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 Characterization of P450 FcpC, the Enzyme Responsible for Bioconversion of Diosgenone to Isonuatigenone in Streptomyces virginiae IBL-14

2009 ◽  
Vol 75 (12) ◽  
pp. 4202-4205 ◽  
Author(s):  
Wei Wang ◽  
Feng-Qing Wang ◽  
Dong-Zhi Wei
Keyword(s):  
Escherichia Coli ◽  
Electron Transfer ◽  
Cytochrome P450 ◽  
Cytochrome P450 Monooxygenase ◽  
P450 Monooxygenase ◽  
Whole Cell ◽  
Electron Transfer Chain ◽  
Streptomyces Virginiae ◽  
Transfer Chain

ABSTRACT A new cytochrome P450 monooxygenase, FcpC, from Streptomyces virginiae IBL-14 has been identified. This enzyme is found to be responsible for the bioconversion of a pyrano-spiro steroid (diosgenone) to a rare nuatigenin-type spiro steroid (isonuatigenone), which is a novel C-25-hydroxylated diosgenone derivative. A whole-cell P450 system was developed for the production of isonuatigenone via the expression of the complete three-component electron transfer chain in an Escherichia coli strain.

Comparison of the structural and kinetic properties of the cytochrome c nitrite reductases from Escherichia coli, Wolinella succinogenes, Sulfurospirillum deleyianum and Desulfovibrio desulfuricans

2006 ◽  
Vol 34 (1) ◽  
pp. 143-145 ◽  
Author(s):  
T.A. Clarke ◽  
A.M. Hemmings ◽  
B. Burlat ◽  
J.N. Butt ◽  
J.A. Cole ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Active Site ◽  
Desulfovibrio Desulfuricans ◽  
Amino Acid Sequences ◽  
Kinetic Properties ◽  
Wolinella Succinogenes ◽  
Nitrite Reductases ◽  
Sulphite Reductase

The recent crystallographic characterization of NrfAs from Sulfurospirillum deleyianum, Wolinella succinogenes, Escherichia coli and Desulfovibrio desulfuricans allows structurally conserved regions to be identified. Comparison of nitrite and sulphite reductase activities from different bacteria shows that the relative activities vary according to organism. By comparison of both amino acid sequences and structures, differences can be identified in the monomer–monomer interface and the active-site channel; these differences could be responsible for the observed variance in substrate activity and indicate that subtle changes in the NrfA structure may optimize the enzyme for different roles.

Characterization of Escherichia coli thioredoxins with altered active site residues

Biochemistry ◽  
1990 ◽  
Vol 29 (15) ◽  
pp. 3701-3709 ◽  
Author(s):  
Florence K. Gleason ◽  
Chang Jin Lim ◽  
Maryam Gerami-Nejad ◽  
James A. Fuchs
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Active Site Residues

scholarly journals Biochemical and Structural Characterization of the Subclass B1 Metallo-β-Lactamase VIM-4

2010 ◽  
Vol 55 (3) ◽  
pp. 1248-1255 ◽  
Author(s):  
Patricia Lassaux ◽  
Daouda A. K. Traoré ◽  
Elodie Loisel ◽  
Adrien Favier ◽  
Jean-Denis Docquier ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Thermal Stability ◽  
Pseudomonas Aeruginosa ◽  
Structural Characterization ◽  
Catalytic Efficiency ◽  
X Ray ◽  
Stabilizing Effect ◽  
Loop 2

ABSTRACTThe metallo-β-lactamase VIM-4, mainly found inPseudomonas aeruginosaorAcinetobacter baumannii, was produced inEscherichia coliand characterized by biochemical and X-ray techniques. A detailed kinetic study performed in the presence of Zn2+at concentrations ranging from 0.4 to 100 μM showed that VIM-4 exhibits a kinetic profile similar to the profiles of VIM-2 and VIM-1. However, VIM-4 is more active than VIM-1 against benzylpenicillin, cephalothin, nitrocefin, and imipenem and is less active than VIM-2 against ampicillin and meropenem. The crystal structure of the dizinc form of VIM-4 was solved at 1.9 Å. The sole difference between VIM-4 and VIM-1 is found at residue 228, which is Ser in VIM-1 and Arg in VIM-4. This substitution has a major impact on the VIM-4 catalytic efficiency compared to that of VIM-1. In contrast, the differences between VIM-2 and VIM-4 seem to be due to a different position of the flapping loop and two substitutions in loop 2. Study of the thermal stability and the activity of the holo- and apo-VIM-4 enzymes revealed that Zn2+ions have a pronounced stabilizing effect on the enzyme and are necessary for preserving the structure.

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 Characterization of Escherichia coli UmuC Active-Site Loops Identifies Variants That Confer UV Hypersensitivity

2011 ◽  
Vol 193 (19) ◽  
pp. 5400-5411 ◽  
Author(s):  
L. A. Hawver ◽  
C. A. Gillooly ◽  
P. J. Beuning
Keyword(s):  
Escherichia Coli ◽  
Active Site
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