scholarly journals Structure of the quinoprotein glucose dehydrogenase of Escherichia coli modelled on that of methanol dehydrogenase from Methylobacterium extorquens

1995 ◽  
Vol 312 (3) ◽  
pp. 679-685 ◽  
Author(s):  
G E Cozier ◽  
C Anthony
Keyword(s):  
Active Site ◽  
Glucose Dehydrogenase ◽  
Ring Structure ◽  
Enzymic Activity ◽  
Methanol Dehydrogenase ◽  
The Novel ◽  
Methylobacterium Extorquens ◽  
Prosthetic Group ◽  
Site Structure ◽  
Proposed Model

The structure of methanol dehydrogenase (MDH) at 0.194 nm (1.94 A) has been used to provide a model structure for part of a membrane quinoprotein glucose dehydrogenase (GDH). The basic superbarrel structure is retained, along with the tryptophan-docking motifs. The active-site regions are similar, but there are important differences, the most important being that GDH lacks the novel disulphide ring structure formed from adjacent cysteines in MDH; in GDH the equivalent region is occupied by His-262. Because of the overall similarities in the active-site region, the mechanism of action of GDH is likely to be similar to that of MDH. The differences in co-ordination to the cation and bonding to the pyrrolo-quinoline quinone (PQQ) in the active site may explain the relative ease of dissociation of the prosthetic group from the holo-GDH. There are considerable differences in the external loops, particularly those involved in formation of the shallow funnel leading to the active site, the configuration of which influences substrate specificity. The proposed model is consistent in many respects with previous proposals for the active-site structure based on the effects of chemical modification on binding of PQQ and enzymic activity.

scholarly journals The role of the novel disulphide ring in the active site of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens

1995 ◽  
Vol 307 (3) ◽  
pp. 735-741 ◽  
Author(s):  
A Avezoux ◽  
M G Goodwin ◽  
C Anthony
Keyword(s):  
Active Site ◽  
Hydrogen Transfer ◽  
Ring Structure ◽  
Methanol Dehydrogenase ◽  
Active Enzyme ◽  
The Novel ◽  
Methylobacterium Extorquens ◽  
Disulphide Bridge ◽  
Prosthetic Group ◽  
Cytochrome Cl

All cysteines in methanol dehydrogenase (MDH) from Methylobacterium extorquens are involved in intra-subunit disulphide bridge formation. One of these is between adjacent cysteine residues which form a novel ring structure in the active site. It is readily reduced, the reduced enzyme being inactive in electron transfer to cytochrome cL. The inactivation is not a result of major structural change or to modification of the prosthetic group pyrrolo-quinoline quinone (PQQ). The reduced enzyme appears to remain active with the artificial electron acceptor phenazine ethosulphate but this is because the dye re-oxidizes the adjacent thiols back to the original disulphide bridge. No free thiols were detected during the reaction cycle with cytochrome cL. Carboxymethylation of the thiols produced by reduction of the novel disulphide ring led to formation of active enzyme. Reconstitution of inactive Ca(2+)-free MDH with Ca2+ led to active enzyme containing the oxidized bridge and reduced quinol, PQQH2, consistent with the conclusion that no hydrogen transfer occurs between these groups in the active site. It is concluded that the disulphide ring in the active site of MDH does not function as a redox component of the reaction. The disulphide ring has no special function in the process of Ca2+ incorporation into the active site. It is suggested that this novel structure might function in the stabilization or protection of the free radical semiquinone form of the prosthetic group (PQQH.) from solvent at the entrance to the active site.

scholarly journals The structure of the quinoprotein alcohol dehydrogenase of Acetobacter aceti modelled on that of methanol dehydrogenase from Methylobacterium extorquens

1995 ◽  
Vol 308 (2) ◽  
pp. 375-379 ◽  
Author(s):  
G E Cozier ◽  
I G Giles ◽  
C Anthony
Keyword(s):  
Alcohol Dehydrogenase ◽  
Active Site ◽  
Mechanisms Of Action ◽  
Methanol Dehydrogenase ◽  
Model Structure ◽  
Methylobacterium Extorquens ◽  
Acetobacter Aceti ◽  
Active Site Region

The 1.94 A structure of methanol dehydrogenase has been used to provide a model structure for part of a membrane quinohaemoprotein alcohol dehydrogenase. The basic superbarrel structure and the active-site region are retained, indicating essentially similar mechanisms of action, but there are considerable differences in the external loops, particularly those involved in formation of the shallow funnel leading to the active site.

The active site structure of the calcium-containing quinoprotein methanol dehydrogenase

Biochemistry ◽  
1993 ◽  
Vol 32 (48) ◽  
pp. 12955-12958 ◽  
Author(s):  
Scott White ◽  
Geoffrey Boyd ◽  
F. Scott Mathews ◽  
Zongxiang Xia ◽  
Weiwen Dai ◽  
...  
Keyword(s):  
Active Site ◽  
Methanol Dehydrogenase ◽  
Site Structure ◽  
Active Site Structure

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

1992 ◽  
Vol 287 (3) ◽  
pp. 709-715 ◽  
Author(s):  
I W Richardson ◽  
C Anthony
Keyword(s):  
Paracoccus Denitrificans ◽  
Molecular Size ◽  
Methanol Dehydrogenase ◽  
Single Atom ◽  
Methylobacterium Extorquens ◽  
Prosthetic Group ◽  
Low Concentrations ◽  
L Proteins ◽  
Group 2 ◽  
Mutant Forms

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.

scholarly journals Characterization of the membrane quinoprotein glucose dehydrogenase from Escherichia coli and characterization of a site-directed mutant in which histidine-262 has been changed to tyrosine

1999 ◽  
Vol 340 (3) ◽  
pp. 639-647 ◽  
Author(s):  
Gyles E. COZIER ◽  
Raff A. SALLEH ◽  
Christopher ANTHONY
Keyword(s):  
Escherichia Coli ◽  
Electron Transfer ◽  
Active Site ◽  
Marked Decrease ◽  
Glucose Dehydrogenase ◽  
Catalytic Efficiency ◽  
Prosthetic Group ◽  
Competitive Inhibitors ◽  
A Site

The requirements for substrate binding in the quinoprotein glucose dehydrogenase (GDH) in the membranes of Escherichia coli are described, together with the changes in activity in a site-directed mutant in which His262 has been altered to a tyrosine residue (H262Y-GDH). The differences in catalytic efficiency between substrates are mainly related to differences in their affinity for the enzyme. Remarkably, it appears that, if a hexose is able to bind in the active site, then it is also oxidized, whereas some pentoses are able to bind (and act as competitive inhibitors), but are not substrates. The activation energies for the oxidation of hexoses and pentoses are almost identical. In a previously published model of the enzyme, His262 is at the entrance to the active site and appears to be important in holding the prosthetic group pyrroloquinoline quinone (PQQ) in place, and it has been suggested that it might play a role in electron transfer from the reduced PQQ to the ubiquinone in the membrane. The H262Y-GDH has a greatly diminished catalytic efficiency for all substrates, which is mainly due to a marked decrease in their affinities for the enzyme, but the rate of electron transfer to oxygen is unaffected. During the processing of the PQQ into the apoenzyme to give active enzyme, its affinity is markedly dependent on the pH, four groups with pK values between pH 7 and pH 8 being involved. Identical results were obtained with H262Y-GDH, showing that His262 it is not directly involved in this process.

scholarly journals Reversible thermal inactivation of the quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. Ca2+ ions are necessary for re-activation

1989 ◽  
Vol 261 (2) ◽  
pp. 415-421 ◽  
Author(s):  
O Geiger ◽  
H Görisch
Keyword(s):  
Acetic Acid ◽  
Active Site ◽  
Protein Concentration ◽  
Thermal Inactivation ◽  
Glucose Dehydrogenase ◽  
Acinetobacter Calcoaceticus ◽  
Pyrroloquinoline Quinone ◽  
Soluble Form ◽  
Activation Process ◽  
Prosthetic Group

The soluble form of the homogeneous quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus is reversibly inactivated at temperatures above 35 degrees C. An equilibrium is established between active and denatured enzyme, this depending on the protein concentration and the inactivation temperature used. Upon thermal inactivation the enzyme dissociates into the prosthetic group pyrroloquinoline quinone and the apo form of glucose dehydrogenase. After inactivation at 50 degrees C active enzyme is re-formed again at 25 degrees C. Ca2+ ions are necessary for the re-activation process. The velocity of re-activation depends on the protein concentration, the concentration of the prosthetic group pyrroloquinoline quinone and the Ca2+ concentration. The apo form of glucose dehydrogenase can be isolated, and in the presence of pyrroloquinoline quinone and Ca2+ active holoenzyme is formed. Even though native glucose dehydrogenase is not inactivated in the presence of EDTA or trans-1,2-diaminocyclohexane-NNN'NH-tetra-acetic acid, Ca2+ stabilizes the enzyme against thermal inactivation. Two Ca2+ ions are found per subunit of glucose dehydrogenase. The data suggest that pyrroloquinoline quinone is bound at the active site via a Ca2+ bridge. Mn2+ and Cd2+ can replace Ca2+ in the re-activation mixture.

scholarly journals Reconstitution of the quinoprotein methanol dehydrogenase from inactive Ca2+-free enzyme with Ca2+, Sr2+ or Ba2+

1996 ◽  
Vol 319 (3) ◽  
pp. 839-842 ◽  
Author(s):  
Matthew G GOODWIN ◽  
Alain AVEZOUX ◽  
Simon L DALES ◽  
Christopher ANTHONY
Keyword(s):  
Activation Energy ◽  
Methanol Dehydrogenase ◽  
Biological Processes ◽  
The Novel ◽  
Methylobacterium Extorquens ◽  
Disulphide Bridge ◽  
Ph Optimum ◽  
Large Conformational Change ◽  
Process Studies ◽  
Time Courses

The reconstitution of active holoenzyme containing calcium from inactive calcium-free methanol dehydrogenase, isolated from a moxA mutant of Methylobacterium extorquens, has a pH optimum of about pH 10, with a well defined pK for the process at pH 9.3. Two Ca2+ ions were irreversibly incorporated per α2β2 tetramer. Calcium could be replaced in the incorporation process by strontium or barium, the affinities for these ions being similar to that for Ca2+. Arrhenius plots for measurement of the activation energy of reconstitution were biphasic; the lower activation energy was typical of most biological processes, while the higher activation energy was at least three times greater, implying the involvement of a large conformational change during incorporation of the cations. The activation energy for incorporation of Ba2+ was considerably higher than that for incorporation of Ca2+. The novel disulphide bridge that is at the active site of the enzyme was not involved in the incorporation process. Studies of the time courses for incorporation of 45Ca2+, production of active enzyme and changes in absorption spectra failed to show any intermediates in the incorporation process.

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