title: Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues.
abstract: 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.
authors: Unilever Research Laboratorium Vlaardingen, The Netherlands.
authors: L G
authors: LG
authors: Frenken
authors: M R
authors: MR
authors: Egmond
authors: A M
authors: AM
authors: Batenburg
authors: J W
authors: JW
authors: Bos
authors: C
authors: C
authors: Visser
authors: C T
authors: CT
authors: Verrips
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
chemical_list: D004269:DNA, Bacterial; D008049:Lipase