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Protein Engineering vol. 8 no. 8 pp. 835-842, 1995
© 1995 Oxford University Press

RESEARCH-ARTICLE

Redesigning the active site of Geotrichum candidum lipase

Joseph D. Schrag1, Thierry Vernet, Louise Laramée, David Y. Thomas, Achim Recktenwald, Monika Okoniewska, Edmund Ziomek, Miroslaw Cygler1 and Annick Thomas-Soumarmon

Biotechnology Research Institute NRC, Montreal, Quebec H4P 2R2, Canada

1To whom correspondence should be addressed

Attempts 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 {alpha}/ß hydrolase fold and suggest that this fold provides a stable protein core for engineering enzymes with unique catalytic properties.

Keywords: catalytic triad/double mutant/G.candidum lipase/human pancreatic lipase/protein design

Received March 10, 1995; revised June 5, 1995; accepted June 20, 1995.


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