Wood S.P., Goodwin P.M., Anthony C.

  Two proteins specifically involved in methanol oxidation in the methylotrophic bacterium Methylobacterium extorquens have been modified by site-directed mutagenesis. Mutation of the proposed active site base (Asp303) to glutamate in methanol dehydrogenase (MDH) gave an active enzyme (D303E-MDH) wi ... >> More

  Two proteins specifically involved in methanol oxidation in the methylotrophic bacterium Methylobacterium extorquens have been modified by site-directed mutagenesis. Mutation of the proposed active site base (Asp303) to glutamate in methanol dehydrogenase (MDH) gave an active enzyme (D303E-MDH) with a greatly reduced affinity for substrate and with a lower activation energy. Results of kinetic and deuterium isotope studies showed that the essential mechanism in the mutant protein was unchanged, and that the step requiring activation by ammonia remained rate limiting. No spectrally detectable intermediates could be observed during the reaction. The X-ray structure, determined to 3 A resolution, of D303E-MDH showed that the position and coordination geometry of the Ca2+ ion in the active site was altered; the larger Glu303 side chain was coordinated to the Ca2+ ion and also hydrogen bonded to the O5 atom of pyrroloquinoline quinone (PQQ). The properties and structure of the D303E-MDH are consistent with the previous proposal that the reaction in MDH is initiated by proton abstraction involving Asp303, and that the mechanism involves a direct hydride transfer reaction. Mutation of the two adjacent cysteine residues that make up the novel disulfide ring in the active site of MDH led to an inactive enzyme, confirming the essential role of this remarkable ring structure. Mutations of cytochrome c(L), which is the electron acceptor from MDH was used to identify Met109 as the sixth ligand to the heme. << Less

  Biochemistry 40:9799-9809(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The active site of methanol dehydrogenase contains a disulphide bridge between adjacent cysteine residues.

  Blake C.C.F., Ghosh M., Harlos K., Avezoux A., Anthony C.

  Adjacent cysteine residues can only form disulphide bridges in a distorted structure containing a cis-peptide link. Such bridges are extremely uncommon, identified so far in the acetyl choline receptor alone where the structure of the bridge is undetermined. Here we present the first molecular des ... >> More

  Adjacent cysteine residues can only form disulphide bridges in a distorted structure containing a cis-peptide link. Such bridges are extremely uncommon, identified so far in the acetyl choline receptor alone where the structure of the bridge is undetermined. Here we present the first molecular description of a disulphide bridge of this type in the quinoprotein methanol dehydrogenase from Methylobacterium extorquens. We show that this structure occurs in close proximity to the pyrrolo-quinoline quinone prosthetic group and a calcium ion in the active site of the enzyme. This unusual disulphide bridge appears to play a role in the electron transfer reaction mediated by methanol dehydrogenase. << Less

  Nat. Struct. Biol. 1:102-105(1994) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The atomic resolution structure of methanol dehydrogenase from Methylobacterium extorquens.

  Williams P.A., Coates L., Mohammed F., Gill R., Erskine P.T., Coker A., Wood S.P., Anthony C., Cooper J.B.

  The crystal structure of methanol dehydrogenase (MDH) from Methylobacterium extorquens has been refined without stereochemical restraints at a resolution of 1.2 A. The high-resolution data have defined the conformation of the tricyclic pyrroloquinoline quinone (PQQ) cofactor ring as entirely plana ... >> More

  The crystal structure of methanol dehydrogenase (MDH) from Methylobacterium extorquens has been refined without stereochemical restraints at a resolution of 1.2 A. The high-resolution data have defined the conformation of the tricyclic pyrroloquinoline quinone (PQQ) cofactor ring as entirely planar. The detailed definition of the active-site geometry has shown many features that are similar to the quinohaemo-protein alcohol dehydrogenases from Comamonas testosteroni and Pseudomonas putida, both of which possess MDH-like and cytochrome c-like domains. Conserved features between the two types of PQQ-containing enzyme suggest a common pathway for electron transfer between MDH and its physiological electron acceptor cytochrome cL. A pathway for proton transfer from the active site to the bulk solvent is also suggested. << Less

  Acta Crystallogr D Biol Crystallogr 61:75-79(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structure and activity of the prosthetic group of methanol dehydrogenase.

  Duine J.A., Frank J., Verwiel P.E.

  The reconstructive ability of the isolated prosthetic group of methanol dehydrogenase with the apoenzyme of glucose dehydrogenase and the results of electron spin resonance measurements suggest that the prosthetic group has not been modified during the isolation. This result, and the properties of ... >> More

  The reconstructive ability of the isolated prosthetic group of methanol dehydrogenase with the apoenzyme of glucose dehydrogenase and the results of electron spin resonance measurements suggest that the prosthetic group has not been modified during the isolation. This result, and the properties of the directly isolated prosthetic group and derivatives, confirm the suggestion that its structure is 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinoline-4,5-dione. From the activity shown by derivatives of the prosthetic group and of structural analogues in the apoenzyme test it is concluded that the o-quinone structure is essential for activity. Hence the trivial name pyrrolo-quinoline quinone would be appropriate. The testing of the analogues also shows that the pyrrolo ring and the 9-carboxylic acid group are not essential for activity as they can be replaced by a pyridinol ring and a 9-hydroxy group respectively. The determination of the molar absorption coefficient of the prosthetic group (18 400 M-1 cm-1 at 249 nm) enables its quantitative anaysis. Thus it could be established that methanol dehydrogenase contains one prosthetic group per enzyme molecule. The consequences of this result in relation to already known properties of this 'quinoprotein' dehydrogenase are discussed. << Less

  Eur J Biochem 108:187-192(1980) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The structure and mechanism of methanol dehydrogenase.

  Anthony C., Williams P.

  This is a review of recent work on methanol dehydrogenase (MDH), a pyrroloquinoline quinone (PQQ)-containing enzyme catalysing the oxidation of methanol to formaldehyde in methylotrophic bacteria. Although it is the most extensively studied of this class of dehydrogenases, it is only recently that ... >> More

  This is a review of recent work on methanol dehydrogenase (MDH), a pyrroloquinoline quinone (PQQ)-containing enzyme catalysing the oxidation of methanol to formaldehyde in methylotrophic bacteria. Although it is the most extensively studied of this class of dehydrogenases, it is only recently that there has been any consensus about its mechanism. This is partly due to recent structural studies on normal and mutant enzymes and partly due to more definitive work on the mechanism of related alcohol and glucose dehydrogenases. This work has also led to conclusions about the subsequent path of electrons and protons during the reoxidation of the reduced quinol form of the prosthetic group. << Less

  Biochim Biophys Acta 1647:18-23(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The interaction of methanol dehydrogenase and its electron acceptor, cytochrome cL in methylotrophic bacteria.

  Cox J.M., Day D.J., Anthony C.

  The interactions of methanol dehydrogenase (MDH, EC1.1.99.8) with its specific electron acceptor cytochrome cL has been investigated in Methylobacterium extorquens and Methylophilus methylotrophus. The MDHs of these two very different methylotrophs have the same alpha 2 beta 2 structure; the inter ... >> More

  The interactions of methanol dehydrogenase (MDH, EC1.1.99.8) with its specific electron acceptor cytochrome cL has been investigated in Methylobacterium extorquens and Methylophilus methylotrophus. The MDHs of these two very different methylotrophs have the same alpha 2 beta 2 structure; the interaction of these MDHs with their specific electron acceptor, cytochrome cL, has been studied using a novel assay system. Electrostatic reactions are involved in 'docking' of the two proteins. EDTA inhibits the reaction by a process involving neither metal chelation nor the 'docking' process. Chemical modification studies showed that the two proteins interact by a 'docking' process involving interactions of lysyl residues on MDH and carboxyl residues on cytochrome cL. When 'zero length', two stage cross-linking was done (with proteins from both bacteria), the alpha-subunits of MDH cross-linked with cytochrome cL by way of lysyl groups on MDH and carboxyl groups on the cytochrome. Tuna mitochondrial cytochrome c provided a model for cytochrome cH which is the electron acceptor for cytochrome cL in the 'methanol oxidase' electron transport chain. Tuna cytochrome c was shown to form crosslinked products with carboxyl-modified cytochrome cL. MDH and tuna cytochrome c competed for the same domain on cytochrome cL. It was concluded that MDH reacts with cytochrome cL by an electrostatic reaction which involves carboxyl groups on cytochrome cL and amino groups on the alpha-subunit of MDH. The same domain on cytochrome cL is involved in subsequent 'docking' with its electron acceptor. << Less

  Biochim. Biophys. Acta 1119:97-106(1992) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Detailed active site configuration of a new crystal form of methanol dehydrogenase from Methylophilus W3A1 at 1.9 A resolution.

  Xia Z.X., He Y.N., Dai W.W., White S.A., Boyd G.D., Mathews F.S.

  The three-dimensional structure of a new crystal form of methanol dehydrogenase from Methylophilus W3A1 has been obtained in the presence of substrate using data recorded at a synchrotron. The structure of this approximately 140 kDa heterotetramer, refined at 1. 9 A resolution, reveals the detaile ... >> More

  The three-dimensional structure of a new crystal form of methanol dehydrogenase from Methylophilus W3A1 has been obtained in the presence of substrate using data recorded at a synchrotron. The structure of this approximately 140 kDa heterotetramer, refined at 1. 9 A resolution, reveals the detailed configuration of its redox cofactor, pyrroloquinoline quinone (PQQ). C4, one of the oxygen-bearing atoms of this orthoquinone is in a planar configuration while C5, which bears the other quinone oxygen, is tetrahedral, suggesting that the PQQ is in the semiquinone redox state. The substrate binding site has been identified close to PQQ and to the side chain of Asp297, the putative active site base. The proximity of the hydroxyl of methanol to C5 of PQQ compared to the greater separation of the substrate methyl group from C5 supports the addition-elimination reaction mechanism involving a hemiketal intermediate. << Less

  Biochemistry 38:1214-1220(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The microbial oxidation of methanol. The prosthetic group of the alcohol dehydrogenase of Pseudomonas sp. M27: a new oxidoreductase prosthetic group.

  Anthony C., Zatman L.J.

  1. The purified alcohol dehydrogenase of Pseudomonas sp. M27, whose action is independent of nicotinamide nucleotides, has absorption peaks at 280mmu and at 350mmu with little or no absorption at or above 450mmu. 2. It does not fluoresce, but green-fluorescent material, diffusible on dialysis, is ... >> More

  1. The purified alcohol dehydrogenase of Pseudomonas sp. M27, whose action is independent of nicotinamide nucleotides, has absorption peaks at 280mmu and at 350mmu with little or no absorption at or above 450mmu. 2. It does not fluoresce, but green-fluorescent material, diffusible on dialysis, is produced when the enzyme is treated with acid or alkali or when it is boiled. 3. Evidence is presented that the enzyme is not a flavoprotein. 4. Kinetic studies show a correlation between enzyme inactivation by acid, alkali or heat and liberation of the fluorescent material. 5. Some purification of the fluorescent material was achieved, but definite identification was not possible; the major component has a fluorescence maximum at about 460mmu with excitation maxima at about 260mmu and 365mmu. 6. Data are given (including absorption and fluorescence spectra) that support the suggestion that the prosthetic group of the enzyme is a pteridine derivative. 7. Possible mechanisms of action of the enzyme are discussed. << Less

  Biochem J 104:960-969(1967) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The microbial oxidation of methanol. 2. The methanol-oxidizing enzyme of Pseudomonas sp. M 27.

  Anthony C., Zatman L.J.

  Biochem J 92:614-621(1964) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.