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J Biol Chem, Vol. 273, Issue 30, 19097-19101, July 24, 1998

Genetically Engineered Zinc-chelating Adenylate Kinase from Escherichia coli with Enhanced Thermal Stability

Véronique Perrier, Simona Burlacu-Miron^§, Serge Bourgeois^¶, Witold K. Surewicz, and Anne-Marie Gilles

From the  Laboratoire de Chimie Structurale des Macromolécules, Institut Pasteur, 75724 Paris Cedex 15, France, ^§ INSERM U 350, Institut Curie-Recherche, 91405 Orsay Cedex, France, ^¶ Laboratoire de Sciences des Sols et Hydrologie, Institut National d'Agronomie, Centre de Grignon, 78850 Thiverval, France, and  Department of Ophthalmology, University of Missouri, Columbia, Missouri 65212

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.

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