title: Cold-active esterase from Psychrobacter sp. Ant300: gene cloning, characterization, and the effects of Gly-->Pro substitution near the active site on its catalytic activity and stability.
abstract: The gene encoding an esterase (PsyEst) of Psychrobacter sp. Ant300, a psychrophilic bacterium isolated from Antarctic soil, was cloned, sequenced, and expressed in Escherichia coli. PsyEst, which is a member of hormone-sensitive lipase (HSL) group of the lipase/esterase family, is a cold-active, themolabile enzyme with high catalytic activity at low temperatures (5-25 degrees C), low activation energy (e.g., 4.6 kcal/mol for hydrolysis of p-nitrophenyl butyrate), and a t(1/2) value of 16 min for thermal inactivation during incubation at 40 degrees C and pH 7.9. A three-dimensional structural model of PsyEst predicted that Gly(244) was located in the loop near the active site of PsyEst and that substitution of this amino-acid residue by proline should potentially rigidify the active-site environment of the enzyme. Thus, we introduced the Gly(244)-->Pro substitution into the enzyme. Stability studies showed that the t(1/2) value for thermal inactivation of the mutant during incubation at 40 degrees C and pH 7.9 was 11.6 h, which was significantly greater than that of the wild-type enzyme. The k(cat)/K(m) value of the mutant was lower for all substrates examined than the value of the wild type. Moreover, this amino-acid substitution caused a shift of the acyl-chain length specificity of the enzyme toward higher preference for short-chain fatty acid esters. All of these observations could be explained in terms of a decrease in active-site flexibility brought about by the mutation and were consistent with the hypothesis that cold activity and thermolability arise from local flexibility around the active site of the enzyme.
authors.affiliation: Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto-Fu, Kyoto 611-0011, Japan.
authors.forename: Ljudmila
authors.initials: L
authors.lastname: Kulakova
authors.forename: Andrey
authors.initials: A
authors.lastname: Galkin
authors.forename: Toru
authors.initials: T
authors.lastname: Nakayama
authors.forename: Tokuzo
authors.initials: T
authors.lastname: Nishino
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Esaki
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D003001:Cloning, Molecular; D003080:Cold Temperature; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D004950:Esterases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D044096:Psychrobacter / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D012988:Soil Microbiology; D013379:Substrate Specificity
chemical_list: D004950:Esterases