Indexed on: 27 Mar '09Published on: 27 Mar '09Published in: Protein engineering, design & selection : PEDS
The sequence of the estA gene (locus SCO 7131) of S. coelicolor A3 (2) suggested that it might differ in substrate specificity from other characterised members of the hormone-sensitive lipase (HSL) family of lipases and esterases. This difference may be attributed to the unique substitutions within the conserved motifs of the family. There was no homologue with any other lipase or esterase to estA in the chromosome of S. avermitilis or other streptomyces species and the sequence showed differences in the conserved motifs from the characterised members of the HSL family. The gene was cloned and expressed as a His-tagged protein in Escherichia coli. The purified enzyme was an esterase, which hydrolyzed the acetate ester of p-nitrophenol, but had little activity on esters with longer side chains, unlike the other characterised bacterial members of the HSL family which showed optimal activity against caproate (C(6)) esters. Site-directed mutagenesis was used to change two amino acids to the consensus for the HSL family. This increased the activity against butyrate and caproate esters. The changes also affected thermostability: in one case increasing stability and in the other case reducing it. A profile was constructed for the HSL family and used to detect 119 members in the protein database. The location of conserved amino acid motifs in a 3-D homology model of the enzyme identified further members of the family with unusual amino acid replacements.