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 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 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.

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.

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.

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

1994 ◽  
Vol 1 (2) ◽  
pp. 102-105 ◽  
Author(s):  
C.C.F. Blake ◽  
M. Ghosh ◽  
K. Harlos ◽  
A. Avezoux ◽  
C. Anthony
Keyword(s):  
Active Site ◽  
Methanol Dehydrogenase ◽  
Cysteine Residues ◽  
Disulphide Bridge

THROMBIN KINETIC PARAMETERS WITH TRIPEPTIDE p-NITROANALIDES UNDER PHYSIOLOGICALLY RELEVANT CONDITIONS

1987 ◽  
Author(s):  
J W Fenton ◽  
J I Witting ◽  
T M Miller
Keyword(s):  
Active Site ◽  
Active Enzyme ◽  
Chromogenic Substrates ◽  
Human Fibrinogen ◽  
Initial Substrate ◽  
Spontaneous Hydrolysis ◽  
Newton Iterations ◽  
Species Variability ◽  
Do So ◽  
Initial Substrate Concentration

The Michaelis-Menten (Km ), catalytic (kcat ), and specificity (kcat/Km ) constants were determined for humSft α- and β-thrombins with the chromogenic substrate S-2238 (H-D-Phe-Pip-Arg-pNA), Chromozym-TH (Tos-Gly-Pro-Arg-pNA), and Spectrozyme-TH (H-D-HHT-Ala-Arg-pNA) in 0.15 M NaCl buffered with 10 mM HEPES ancTlO mM Tris-HCT at pH 7.4, 37°C. Spontaneous hydrolysis was insignificant for these substrates. Both S-2238 and Spectrozyme-TH exhibited limiting solubilities at ∼35 μM, while Chromozym-TH did not do so up to 50 μM. From initial estimates, Km's and k t's were refined by computer Gause-Newton iterations or nonlinearneast-square fits for the concentration of p-nitroanalide formed per second versus the initial substrate concentration. No major differences were found between a-thrombin (99% α, 91% esterolytically active enzyme, and > 3,500 kilo-U.S. clotting units/g with fibrinogen) and Yγ-thrombin (98% γ. 89% active enzyme, and < 10 kilo-units/g). For α- versus γ-thrombin and the three substrates, respectively, the Km 's were 6.75 ± 0.13 vs 7.62 ± 0.30, 18.4 ± 0.4 vs 23.0 ± 0.3, and 2.53 ± 0.02 vs 3.85 ± 0.51 μM; the kcat> s were 125 ± 1 vs 134 ± 2, 181 ± 2 vs 130 ± 1, and 35.5 ± 0.2 vs 52.4 ± 2.0 s−1 ; and the kcat/Km 's were 18.5 vs 17.6, 9.84 vs 5.65, and 10.1 vs 13.6 s−1 μm . These values closely approximate those of 7.2 ± 0.9 μM, 84 ± 4 s−1 , and 11.7 s−1 μM−1 determined by Higgins, Lewis, and Shafer (J. Biol. Chem. 258:9276-9282, 1983) for the Aα cleavage of human fibrinogen by human α-thrombin under physiologically relevant conditions. Thus, these chromogenic substrates have thrombin specificities similar to that of fibrinogen, although their amidolytic activities are independent of additional active-site regions required for fibrinogen clotting activity (α vs γ-thrombin). Fibrinogen interactions with such active-site regions might account for why the fibrinogen Aa site is an atypical thrombin susceptible bond and the high species variability of fibrinopeptides. (Supported in part by NIH grant HL-13160).

scholarly journals Halogen Bond Interactions of Novel HIV-1 Protease Inhibitors (PI) (GRL-001-15 and GRL-003-15) with the Flap of Protease Are Critical for Their Potent Activity against Wild-Type HIV-1 and Multi-PI-Resistant Variants

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Shin-ichiro Hattori ◽  
Hironori Hayashi ◽  
Haydar Bulut ◽  
Kalapala Venkateswara Rao ◽  
Prasanth R. Nyalapatla ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Protease Inhibitors ◽  
Active Site ◽  
Fluorine Atom ◽  
Halogen Bond ◽  
The Novel ◽  
Wild Type ◽  
Flap Region ◽  
Hiv 1

ABSTRACTWe generated two novel nonpeptidic HIV-1 protease inhibitors (PIs), GRL-001-15 and GRL-003-15, which contain unique crown-like tetrahydropyranofuran (Crn-THF) and P2′-cyclopropyl-aminobenzothiazole (Cp-Abt) moieties as P2 and P2′ ligands, respectively. GRL-001-15 and GRL-003-15 havemeta-monofluorophenyl andpara-monofluorophenyl at the P1 site, respectively, exert highly potent activity against wild-type HIV-1 with 50% effective concentrations (EC50s) of 57 and 50 pM, respectively, and have favorable cytotoxicity profiles with 50% cytotoxic concentrations (CC50s) of 38 and 11 μM, respectively. The activity of GRL-001-15 against multi-PI-resistant HIV-1 variants was generally greater than that of GRL-003-15. The EC50of GRL-001-15 against an HIV-1 variant that was highly resistant to multiple PIs, including darunavir (DRV) (HIV-1DRVRP30), was 0.17 nM, and that of GRL-003-15 was 3.3 nM, while DRV was much less active, with an EC50of 216 nM. The emergence of HIV-1 variants resistant to GRL-001-15 and GRL-003-15 was significantly delayed compared to that of variants resistant to selected PIs, including DRV. Structural analyses of wild-type protease (PRWT) complexed with the novel PIs revealed that GRL-001-15’smeta-fluorine atom forms halogen bond interactions (2.9 and 3.0 Å) with Gly49 and Ile50, respectively, of the protease flap region and with Pro81′ (2.7 and 3.2 Å), which is located close to the protease active site, and that two fluorine atoms of GRL-142-13 form multiple halogen bond interactions with Gly49, Ile50, Pro81′, Ile82′, and Arg8′. In contrast, GRL-003-15 forms halogen bond interactions with Pro81′ alone, suggesting that the reduced antiviral activity of GRL-003-15 is due to the loss of the interactions with the flap region.

scholarly journals Functional investigation of methanol dehydrogenase-like protein XoxF in Methylobacterium extorquens AM1

Microbiology ◽  
2010 ◽  
Vol 156 (8) ◽  
pp. 2575-2586 ◽  
Author(s):  
Sabrina Schmidt ◽  
Philipp Christen ◽  
Patrick Kiefer ◽  
Julia A. Vorholt
Keyword(s):  
Growth Parameters ◽  
Methanol Dehydrogenase ◽  
Plant Colonization ◽  
Kinetic Properties ◽  
Wild Type ◽  
Methylobacterium Extorquens ◽  
Experimental Conditions ◽  
Dependent Protein ◽  
One Carbon Metabolism ◽  
Functional Investigation

Methanol dehydrogenase-like protein XoxF of Methylobacterium extorquens AM1 exhibits a sequence identity of 50 % to the catalytic subunit MxaF of periplasmic methanol dehydrogenase in the same organism. The latter has been characterized in detail, identified as a pyrroloquinoline quinone (PQQ)-dependent protein, and shown to be essential for growth in the presence of methanol in this methylotrophic model bacterium. In contrast, the function of XoxF in M. extorquens AM1 has not yet been elucidated, and a phenotype remained to be described for a xoxF mutant. Here, we found that a xoxF mutant is less competitive than the wild-type during colonization of the phyllosphere of Arabidopsis thaliana, indicating a function for XoxF during plant colonization. A comparison of the growth parameters of the M. extorquens AM1 xoxF mutant with those of the wild-type during exponential growth revealed a reduced methanol uptake rate and a reduced growth rate for the xoxF mutant of about 30 %. Experiments with cells starved for carbon revealed that methanol oxidation in the xoxF mutant occurs less rapidly compared with the wild-type, especially in the first minutes after methanol addition. A distinct phenotype for the xoxF mutant was also observed when formate and CO2 production were measured after the addition of methanol or formaldehyde to starved cells. The wild-type, but not the xoxF mutant, accumulated formate upon substrate addition and had a 1 h lag in CO2 production under the experimental conditions. Determination of the kinetic properties of the purified enzyme showed a conversion capacity for both formaldehyde and methanol. The results suggest that XoxF is involved in one-carbon metabolism in M. extorquens AM1.

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