Redesigning the active site of Geotrichum candidum lipase

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DOIResolve DOI: http://doi.org/10.1093/protein/8.8.835
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TypeArticle
Journal titleProtein Engineering
Volume8
Issue8
Pages835842; # of pages: 8
Subjectpha; catalytic triad; double mutant; G.candidum lipase; human pancreatic lipase; protein design
AbstractAttempts to engineer enzymes with unique catalytic properties have largely focused on altering the existing specificities by reshaping the substrate binding pockets. Few experiments have aimed at modifying the configuration of the residues essential for catalysis. The difference in the topological location of the triad acids of Geotrichum candidum lipase (GCL) and the catalytic domain of human pancreatic lipase (HPL), despite great similarities in their topologies and 3-D structures, suggest that these are related enzymes whose catalytic triads have been rearranged in the course of evolution (Schrag et aL, 1992). In this study we prepared a double mutant GCL in which the catalytic triad acid is shifted to the position equivalent to the location of the triad acid of HPL. The double mutant maintains ∼10% of the wild type activity against triglycerides and the fluorogenic ester 4-methylumbelliferyl-oleate. The only significant differences between the 3-D structures of the double mutant and wild type GCL are at the mutated sites. Even the water structure in the region of the triad is unchanged. The hydrogen bonding pattern of the catalytic triad of the double mutant is very similar to that of pancreatic lipase. The acid of the double mutant is stabilized by only two hydrogen bonds, whereas three hydrogen bonds are observed in the wild type enzyme. These results strongly support the hypothesis that the pancreatic Upases are evolutionary switchpoints between the two observed arrangements of the catalytic triads supported by the α/β hydrolase fold and suggest that this fold provides a stable protein core for engineering enzymes with unique catalytic properties.
Publication date
LanguageEnglish
AffiliationNRC Biotechnology Research Institute; National Research Council Canada
Peer reviewedNo
NRC number38539
NPARC number3539942
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Record identifierb68a238c-5056-4ea5-809e-6e1fe9ab1330
Record created2009-03-01
Record modified2016-05-09
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