Redesigning the active site of Geotrichum candidum lipase

Abstract

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 approximately 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 lipases are evolutionary switchpoints between the two observed arrangements of the catalytic triads supported by the alpha/beta hydrolase fold and suggest that this fold provides a stable protein core for engineering enzymes with unique catalytic properties.

Repositioning the Catalytic Triad Aspartic Acid of Haloalkane Dehalogenase: Effects on Stability, Kinetics, and Structure

“…Moreover, in the model the O 1 atom of Glu148 appears to be located at about the same position as Asp260O δ2 in the wild-type structure (Figure 7). This means that the indole ring of His289 is typically in the same plane as in the wildtype structure and that only subtle changes in hydrogen bonding and stabilization of the triad residues account for the differences in kinetics, as was also the case for similar mutants of Geotrichum candidum lipase (Schrag et al, 1994).…”

Microbial modulation of aromatic esters in wine: Current knowledge and future prospects

“…Rational Protein Design, using computer modelling, to understand the enantioselectivity and substrate specificity of esterases and other wine-related enzymes, to design enzymes with improved activity and selectivity. Directed evolution to alter substrate specificity or improve stability and activity of microbial esterases (Neuenschwander, Butz, Heintz, Kast, & Hilvert, 2007;Schrag et al, 1995).…”

Bacterial Biocatalysts: Molecular Biology, Three-Dimensional Structures, and Biotechnological Applications of Lipases

“…However, the topological position of the catalytic acid seems to be variable: in pancreatic lipase it is positioned after strand 6 (163). This observation prompted Schrag et al (131) to redesign the active site of G. candidum lipase by shifting the position of the catalytic acid from strand 7 to strand 6. The double mutant retained ∼10% of the wild-type activity, confirming the variability of the position of the catalytic acid.…”