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C Anthony

Publications and source records attributed to C Anthony.

At least 55 records · Page 3Linked to original sources
PubMed1994

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

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.

Alcohol Oxidoreductases↗
PubMed1992

Crystallization and preliminary crystallographic investigation of methanol dehydrogenase from Methylobacterium extorquens AM1.

Single crystals of methanol dehydrogenase (MDH) from Methylobacterium extorquens AM1 have been grown by the vapour diffusion method. These crystals diffract to beyond 2 A resolution and are suitable for X-ray crystallography. They belong to the orthorhombic space group P2(1)2(1)2(1) and have the following unit cell parameters: a = 66.79 A, b = 108.9 A, c = 188.9 A. One asymmetric unit contains an alpha 2 beta 2 tetramer of MDH and the location of the non-crystallographic 2-fold symmetry axis of this tetramer is defined by the paired positions of the binding sites of heavy atoms in four MDH-derivatives.

Alcohol Oxidoreductases↗
PubMed1992

Characterization of mutant forms of the quinoprotein methanol dehydrogenase lacking an essential calcium ion.

Methanol dehydrogenase (MDH) from Methylobacterium extorquens, Methylophilus methylotrophus, Paracoccus denitrificans and Hyphomicrobium X all contained a single atom of Ca2+ per alpha 2 beta 2 tetramer. The role of Ca2+ was investigated using the MDH from Methylobacterium extorquens. This was shown to be similar to the MDH from Hyphomicrobium X in having 2 mol of prosthetic group (pyrroloquinoline quinine; PQQ) per mol of tetramer, the PQQ being predominantly in the semiquinone form. MDH isolated from the methanol oxidation mutants MoxA-, K- and L- contained no Ca2+. They were identical with the enzyme isolated from wild-type bacteria with respect to molecular size, subunit configuration, pI, N-terminal amino acid sequence and stability under denaturing conditions (low pH, high urea and high guanidinium chloride) and in the nature and content of the prosthetic group (2 mol of PQQ per mol of MDH). They differed in their lack of Ca2+, the oxidation state of the extracted PQQ (fully oxidized), absence of the semiquinone form of PQQ in the enzyme, reactivity with the suicide inhibitor cyclopropanol and absorption spectrum, which indicated that PQQ is bound differently from that in normal MDH. Incubation of MDH from the mutants in calcium salts led to irreversible time-dependent reconstitution of full activity concomitant with restoration of a spectrum corresponding to that of fully reduced normal MDH. It is concluded that Ca2+ in MDH is directly or indirectly involved in binding PQQ in the active site. The MoxA, K and L proteins may be involved in maintaining a high Ca2+ concentration in the periplasm. It is more likely, however, that they fill a 'chaperone' function, stabilizing a configuration of MDH which permits incorporation of low concentrations of Ca2+ into the protein.

Alcohol Oxidoreductases↗
PubMed1992

The mechanism of inhibition by EDTA and EGTA of methanol oxidation by methylotrophic bacteria.

Ethyleneglycol (aminoethylether) tetra-acetic acid (EGTA) was shown to be a potent competitive inhibitor of electron transfer between methanol dehydrogenase (MDH) and its electron acceptor cytochrome cL. Addition of Ca2+ ions relieved the inhibition by removal of the inhibitory EGTA. Removal of EGTA by gel filtration completely relieved the inhibition. EGTA did not remove the tightly bound Ca2+ present in the MDH. Indo-1, a fluorescent analogue of EGTA, bound tightly to MDH in a 1:1 ratio but not to cytochrome cL; binding was prevented by EGTA. It was concluded that EGTA inhibits methanol oxidation by binding to lysyl or arginyl residues on MDH thus preventing docking with cytochrome cL.

Alcohol Oxidoreductases↗
PubMed1992

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

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.

Alcohol Oxidoreductases↗
PubMed1991

The interaction of methanol dehydrogenase and cytochrome cL in the acidophilic methylotroph Acetobacter methanolicus.

The quinoprotein methanol dehydrogenase (MDH) of Acetobacter methanolicus has an alpha 2 beta 2 structure. By contrast with other MDHs, the beta-subunit (approx. 8.5 kDa) does not contain the five lysine residues previously proposed to be involved in ionic interactions with the electron acceptor cytochrome cL. That electrostatic interactions are involved was confirmed by the demonstration that methanol:cytochrome cL oxidoreductase activity was inhibited by high ionic strength (I), the strength of interaction being inversely related to the square root of I. Specific modifiers of arginine residues on MDH inhibited this reaction but not the dye-linked MDH activity. Modification of lysine residues on MDH that altered its charge had no effect on the dye-linked activity but inhibited reaction with cytochrome cL. When the charge was retained on modification of lysine residues, little effect on either activity was observed. Cross-linking experiments confirmed that lysine residues on the alpha-subunit, but not the beta-subunit, are involved in the 'docking' process between the proteins.

Acetobacter↗
PubMed1991

The periplasmic modifier protein for methanol dehydrogenase in the methylotrophs Methylophilus methylotrophus and Paracoccus denitrificans.

A modifier protein (M-protein), which increases the affinity of methanol dehydrogenase (MDH) for alcohols but decreases its affinity for formaldehyde, has been partially purified from Methylophilus methylotrophus and Paracoccus denitrificans. Analysis was complicated by non-protein factors in bacterial extracts that are able to mimic M-protein in one of its functions-that of increasing the activity of MDH with butane-1,3-diol in the dye-linked assay system. The 67 kDa polypeptide, previously identified as a subunit of the M-protein, is an unrelated cytoplasmic protein. The M-protein is exclusively periplasmic and is a multimeric protein with subunits of 45 kDa. The M-protein is active in the 'physiological' assay system with the specific cytochrome c electron acceptor for MDH, lowering its affinity for formaldehyde. It has its maximum effect when the ratio of M-protein:MDH is 1:5 but its concentration in the periplasm is much lower than 20% of that of MDH.

Alcohol Oxidoreductases↗
PubMed1990

Nucleotide sequence of the Methylobacterium extorquens AM1 moxF and moxJ genes involved in methanol oxidation.

The nucleotide sequence has been determined for two genes involved in methanol oxidation in the facultative methylotroph, Methylobacterium extorquens AM1. The two genes are moxF, encoding the 66-kDa subunit of the methanol dehydrogenase and moxJ, located immediately downstream from moxF, which encodes a 30-kDa protein with unknown function. This information completes the sequence of the 5.86-kb XhoI-SalI fragment containing the moxFJGI region in M. extorquens AM1, and the structure of this gene cluster is presented. Evidence is presented that moxJ is also present in Paracoccus denitrificans. The aa sequence of MoxJ has provided little information concerning its function, but it does appear to contain a signal sequence suggesting a periplasmic location.

Alcohol Oxidoreductases↗
PubMed1990

The purification, characterization and role of the d-type cytochrome oxidase of Klebsiella pneumoniae during nitrogen fixation.

Klebsiella pneumoniae synthesized only b-type and d-type cytochromes under the wide range of growth conditions tested, and reaction with CO revealed two potential oxidases. The o-type oxidase was produced only in the presence of O2 and appeared to be repressed by glucose. The d-type oxidase was, by contrast, produced only in the absence of measurable O2 (less than 1 microM), and was the only oxidase expressed in nitrogen-fixing conditions. It was extracted from the membrane, purified and shown to be similar to that from E. coli in being a heterodimer (subunits of Mr 52,000 and 35,000), in containing two distinguishable b haems and haem d (one or two molecules per molecule of oxidase), and in being able to react with O2 to give a stable oxygenated intermediate. The purified d-type cytochrome oxidase had a very high affinity for O2 (Km 20 nM; measured by the spectral properties of leghaemoglobin). It is proposed that this provides a role for this oxidase in lowering the O2 concentration to allow nitrogenase synthesis and function, and to provide a terminal oxidase to permit electron-transport-coupled ATP synthesis which supports the increase in efficiency of nitrogen fixation observed under microaerobic conditions.

Amino Acids↗
PubMed1990

Characterization of a novel soluble c-type cytochrome in a moxD mutant of Methylobacterium extorquens AM1.

Methylobacterium extorquens AM1 contains a novel c-type cytochrome, called cytochrome c-553, previously thought to be a precursor of the electron acceptor (cytochrome cL) for methanol dehydrogenase. Its amino acid composition and serological characteristics show that it has no structural relationship to cytochrome cL. It usually comprises less than 5% of the total c-type cytochromes. In a moxD mutant, which contains neither methanol dehydrogenase nor cytochrome cL, it comprises 30% of the soluble cytochrome and it has been purified and characterized from that mutant. Cytochrome c-553 is large (Mr 23,000), acidic and monohaem, with a redox potential of 194 mV. It reacts rapidly and completely with CO but is not autoxidizable. It is not autoreducible, and it is not an electron acceptor from methanol dehydrogenase or methylamine dehydrogenase, nor an important electron donor to the oxidase. It is able to accept electrons from cytochrome cL and to donate electrons to cytochrome cH. It is present in the soluble fraction (presumably periplasmic) and membrane fraction of wild-type bacteria during growth on a wide range of growth substrates, but its function in these bacteria or in the moxD mutant has not been determined.

Amino Acids↗
PubMed1990

Roles for enteric d-type cytochrome oxidase in N2 fixation and microaerobiosis.

Escherichia coli strains that lacked the d-type cytochrome oxidase, the terminal oxidase with a high affinity for O2, grew anaerobically as well as the wild type did and were not impaired in the ability to evolve H2 from either glucose or formate. The anaerobic synthesis and activity of nitrogenase in transconjugants of these strains carrying Klebsiella pneumoniae nif genes were also normal. However, the behavior towards O2 of anaerobically grown bacteria lacking the d-type oxidase differed from that of the wild type in the following ways: the potential O2 uptake was lower, H2 evolution and nitrogenase activity supported by fermentation were more strongly inhibited by O2, and microaerobic O2-dependent nitrogenase activity in the absence of a fermentable carbon source did not occur. These results show that the d-type oxidase serves two functions in enteric bacteria--to conserve energy under microaerobic conditions and to protect anaerobic processes from inhibition by O2.

Aerobiosis↗
PubMed1989

The 'methylamine oxidase' system of an obligate methylotroph.

The terminal respiratory oxidase was solubilized from membranes of organism 4025, an obligate methylotroph. The partially purified oxidase is probably a cytochrome co. It does not oxidize amicyanin, but it oxidizes 'azurin' and cytochromes cH and cL. By using a complete 'methylamine oxidase' system reconstituted from pure methylamine dehydrogenase, purified oxidase and soluble blue copper proteins and cytochromes, it was confirmed that amicyanin is essential for methylamine oxidation; it could not be replaced by 'azurin' or cytochrome cH or cL. It was shown that the usual mediator between amicyanin and the oxidase is cytochrome cH, with 'azurin' able to replace it during growth at the high copper concentrations required for optimum growth of this unusual methylotroph.

Azurin↗