A multifaceted approach is adopted to characterize EstA from Aspergillus niger (Bourne et al., 2004). Collectively, biophysical, bioinformatic, and biochemical analyses identify EstA as the lead member of a new class of fungal esterases within the superfamily of alpha/beta-hydrolases.
Title: Structure and conformational flexibility of Candida rugosa lipase Cygler M, Schrag JD Ref: Biochimica & Biophysica Acta, 1441:205, 1999 : PubMed
Three-dimensional structures of a number of lipases determined in the past decade have provided a solid structural foundation for our understanding of lipase function. The structural studies of Candida rugosa lipase summarized here have addressed many facets of interfacial catalysis. These studies have revealed a fold and catalytic site common to other lipases. Different conformations likely to correlate with interfacial activation of the enzyme were observed in different crystal forms. The structures of enzyme-inhibitor complexes have identified the binding site for the scissile fatty acyl chain, provided the basis for molecular modeling of triglyceride binding and provided insight into the structural basis of the common enantiopreferences shown by lipases.
Title: Structure as basis for understanding interfacial properties of lipases Cygler M, Schrag JD Ref: Methods Enzymol, 284:3, 1997 : PubMed
Title: High-level production of recombinant Geotrichum candidum lipases in yeast Pichia pastoris Holmquist M, Tessier DC, Cygler M Ref: Protein Expr Purif, 11:35, 1997 : PubMed
We describe the heterologous high-level expression of the two Geotrichum candidum lipase (GCL) isoenzymes from strain ATCC 34614 in the methylotrophic yeast Pichia pastoris. The lipase cDNAs were placed under the control of the methanol-inducible alcohol oxidase promoter. The lipases expressed in P. pastoris were fused to the alpha-factor secretion signal peptide of Saccharomyces cerevisiae and were secreted into the culture medium. Cultures of P. pastoris expressing lipase accumulated active recombinant enzyme in the supernatant to levels of approximately 60 mg/L virtually free from contaminating proteins. This yield exceeds that previously reported with S. cerevisiae by a factor of more than 60. Recombinant GCL I and GCL II had molecular masses of approximately 63 and approximately 66 kDa, respectively, as determined by SDS-PAGE. The result of endoglucosidase H digestion followed by Western blot analysis of the lipases suggested that the enzymes expressed in P. pastoris received N-linked high-mannose-type glycosylation to an extent, 6-8% (w/w), similar to that in G. candidum. The specific activities and substrate specificities of both recombinant lipases were determined and were found to agree with what has been reported for the enzymes isolated from the native source.
Title: Lipases and alpha/beta hydrolase fold Schrag JD, Cygler M Ref: Methods Enzymol, 284:85, 1997 : PubMed
The three-dimensional structures of more than 20 representatives of the / hydrolase fold are now known and many more members have been identified by sequence and secondary structure comparisons. The fold is proving to be a common and stable way to assemble a wide variety of catalytic activities. With emphasis on lipases, this chapter reviews the features of this fold and the resources used to identify similarities in the rapidly growing number of enzymes and proteins that share this fold. The enzymes in this fold family include peroxidases, proteases, lipases, esterases, dehalogenases, and epoxide hydrolases. This fold is versatile in terms of the identities of catalytic residues and in their locations. The amino acids thus far observed as catalytic nucleophiles are serine, cysteine, and aspartate and both glutamate and aspartate have been observed as the catalytic acid. Although the acid is generally located after strand beta7, functional triads can also be constructed with the acid located after strand beta6. This fold family is also known to include proteins with no catalytic activity.
BACKGROUND:
The interfacial activation of lipases results primarily from conformational changes in the enzymes which expose the active site and provide a hydrophobic surface for interaction with the lipid substrate. Comparison of the crystallization conditions used and the structures observed for a variety of lipases suggests that the enzyme conformation is dependent on solution conditions. Pseudomonas cepacia lipase (PCL) was crystallized in conditions from which the open, active conformation of the enzyme was expected. Its three-dimensional structure was determined independently in three different laboratories and was compared with the previously reported closed conformations of the closely related lipases from Pseudomonas glumae (PGL) and Chromobacterium viscosum (CVL). These structures provide new insights into the function of this commercially important family of lipases.
RESULTS:
The three independent structures of PCL superimpose with only small differences in the mainchain conformations. As expected, the observed conformation reveals a catalytic site exposed to the solvent. Superposition of PCL with the PGL and CVL structures indicates that the rearrangement from the closed to the open conformation involves three loops. The largest movement involves a 40 residue stretch, within which a helical segment moves to afford access to the catalytic site. A hydrophobic cleft that is presumed to be the lipid binding site is formed around the active site.
CONCLUSIONS:
The interfacial activation of Pseudomonas lipases involves conformational rearrangements of surface loops and appears to conform to models of activation deduced from the structures of fungal and mammalian lipases. Factors controlling the conformational rearrangement are not understood, but a comparison of crystallization conditions and observed conformation suggests that the conformation of the protein is determined by the solution conditions, perhaps by the dielectric constant.
Title: Crystal structure of Kex1deltap, a prohormone-processing carboxypeptidase from Saccharomyces cerevisiae Shilton BH, Thomas DY, Cygler M Ref: Biochemistry, 36:9002, 1997 : PubMed
Kex1p is a prohormone-processing serine carboxypeptidase found in Saccharomyces cerevisiae. In contrast to yeast serine carboxypeptidase (CPD-Y) and wheat serine carboxypeptidase II (CPDW-II), Kex1p displays a very narrow specificity for lysyl or arginyl residues at the C-terminus of the substrate. The structure of Kex1Deltap, an enzyme that lacks the acidic domain and membrane-spanning portion of Kex1p, has been solved by a combination of molecular replacement and multiple isomorphous replacement and refined to a resolution of 2.4 A. The S1' site of Kex1Deltap is sterically restricted compared to those from CPD-Y or CPDW-II; it also contains two acidic groups that are well positioned to interact with the basic group of a lysine or arginine side chain. The high specificity of Kex1p can therefore be explained by a combination of steric and electronic factors. The structure of the S1 site of Kex1Deltap is also well suited for binding of a lysine or arginine side chain, and the enzyme may therefore exhibit a preference for these residues at P1.
A lipase was isolated from Penicillium sp. strain UZLM-4 and characterized. This lipase has a molecular weight of 27,344 (determined by mass spectrometry) and hydrolyzes triglycerides in preference to mono- and diglyceride substrates. Among various triglyceride substrates, tributyrin is hydrolyzed about four times faster than any other tested. The lipase has a preference for hydrolysis at the 1,3 positions of the lipids and shows a weak stereoselectivity for the S enantiomer. Unlike most other lipases, this lipase is stable and has a high activity at low surface pressures (5-10 mN/m).
Title: Crystallization of a soluble form of the Kex1p serine carboxypeptidase from Saccharomyces cerevisiae Shilton BH, Li Y, Tessier D, Thomas DY, Cygler M Ref: Protein Science, 5:395, 1996 : PubMed
A soluble form of the killer factor and prohormone-processing carboxypeptidase, "Kex1 delta p," from Saccharomyces cerevisiae, has been crystallized in 17-22% poly(enthylene glycol) methyl ether (average M(r) = 5,000), 100 mM ammonium acetate, 5% glycerol, pH 6.5, at 20 degrees C. A native data set (2.8 A resolution) and four derivative data sets (3.0-3.2 A resolution) were collected at the Photon Factory (lambda = 1.0 A). The crystals belong to space group P2(1)2(1)2(1) with a =56.6 A, b = 84.0 A, c = 111.8 A. Freezing a Kex1 delta p crystal has facilitated the collection of a 2.4-A data set using a rotating anode source (lambda = 1.5418 A). Molecular replacement models have been built based on the structures of wheat serine carboxypeptidase (CPDW-II; Liao DI et al., 1992, Biochemistry 31:9796-9812) and yeast carboxypeptidase Y.
Title: Expression and characterization of Geotrichum candidum lipase I gene. Comparison of specificity profile with lipase II Bertolini MC, Schrag JD, Cygler M, Ziomek E, Thomas DY, Vernet T Ref: European Journal of Biochemistry, 228:863, 1995 : PubMed
Despite tremendous progress in the elucidation of three-dimensional structures of lipases, the molecular basis for their observed substrate preference is not well understood. In an effort to correlate the lipase structure with its substrate preference and to clarify the contradicting reports in the literature, we have compared the enzymic characteristics of two closely related recombinant lipases from the fungus Geotrichum candidum. These enzymes were expressed in the yeast Saccharomyces cerevisiae as fusions with an N-terminal poly(His) tag and were purified in a single step by metal-affinity chromatography. Their specific activities against a series of triacylglycerol substrates were compared using a titrimetric assay. The substrates varied in fatty acyl chain length, number of double bonds and their position along the chain. G. candidum lipases I and II (GCL I and GLC II) are markedly different with respect to their substrate preferences. For unsaturated substrates having long fatty acyl chains (C18:2 cis-9, cis-12 and C18:3 cis-9, cis-12, cis-15), GCL I showed higher specific activity than GCL II, whereas GCL II showed higher specific activity against saturated substrates having short fatty acid chains (C8, C10, C12 and C14). We have constructed a hybrid molecule containing the N-terminal portion of GCL I (including the flap covering the active site) linked to the C-terminal portion of GCL II. The hybrid molecule showed a substrate preference pattern identical to that of GCL II. These results indicate that sequence variation within the N-terminal 194 amino acids of G. candidum lipases do not contribute to the observed variation in efficiency by which the lipases hydrolyze their substrates. Moreover, it also shows that the flap region in GCL is not directly involved in substrate differentiation, even though this region is thought to be involved in recognition of the interface and in the activation of the enzyme.
Title: Structural determinants defining common stereoselectivity of lipases toward secondary alcohols Cygler M, Grochulski P, Schrag JD Ref: Can J Microbiol, 41 Suppl 1:289, 1995 : PubMed
In this review we summarize some aspects of the enantiopreference of the lipase from Candida rugosa following structural analysis of complexes of this lipase with two enantiomers of an analog of a tetrahedral intermediate in the hydrolysis of simple esters. The analysis of the molecular basis of the enantiomeric differentiation suggests that these results can be generalized to a large class of lipases and esterases. We also summarize our experiments on identification of the key regions in the lipases from Geotrichum candidum lipase responsible for differentiation between fatty acyl chains.
Title: Substrate Binding Site and the Role of the FLAP Loop in Candida rugosa Lipase, A Close Relative of Acetylcholinesterase Cygler M, Grochulski P, Schrag JD Ref: In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases, (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp.:71, 1995 : PubMed
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.
The fungus Geotrichum candidum produces extracellular lipases. Purification and characterization of different lipase isoforms from various G. candidum strains is difficult due to the close physical and biochemical properties of the isoforms. Consequently, the characterization of these enzymes and their substrate specificities has been difficult. We have determined the lipase genes present in four strains of G. candidum (ATCC 34614, NRCC 205002, NRRL Y-552 and NRRL Y-553) by molecular cloning and DNA sequencing. Each strain contains two genes similar to the previously identified lipase I and lipase II cDNAs. Our data suggest that no other related lipase genes are present in these strains. Each lipase-gene family shows sequence variation (polymorphism) that is confirmed by Southern-blot analysis. This polymorphism and the sequence differences between lipase I and lipase II have been localized within the previously determined three-dimensional structure of lipase II. Although most of the amino acid substitutions are located on the protein surface, some are present in structural features possibly involved in determining substrate specificity.
The structures of Candida rugosa lipase-inhibitor complexes demonstrate that the scissile fatty acyl chain is bound in a narrow, hydrophobic tunnel which is unique among lipases studied to date. Modeling of triglyceride binding suggests that the bound lipid must adopt a "tuning fork" conformation. The complexes, analogs of tetrahedral intermediates of the acylation and deacylation steps of the reaction pathway, localize the components of the oxyanion hole and define the stereochemistry of ester hydrolysis. Comparison with other lipases suggests that the positioning of the scissile fatty acyl chain and ester bond and the stereochemistry of hydrolysis are the same in all lipases which share the alpha/beta-hydrolase fold.
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 do 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.
Based on the recently determined X-ray structures of Torpedo californica acetylcholinesterase and Geotrichum candidum lipase and on their three-dimensional superposition, an improved alignment of a collection of 32 related amino acid sequences of other esterases, lipases, and related proteins was obtained. On the basis of this alignment, 24 residues are found to be invariant in 29 sequences of hydrolytic enzymes, and an additional 49 are well conserved. The conservation in the three remaining sequences is somewhat lower. The conserved residues include the active site, disulfide bridges, salt bridges, and residues in the core of the proteins. Most invariant residues are located at the edges of secondary structural elements. A clear structural basis for the preservation of many of these residues can be determined from comparison of the two X-ray structures.
Title: Enzymes. Snapshots along the pathway [news; comment] Cygler M Ref: Nature, 363:674, 1993 : PubMed
The structure of the Candida rugosa lipase determined at 2.06-A resolution reveals a conformation with a solvent-accessible active site. Comparison with the crystal structure of the homologous lipase from Geotrichum candidum, in which the active site is covered by surface loops and is inaccessible from the solvent, shows that the largest structural differences occur in the vicinity of the active site. Three loops in this region differ significantly in conformation, and the interfacial activation of these lipases is likely to be associated with conformational rearrangements of these loops. The "open" structure provides a new image of the substrate binding region and active site access, which is different from that inferred from the structure of the "closed" form of the G. candidum lipase.
Title: 1.8 A refined structure of the lipase from Geotrichum candidum Schrag JD, Cygler M Ref: Journal of Molecular Biology, 230:575, 1993 : PubMed
A lipase from the fungus Geotrichum candidum is one of only three interfacially activated lipases whose structures have been reported to date. We have previously reported the partially refined 2.2 A structure of this enzyme. We have subsequently extended the resolution and here report the fully refined 1.8 A structure of this lipase. The structure observed in the crystal is apparently not the lipolytic conformation, as the active site is not accessible from the surface of the molecule. A single large cavity is found in the interior of the molecule and extends from the catalytic Ser to two surface helices, suggesting that this face may be the region that interacts with the lipid interface. The mobility of local segments on this face is indicated by temperature factors larger than elsewhere in the molecule and by the observation of several residues whose side-chains are discretely disordered. These observations strongly suggest that this portion of the molecule is involved in interfacial and substrate binding, but the exact nature of the conformational changes induced by binding to the lipid interface can not be determined.
The three-dimensional structure of lipase II of Geotrichum candidum strain ATCC34614 (GCL II) has provided insights with respect to the nature of the catalytic machinery of lipases. To support these structural observations, we have carried out an analysis of GCL II by mutagenesis. The gene encoding lipase II of Geotrichum candidum strain ATCC34614 (GCL II) was amplified using the polymerase chain reaction, cloned, and sequenced. The intronless lipase gene was expressed and secreted from Saccharomyces cerevisiae at approximately 5 mg/liter of culture. Recombinant GCL II was purified by immunoaffinity chromatography and characterized using a combination of substrates and independent analytical methods. The recombinant enzyme and the enzyme isolated from its natural source have comparable specific activities against triolein of about 1000 mumol of oleic acid released/min/mg of protein. The putative catalytic triad Ser217-His463-Glu354 was probed by site-directed mutagenesis. The substitution of Ser217 by either Cys or Thr and of His463 by Ala led to a complete elimination of the activity against both triolein and tributyrin. Substitution of Glu354 by either Ser, Ala or Gln renders the enzyme inactive and also perturbs the enzyme stability. However, the enzyme with the conservative replacement Glu354 Asp is stable and displays only a small decrease of triolein activity but a 10-fold decrease in activity against tributyrin. There was no appreciable difference in esterase activity between the native, recombinant wild type, and Glu354 Asp mutant. These results confirm that the triad formed by Ser217-Glu354-His463 is essential for catalytic activity. They also show that the active site of GCL II is more tolerant to a conservative change of the carboxylic side chain within the triad than are other hydrolases with similar catalytic triads.
Title: Advances in structural understanding of lipases Cygler M, Schrag JD, Ergan F Ref: Biotechnol Genet Eng Rev, 10:143, 1992 : PubMed
Title: Sequence Alignment of Esterases and Lipases Based on 3-D Structures of Two Members of This Family Cygler M, Schrag JD Ref: In Multidisciplinary approaches to cholinesterase functions - Proceedings of Fourth International Meeting on Cholinesterases, (Shafferman, A. and Velan, B., Eds) Plenum Press, New York:109, 1992 : PubMed
We have identified a new protein fold--the alpha/beta hydrolase fold--that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta sheet, not barrel, of eight beta-sheets connected by alpha-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the alpha/beta hydrolase fold enzymes.
Title: Pancreatic lipases: evolutionary intermediates in a positional change of catalytic carboxylates? Schrag JD, Winkler FK, Cygler M Ref: Journal of Biological Chemistry, 267:4300, 1992 : PubMed
Comparison of the fold of lipases from Geotrichum candidum and from human pancreas identified a high degree of similarity which was not expected on the basis of their amino acid sequences. Although both enzymes utilize a serine protease-like catalytic triad, they differ in the topological position of the acid. We speculate that these proteins are evolutionarily related and that the pancreatic lipase is an evolutionary intermediate in the pathway of migration of the catalytic acid to a new position within the fold.
Title: Ser-His-Glu triad forms the catalytic site of the lipase from Geotrichum candidum Schrag JD, Li YG, Wu S, Cygler M Ref: Nature, 351:761, 1991 : PubMed
The Ser-His-Asp triad is a well known structural feature of the serine proteases. It has also been directly observed in the catalytic sites of two lipases, whose high-resolution three-dimensional structures have been determined 1,2. Lipases show a wide variety of sizes, substrate and positional specificities, and catalytic rates 3. They achieve maximal catalytic rates at oil-water interfaces. The fungus Geotrichum candidum produces several different forms of lipases, two of which have been purified to homogeneity 4,5. Two lipase genes have been identified, cloned and sequenced 6,7. Both code for proteins of 544 amino acids with a total relative molecular mass of about 60,000 (Mr 60K). The two forms are 86% identical. Their isoelectric points differ slightly, being between 4.3 and 4.6. About 7% of the total Mr is carbohydrate. Until now, only a low resolution structure of GCL has been reported 8, but no high resolution structure has followed. We now report the three-dimensional structure of a lipase from G. candidum (GCL) at 2.2 A resolution. Unlike the other lipases and serine proteases, the catalytic triad of GCL is Ser-His-Glu, with glutamic acid replacing the usual aspartate. Although the sequence similarity with the other two lipases is limited to the region near the active-site serine, there is some similarity in their three-dimensional structures. The GCL is also an alpha/beta protein with a central mixed beta sheet whose topology is similar to that of the N-terminal domain of human pancreatic lipase. As in the other lipases 1,2, the catalytic site is buried under surface loops. Sequence comparisons with proteins from the cholinesterase family suggest that they also contain the Ser-His-Glu triad.
