Identification of the histidine and aspartic acid residues essential for enzymatic activity of a family I.3 lipase by site‐directed mutagenesis

[摘要]

A lipase from Pseudomonas sp. MIS38 (PML) is a member of the lipase family I.3. We analyzed the roles of the five histidine residues (His³⁰, His²⁷⁴, His²⁹¹, His³¹³, and His³⁶⁵) and five acidic amino acid residues (Glu²⁵³, Asp²⁵⁵, Asp²⁶², Asp²⁷⁵, and Asp²⁹⁰), which are fully conserved in the amino acid sequences of family I.3 lipases, by site-directed mutagenesis. We showed that the mutation of His³¹³ or Asp²⁵⁵ to Ala almost fully inactivated the enzyme, whereas the mutations of other residues to Ala did not seriously affect the enzymatic activity. Measurement of the far- and near-UV circular dichroism spectra suggests that inactivation by the mutation of His³¹³ or Asp²⁵⁵ is not due to marked changes in the tertiary structure. We propose that His³¹³ and Asp²⁵⁵, together with Ser²⁰⁷, form a catalytic triad in PML.

[学科分类] 生物化学/生物物理

[关键词] Lipase;Site-directed mutagenesis;Catalytic triad;Active-site;Pseudomonas;PML;Pseudomonas sp. MIS38 lipase;CD;circular dichroism