No abstract

Lipases are hydrolytic enzymes which break down triacylglycerides into free fatty acids and glycerols. They have been classified as serine hydrolases owing to their inhibition by diethyl p-nitrophenyl phosphate. Lipase activity is greatly increased at the lipid-water interface, a phenomenon known as interfacial activation. X-ray analysis has revealed the atomic structures of two triacylglycerol lipases, unrelated in sequence: the human pancreatic lipase (hPL)4, and an enzyme isolated from the fungus Rhizomucor (formerly Mucor) miehei (RmL). In both enzymes the active centres contain structurally analogous Asp-His-Ser triads (characteristic of serine proteinases), which are buried completely beneath a short helical segment, or 'lid'. Here we present the crystal structure (at 3 A resolution) of a complex of R. miehei lipase with n-hexylphosphonate ethyl ester in which the enzyme's active site is exposed by the movement of the helical lid. This movement also increases the nonpolarity of the surface surrounding the catalytic site. We propose that the structure of the enzyme in this complex is equivalent to the activated state generated by the oil-water interface.

Many lipases are potent catalysts of stereoselective reactions and are therefore of interest for use in chemical synthesis. The crystal structures of lipases show a large variation in the shapes of their active site environments that may explain the large variation in substrate specificity of these enzymes. We have determined the three-dimensional structure of Candida antarctica lipase B (CALB) cocrystallized with the detergent Tween 80. In another crystal form, the structure of the enzyme in complex with a covalently bound phosphonate inhibitor has been determined. In both structures, the active site is exposed to the external solvent. The potential lid-forming helix alpha 5 in CALB is well-ordered in the Tween 80 structure and disordered in the inhibitor complex. The tetrahedral intermediates of two chiral substrates have been modeled on the basis of available structural and biochemical information. The results of this study provide a structural explanation for the high stereoselectivity of CALB toward many secondary alcohols.

The already known X-ray structures of lipases provide little evidence about initial, discrete structural steps occurring in the first phases of their activation in the presence of lipids (process referred to as interfacial activation). To address this problem, five new Thermomyces (formerly Humicola) lanuginosa lipase (TlL) crystal structures have been solved and compared with four previously reported structures of this enzyme. The bias coming from different crystallization media has been minimized by the growth of all crystals under the same crystallization conditions, in the presence of detergent/lipid analogues, with low or high ionic strength as the only main variable. Resulting structures and their characteristic features allowed the identification of three structurally distinct species of this enzyme: low activity form (LA), activated form (A), and fully Active (FA) form. The isomerization of the Cys268-Cys22 disulfide, synchronized with the formation of a new, short alpha(0) helix and flipping of the Arg84 (Arginine switch) located in the lid's proximal hinge, have been postulated as the key, structural factors of the initial transitions between LA and A forms. The experimental results were supplemented by theoretical calculations. The magnitude of the activation barrier between LA (ground state) and A (end state) forms of TlL (10.6 kcal/mol) is comparable to the enthalpic barriers typical for ring flips and disulfide isomerizations at ambient temperatures. This suggests that the sequence of the structural changes, as exemplified in various TlL crystal structures, mirror those that may occur during interfacial activation.

A simple and versatile method for the preparation of functional enzyme-gold nanoparticle conjugates using "click" chemistry has been developed. In a copper-catalyzed 1,2,3-triazole cycloaddition, an acetylene-functionalized Thermomyces lanuginosus lipase has been attached to azide-functionalized water-soluble gold nanoparticles under retention of enzymatic activity. The products have been characterized by gel electrophoresis and a fluorometric lipase activity assay. It is estimated that the equivalent of approximately seven fully active lipase molecules are attached to each nanoparticle.

Phospholipids are present in all living organisms. They are a major component of all biological membranes, along with glycolipids and cholesterol. Enzymes aimed at modifying phospholipids, namely, phospholipases, are consequently widespread in nature, playing very diverse roles from aggression in snake venom to signal transduction and digestion in humans. In this review, we give a general overview of phospholipases A1, A2, C and D from a sequence and structural perspective and their industrial application. The use of phospholipases in industrial processes has grown hand-in-hand with our ability to clone and express the genes in microbial hosts with commercially attractive amounts. Further, the use in industrial processes is increasing by optimizing the enzymes by protein engineering. Here, we give a perspective on the work done to date to express phospholipases in heterologous hosts and the efforts to optimize them by protein engineering. We will draw attention to the industrial processes where phospholipases play a key role and show how the use of a phospholipase for oil degumming leads to substantial environmental benefits. This illustrates a very general trend: the use of enzymes as an alternative to chemical processes to make products often provides a cleaner solution for the industrial processes. In a world with great demands on non-polluting, energy saving technical solutions--white biotechnology is a strong alternative.

The crystal structure of a novel esterase from Streptomyces scabies, a causal agent of the potato scab disease, was solved at 2.1 A resolution. The tertiary fold of the enzyme is substantially different from that of the alpha/beta hydrolase family and unique among all known hydrolases. The active site contains a dyad of Ser 14 and His 283, closely resembling two of the three components of typical Ser-His-Asp(Glu) triads from other serine hydrolases. Proper orientation of the active site imidazol is maintained by a hydrogen bond between the N delta-H group and a main chain oxygen. Thus, the enzyme constitutes the first known natural variation of the chymotrypsin-like triad in which a carboxylic acid is replaced by a neutral hydrogen-bond acceptor.

The best of both worlds. Long molecular dynamics (MD) simulations of Candida antarctica lipase B (CALB) confirmed the function of helix alpha5 as a lid structure. Replacement of the helix with corresponding lid regions from CALB homologues from Neurospora crassa and Gibberella zeae resulted in new CALB chimeras with novel biocatalytic properties. The figure shows a snapshot from the MD simulation. The Candida antarctica lipase B (CALB) has found very extensive use in biocatalysis reactions. Long molecular dynamics simulations of CALB in explicit aqueous solvent confirmed the high mobility of the regions lining the channel that leads into the active site, in particular, of helices alpha5 and alpha10. The simulation also confirmed the function of helix alpha5 as a lid of the lipase. Replacing it with corresponding lid regions from the CALB homologues from Neurospora crassa and Gibberella zeae resulted in two new CALB mutants. Characterization of these revealed several interesting properties, including increased hydrolytic activity on simple esters, specifically substrates with C(alpha) branching on the carboxylic side, and much increased enantioselectivity in hydrolysis of racemic ethyl 2-phenylpropanoate (E>50), which is a common structure of the profen drug family.