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PEDS Advance Access originally published online on March 25, 2009
Protein Engineering Design and Selection 2009 22(6):333-339; doi:10.1093/protein/gzp009
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© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Mining the genome sequence for novel enzyme activity: characterisation of an unusual member of the hormone-sensitive lipase family of esterases from the genome of Streptomyces coelicolor A3 (2)

Sameh H. Soror1,2,3, Ren Rao1 and John Cullum1

1LB Genetik, University of Kaiserslautern, Postfach 3049, 67653 Kaiserslautern, Germany 2Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Helwan Ain Helwan, University Campus, 11795 Cairo, Egypt

3 To whom correspondence should be addressed. E-mail: shamdy26{at}hotmail.com

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 (C6) 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.

Keywords: (HSL) family/Streptomyces coelicolor/esterases/genome sequence/thermostability

Received April 30, 2008; revised February 6, 2009; accepted February 27, 2009.


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