Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues.
AUTOR(ES)
Frenken, L G
RESUMO
The lipA gene encoding the extracellular lipase produced by Pseudomonas glumae PG1 was cloned and characterized. A sequence analysis revealed an open reading frame of 358 codons encoding the mature lipase (319 amino acids) preceded by a rather long signal sequence of 39 amino acids. As a first step in structure-function analysis, we determined the Ser-Asp-His triad which makes up the catalytic site of this lipase. On the basis of primary sequence homology with other known Pseudomonas lipases, a number of putative active site residues located in conserved areas were found. To determine the residues actually involved in catalysis, we constructed a number of substitution mutants for conserved Ser, Asp, and His residues. These mutant lipases were produced by using P. glumae PG3, from which the wild-type lipase gene was deleted by gene replacement. By following this approach, we showed that Ser-87, Asp-241, and His-285 make up the catalytic triad of the P. glumae lipase. This knowledge, together with information on the catalytic mechanism and on the three-dimensional structure, should facilitate the selection of specific modifications for tailoring this lipase for specific industrial applications.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=183182Documentos Relacionados
- Active site analysis and stabilization of sarcosine oxidase by the substitution of cysteine residues.
- Tolerance of 5-fluorodeoxyuridine resistant human thymidylate synthases to alterations in active site residues.
- Nitrilase in biosynthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile: cloning of the Alcaligenes gene and site-directed mutagenesis of cysteine residues.
- Cloning, sequence, and expression of a lipase gene from Pseudomonas cepacia: lipase production in heterologous hosts requires two Pseudomonas genes.
- Ligation-independent cloning of PCR products with primers containing nonbase residues.