title: Engineering the properties of a cold active enzyme through rational redesign of the active site.
abstract: In an effort to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side-chain flexibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Trp260 and Ala219 of the catalytic site and His135 of the Mg2+ binding site. The replacement of Trp260 by Lys in mutant W260K, resulted in an enzyme less active than the wild-type in the temperature range 5-25 degrees C. The additional replacement of Ala219 by Asn in the double mutant W260K/A219N, resulted in a drastic increase in the energy of activation, which was reflected in a considerably decreased activity at temperatures of 5-15 degrees C and a significantly increased activity at 20-25 degrees C. Further substitution of His135 by Asp in the triple mutant W260K/A219N/H135D restored a low energy of activation. In addition, the His135-->Asp replacement in mutants H135D and W260K/A219N/H135D resulted in considerable stabilization. These results suggest that the psychrophilic character of mutants can be established or masked by very slight variations of the wild-type sequence, which may affect active site flexibility through changes in various conformational constraints.
authors.affiliation: Institute of Molecular Biology and Biotechnology (IMBB), Enzyme Technology Division, Heraklion 711 10, Crete, Greece.
authors.forename: I
authors.initials: I
authors.lastname: Tsigos
authors.forename: K
authors.initials: K
authors.lastname: Mavromatis
authors.forename: M
authors.initials: M
authors.lastname: Tzanodaskalaki
authors.forename: C
authors.initials: C
authors.lastname: Pozidis
authors.forename: M
authors.initials: M
authors.lastname: Kokkinidis
authors.forename: V
authors.initials: V
authors.lastname: Bouriotis
mesh_terms: D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013696:Temperature
chemical_list: D017931:DNA Primers; D000469:Alkaline Phosphatase