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

Ljudmila Kulakova, Andrey Galkin, Toru Nakayama, Tokuzo Nishino, Nobuyoshi Esaki

Research output: Contribution to journalArticlepeer-review

97 Citations (Scopus)

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°C), low activation energy (e.g., 4.6 kcal/mol for hydrolysis of p-nitrophenyl butyrate), and a t1/2 value of 16 min for thermal inactivation during incubation at 40°C and pH 7.9. A three-dimensional structural model of PsyEst predicted that Gly244 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 Gly244→Pro substitution into the enzyme. Stability studies showed that the t1/2 value for thermal inactivation of the mutant during incubation at 40°C and pH 7.9 was 11.6 h, which was significantly greater than that of the wild-type enzyme. The kcat/Km 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.

Original languageEnglish
Pages (from-to)59-65
Number of pages7
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1696
Issue number1
DOIs
Publication statusPublished - 2004 Jan 14

Keywords

  • Esterase
  • Psychrobacter sp.
  • Substitution

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology

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