Two conformational states of Candida rugosa lipase

Abstract

The structure of Candida rugosa lipase in a new crystal form has been determined and refined at 2.1 A resolution. The lipase molecule was found in an inactive conformation, with the active site shielded from the solvent by a part of the polypeptide chain-the flap. Comparison of this structure with the previously determined "open" form of this lipase, in which the active site is accessible to the solvent and presumably the substrate, shows that the transition between these 2 states requires only movement of the flap. The backbone NH groups forming the putative oxyanion hole d o not change position during this rearrangement, indicating that this feature is preformed in the inactive state. The 2 lipase conformations probably correspond to states at opposite ends of the pathway of interfacial activation. Quantitative analysis indicates a large increase of the hydrophobic surface in the vicinity of the active site. The flap undergoes a flexible rearrangement during which some of its secondary structure refolds. The interactions of the flap with the rest of the protein change from mostly hydrophobic in the inactive form to largely hydrophilic in the "open" conformation. Although the flap movement cannot be described as a rigid body motion, it has very definite hinge points at Glu 66 and at Pro 92. The rearrangement is accompanied by a cis-trans isomerization of this proline, which likely increases the energy required for the transition between the 2 states, and may play a role in the stabilization of the active conformation at the water/lipid interface. Carbohydrate attached at Asn 351 also provides stabilization for the open conformation of the flap. Keywords: crystallography; interfacial activation; lipasesLipases of known 3-dimensional structure show significant similarities in their topologies and conform in full or in part to the a/fl hydrolase fold ( Ollis et al., 1992). The catalytic machinery of lipases consists of a serine protease-like triad, Ser-His-Asp/Glu, and their hydrolysis of ester bonds of triacylglycerols is thought to involve an enzymatic mechanism similar to that of the serine proteases (Chapus et al., 1988). The activity of lipases is dramatically enhanced by the presence of a lipid/water interface (Desnuelle, 1972), and it is now well accepted that the phenomenon of interfacial activation involves a conformational change in the enzyme. This has been documented for 2 lipases, one from Rhizomucor miehei (Brzozowski et ai., 199i) and another from human pancreas (van Tilbeurgh et al., 1993), in which binding of an inhibitor or substrate analog causes a conformational rearrangement of 1 or more loops near the active site, exposing the serine nucleophile to the solvent and creating also an oxyanion hole in the process.A similar rearrangement was also proposed for larger lipases from Geotrichum candidurn (GCL;Schrag & Cygler, 1993) Candida rugosa (CRL; Grochubki et ai., 1993). These 2 enzymes show -40% amino acid sequence identity, and their overall 3D structures are very similar (Grochulski ...

Frozen Microemulsions for MAPLE Immobilization of Lipase

“…Moreover, in the spectrum of M-CRL2 the amide III band is partially overlapped with the amide II one consequently, to avoid errors in the baseline correction, these two bands were included in the fitting procedure. CRL conformation was determined by X-ray diffraction on lipase crystals [29,30]. XRD data revealed in the CRL open conformation a content 30% for α-helices and 12% for β-sheets.…”

A structural basis for enantioselective inhibition of Candida rugosa lipase by long‐chain aliphatic alcohols

“…This means that a long-chain aliphatic alcohol substrate could bind to the tunnel of the active site of C. rugosa lipase. In fact, the electron density seen at the entrance of the tunnel by X-ray crystallographic analysis of the open lipase was assigned to 2-methyl-2,4-pentanediol, which was used as a precipitating agent (Grochulski et al, 1994b). In the X-ray structure of the related Geotrichum candidum lipase, an unidentified organic molecule was bound in the active site tunnel .…”

“…All water molecules bound to the lipase according to the X-ray structure were included in the calculations, except one water molecule, which was removed to permit binding of substrate into the active site of the enzyme. The carbohydrate moieties covalently linked to Asn 314 and Asn 351 (Grochulski et al, 1994b) were excluded in the calculations.…”

Production of Oleic Acid Ethyl Ester Catalyzed by Crude Rice Bran (Oryza sativa) Lipase in a Modified Fed-batch System: A Problem and its Solution

“…This ratio (2.05:1) was the optimum ratio which produced the highest yield [5]. Since water (a by product of the reaction) could reduce the reaction rate, the system was also including an addition of zeolite powder into the reaction system to absorb the water [9,10,11,12,13]. The ester conversion was then evaluated.…”