Site-directed mutagenesis of Escherichia coli ornithine transcarbamoylase: role of arginine-57 in substrate binding and catalysis

Biochemistry ◽  
1988 ◽  
Vol 27 (24) ◽  
pp. 8823-8832 ◽  
Author(s):  
Lawrence C. Kuo ◽  
Arthur W. Miller ◽  
Sunjoo Lee ◽  
Cheryl Kozuma
Keyword(s):  
Escherichia Coli ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Ornithine Transcarbamoylase

scholarly journals Studies on the mechanism of hydroxymethylbilane synthase concerning the role of arginine residues in substrate binding

1991 ◽  
Vol 275 (2) ◽  
pp. 447-452 ◽  
Author(s):  
M Lander ◽  
A R Pitt ◽  
P R Alefounder ◽  
D Bardy ◽  
C Abell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Kinetic Parameters ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Side Chains ◽  
Enzyme Substrate ◽  
Arginine Residues

The role of conserved arginine residues in hydroxymethylbilane synthase was investigated by replacing these residues in the enzyme from Escherichia coli with leucine residues by using site-directed mutagenesis. The kinetic parameters for these mutant enzymes and studies on the formation of intermediate enzyme-substrate complexes indicate that several of these arginine residues are involved in binding the carboxylate side chains of the pyrromethane cofactor and the growing oligopyrrole chain.

Role of asparagine 152 in catalysis of .beta.-lactam hydrolysis by Escherichia coli AmpC .beta.-lactamase studied by site-directed mutagenesis

Biochemistry ◽  
1995 ◽  
Vol 34 (23) ◽  
pp. 7757-7764 ◽  
Author(s):  
Alain Dubus ◽  
Staffan Normark ◽  
Malgosia Kania ◽  
Malcolm G. P. Page
Keyword(s):  
Escherichia Coli ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Beta Lactamase ◽  
Beta Lactam

scholarly journals The role of cysteine residues 129 and 329 in Escherichia coli K1 CMP-NeuAc synthase

1993 ◽  
Vol 295 (2) ◽  
pp. 485-491 ◽  
Author(s):  
G Zapata ◽  
P P Roller ◽  
J Crowley ◽  
W F Vann
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Acetylneuraminic Acid ◽  
Denatured States ◽  
Escherichia Coli K1

N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.

scholarly journals Cloning, Expression, and Site-Directed Mutagenesis of the Propene Monooxygenase Genes from Mycobacterium sp. Strain M156

2005 ◽  
Vol 71 (4) ◽  
pp. 1909-1914 ◽  
Author(s):  
Chan K. Chan Kwo Chion ◽  
Sarah E. Askew ◽  
David J. Leak
Keyword(s):  
Mutational Analysis ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Active Site Residues ◽  
Styrene Oxidation ◽  
Overlapping Reading Frames ◽  
Reading Frames

ABSTRACT Propene monooxygenase has been cloned from Mycobacterium sp. strain M156, based on hybridization with the amoABCD genes of Rhodococcus corallinus B276. Sequencing indicated that the mycobacterial enzyme is a member of the binuclear nonheme iron monooxygenase family and, in gene order and sequence, is most similar to that from R. corallinus B-276. Attempts were made to express the pmoABCD operon in Escherichia coli and Mycobacterium smegmatis mc2155. In the former, there appeared to be a problem resolving overlapping reading frames between pmoA and -B and between pmoC and -D, while in the latter, problems were encountered with plasmid instability when the pmoABCD genes were placed under the control of the hsp60 heat shock promoter in the pNBV1 vector. Fortuitously, constructs with the opposite orientation were constitutively expressed at a level sufficient to allow preliminary mutational analysis. Two PMO active-site residues (A94 and V188) were targeted by site-directed mutagenesis to alter their stereoselectivity. The results suggest that changing the volume occupied by the side chain at V188 leads to a systematic alteration in the stereoselectivity of styrene oxidation, presumably by producing different orientations for substrate binding during catalysis. Changing the volume occupied by the side chain at A94 produced a nonsystematic change in stereoselectivity, which may be attributable to the role of this residue in expansion of the binding site during substrate binding. Neither set of mutations changed the enzyme's specificity for epoxidation.

Investigation of the functional role of tryptophan-22 in Escherichia coli dihydrofolate reductase by site-directed mutagenesis

Biochemistry ◽  
1991 ◽  
Vol 30 (46) ◽  
pp. 11092-11103 ◽  
Author(s):  
Mark S. Warren ◽  
Katherine A. Brown ◽  
Martin F. Farnum ◽  
Elizabeth E. Howell ◽  
Joseph Kraut
Keyword(s):  
Escherichia Coli ◽  
Dihydrofolate Reductase ◽  
Functional Role ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Site-directed mutagenesis on TEM-1 ß-lactamase: role of Glul66 in catalysis and substrate binding

1991 ◽  
Vol 4 (7) ◽  
pp. 805-810 ◽  
Author(s):  
Myriam Delaire ◽  
Francoise Lenfant ◽  
Roger Labia ◽  
Jean-Michel Masson
Keyword(s):  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis
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