Genetically engineered zinc-chelating adenylate kinase from Escherichia coli with enhanced thermal stability.
In contrast with adenylate kinase from Gram-negative bacteria, the enzyme from Gram-positive organisms harbors a structural Zn2+ bound to 3 or 4 Cys residues in the structural motif Cys-X2-Cys-X16-Cys-X2-Cys/Asp. Site-directed mutagenesis of His126, Ser129, Asp146, and Thr149 (corresponding to Cys130, Cys133, Cys150, and Cys153 in adenylate kinase from Bacillus stearothermophilus) in Escherichia coli adenylate kinase was undertaken for determining whether the presence of Cys residues is the only prerequisite to bind zinc or (possible) other cations. A number of variants of adenylate kinase from E. coli, containing 1-4 Cys residues were obtained, purified, and analyzed for metal content, structural integrity, activity, and thermodynamic stability. All mutants bearing 3 or 4 cysteine residues acquired zinc binding properties. Moreover, the quadruple mutant exhibited a remarkably high thermal stability as compared with the wild-type form with preservation of the kinetic parameters of the parent enzyme.
Laboratoire de Chimie Structurale des Macromolécules, Institut Pasteur, 75724 Paris Cedex 15, France.
V
V
Perrier
S
S
Burlacu-Miron
S
S
Bourgeois
W K
WK
Surewicz
A M
AM
Gilles
D000263:Adenylate Kinase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002384:Catalysis; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005818:Genetic Engineering; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D013054:Spectrophotometry, Atomic; D013696:Temperature; D014357:Trypsin / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
D010446:Peptide Fragments; D004228:Dithionitrobenzoic Acid; D000263:Adenylate Kinase; D014357:Trypsin; D015032:Zinc; D003545:Cysteine