Schrag JD


Full name : Schrag Joseph D

First name : Joseph D

Mail : National Research Council Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, Quebec, H4P 2R2

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References (28)

Title : Different active-site loop orientation in serine hydrolases versus acyltransferases - Jiang_2011_Chembiochem_12_768
Author(s) : Jiang Y , Morley KL , Schrag JD , Kazlauskas RJ
Ref : Chembiochem , 12 :768 , 2011
Abstract : Acyl transfer is a key reaction in biosynthesis, including synthesis of antibiotics and polyesters. Although researchers have long recognized the similar protein fold and catalytic machinery in acyltransferases and hydrolases, the molecular basis for the different reactivity has been a long-standing mystery. By comparison of X-ray structures, we identified a different oxyanion-loop orientation in the active site. In esterases/lipases a carbonyl oxygen points toward the active site, whereas in acyltransferases a NH of the main-chain amide points toward the active site. Amino acid sequence comparisons alone cannot identify such a difference in the main-chain orientation. To identify how this difference might change the reaction mechanism, we solved the X-ray crystal structure of Pseudomonas fluorescens esterase containing a sulfonate transition-state analogue bound to the active-site serine. This structure mimics the transition state for the attack of water on the acyl-enzyme and shows a bridging water molecule between the carbonyl oxygen mentioned above and the sulfonyl oxygen that mimics the attacking water. A possible mechanistic role for this bridging water molecule is to position and activate the attacking water molecule in hydrolases, but to deactivate the attacking water molecule in acyl transferases.
ESTHER : Jiang_2011_Chembiochem_12_768
PubMedSearch : Jiang_2011_Chembiochem_12_768
PubMedID: 21351219
Gene_locus related to this paper: psefl-este

Title : Switching catalysis from hydrolysis to perhydrolysis in Pseudomonas fluorescens esterase - Yin_2010_Biochemistry_49_1931
Author(s) : Yin DL , Bernhardt P , Morley KL , Jiang Y , Cheeseman JD , Purpero V , Schrag JD , Kazlauskas RJ
Ref : Biochemistry , 49 :1931 , 2010
Abstract : Many serine hydrolases catalyze perhydrolysis, the reversible formation of peracids from carboxylic acids and hydrogen peroxide. Recently, we showed that a single amino acid substitution in the alcohol binding pocket, L29P, in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation >100-fold [Bernhardt et al. (2005) Angew. Chem., Int. Ed. 44, 2742]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two X-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of epsilon-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction, hydrolysis of peracetic acid to acetic acid and hydrogen peroxide, occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can the forward reaction. Consistent with this prediction, two variants with additional amino acid substitutions showed 2-fold higher k(cat), but K(m) also increased so the specificity constant, k(cat)/K(m), remained similar. Third, how does the L29P substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate) but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of epsilon-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the active site blocks access for larger alcohol moieties but binds epsilon-caprolactone more tightly. These results are consistent with the natural function of perhydrolases being either hydrolysis of peroxycarboxylic acids or hydrolysis of lactones.
ESTHER : Yin_2010_Biochemistry_49_1931
PubMedSearch : Yin_2010_Biochemistry_49_1931
PubMedID: 20112920
Gene_locus related to this paper: psefl-este

Title : Mirror-image packing in enantiomer discrimination molecular basis for the enantioselectivity of B.cepacia lipase toward 2-methyl-3-phenyl-1-propanol - Mezzetti_2005_Chem.Biol_12_427
Author(s) : Mezzetti A , Schrag JD , Cheong CS , Kazlauskas RJ
Ref : Chemical Biology , 12 :427 , 2005
Abstract : Synthetic chemists often exploit the high enantioselectivity of lipases to prepare pure enantiomers of primary alcohols, but the molecular basis for this enantioselectivity is unknown. The crystal structures of two phosphonate transition-state analogs bound to Burkholderia cepacia lipase reveal this molecular basis for a typical primary alcohol: 2-methyl-3-phenyl-1-propanol. The enantiomeric alcohol moieties adopt surprisingly similar orientations, with only subtle differences that make it difficult to predict how to alter enantioselectivity. These structures, along with a survey of previous structures of enzyme bound enantiomers, reveal that binding of enantiomers does not involve an exchange of two substituent positions as most researchers assumed. Instead, the enantiomers adopt mirror-image packing, where three of the four substituents at the stereocenter lie in similar positions. The fourth substituent, hydrogen, points in opposite directions.
ESTHER : Mezzetti_2005_Chem.Biol_12_427
PubMedSearch : Mezzetti_2005_Chem.Biol_12_427
PubMedID: 15850979
Gene_locus related to this paper: burce-lipaa

Title : Structure of an aryl esterase from Pseudomonas fluorescens - Cheeseman_2004_Acta.Crystallogr.D.Biol.Crystallogr_60_1237
Author(s) : Cheeseman JD , Tocilj A , Park S , Schrag JD , Kazlauskas RJ
Ref : Acta Crystallographica D Biol Crystallogr , 60 :1237 , 2004
Abstract : The structure of PFE, an aryl esterase from Pseudomonas fluorescens, has been solved to a resolution of 1.8 A by X-ray diffraction and shows a characteristic alpha/beta-hydrolase fold. In addition to catalyzing the hydrolysis of esters in vitro, PFE also shows low bromoperoxidase activity. PFE shows highest structural similarity, including the active-site environment, to a family of non-heme bacterial haloperoxidases, with an r.m.s. deviation in 271 C(alpha) atoms between PFE and its five closest structural neighbors averaging 0.8 A. PFE has far less similarity (r.m.s. deviation in 218 C(alpha) atoms of 5.0 A) to P. fluorescens carboxyl esterase. PFE favors activated esters with small acyl groups, such as phenyl acetate. The X-ray structure of PFE reveals a significantly occluded active site. In addition, several residues, including Trp28 and Met95, limit the size of the acyl-binding pocket, explaining its preference for small acyl groups.
ESTHER : Cheeseman_2004_Acta.Crystallogr.D.Biol.Crystallogr_60_1237
PubMedSearch : Cheeseman_2004_Acta.Crystallogr.D.Biol.Crystallogr_60_1237
PubMedID: 15213385
Gene_locus related to this paper: psefl-este

Title : Defining substrate characteristics from 3D structure\; perspective on EstA structure - Schrag_2004_Structure.(Camb)_12_521
Author(s) : Schrag JD , Cygler M
Ref : Structure , 12 :521 , 2004
Abstract : 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.
ESTHER : Schrag_2004_Structure.(Camb)_12_521
PubMedSearch : Schrag_2004_Structure.(Camb)_12_521
PubMedID: 15062074
Gene_locus related to this paper: aspni-EstA

Title : Structure and conformational flexibility of Candida rugosa lipase - Cygler_1999_Biochim.Biophys.Acta_1441_205
Author(s) : Cygler M , Schrag JD
Ref : Biochimica & Biophysica Acta , 1441 :205 , 1999
Abstract : 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.
ESTHER : Cygler_1999_Biochim.Biophys.Acta_1441_205
PubMedSearch : Cygler_1999_Biochim.Biophys.Acta_1441_205
PubMedID: 10570248

Title : Quaternary Structure of Tetrameric Acetylcholinesterase -
Author(s) : Raves ML , Giles K , Schrag JD , Schmid MF , Phillips JN, Jr. , Chiu W , Howard AJ , Silman I , Sussman JL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :351 , 1998
Gene_locus related to this paper: eleel-ACHE

Title : Structure as basis for understanding interfacial properties of lipases -
Author(s) : Cygler M , Schrag JD
Ref : Methods Enzymol , 284 :3 , 1997
PubMedID: 9379943

Title : Lipases and alpha\/beta hydrolase fold - Schrag_1997_Methods.Enzymol_284_85
Author(s) : Schrag JD , Cygler M
Ref : Methods Enzymol , 284 :85 , 1997
Abstract : 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.
ESTHER : Schrag_1997_Methods.Enzymol_284_85
PubMedSearch : Schrag_1997_Methods.Enzymol_284_85
PubMedID: 9379946

Title : The open conformation of a Pseudomonas lipase - Schrag_1997_Structure_5_187
Author(s) : Schrag JD , Li Y , Cygler M , Lang D , Burgdorf T , Hecht HJ , Schmid RD , Schomburg D , Rydel TJ , Oliver JD , Strickland LC , Dunaway CM , Larson SB , Day J , McPherson A
Ref : Structure , 5 :187 , 1997
Abstract : 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.
ESTHER : Schrag_1997_Structure_5_187
PubMedSearch : Schrag_1997_Structure_5_187
PubMedID: 9032074
Gene_locus related to this paper: burce-lipaa

Title : Purification and characterization of a Penicillium sp. lipase which discriminates against diglycerides - Gulomova_1996_Lipids_31_379
Author(s) : Gulomova K , Ziomek E , Schrag JD , Davranov K , Cygler M
Ref : Lipids , 31 :379 , 1996
Abstract : 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).
ESTHER : Gulomova_1996_Lipids_31_379
PubMedSearch : Gulomova_1996_Lipids_31_379
PubMedID: 8743049

Title : Structural determinants defining common stereoselectivity of lipases toward secondary alcohols - Cygler_1995_Can.J.Microbiol_41 Suppl 1_289
Author(s) : Cygler M , Grochulski P , Schrag JD
Ref : Can J Microbiol , 41 Suppl 1 :289 , 1995
Abstract : 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.
ESTHER : Cygler_1995_Can.J.Microbiol_41 Suppl 1_289
PubMedSearch : Cygler_1995_Can.J.Microbiol_41 Suppl 1_289
PubMedID: 7606666

Title : Redesigning the active site of Geotrichum candidum lipase - Schrag_1995_Protein.Eng_8_835
Author(s) : Schrag JD , Vernet T , Laramee L , Thomas DY , Recktenwald A , Okoniewska M , Ziomek E , Cygler M
Ref : Protein Engineering , 8 :835 , 1995
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.
ESTHER : Schrag_1995_Protein.Eng_8_835
PubMedSearch : Schrag_1995_Protein.Eng_8_835
PubMedID: 8637854

Title : Substrate Binding Site and the Role of the FLAP Loop in Candida rugosa Lipase, A Close Relative of Acetylcholinesterase -
Author(s) : 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

Title : Expression and characterization of Geotrichum candidum lipase I gene. Comparison of specificity profile with lipase II - Bertolini_1995_Eur.J.Biochem_228_863
Author(s) : Bertolini MC , Schrag JD , Cygler M , Ziomek E , Thomas DY , Vernet T
Ref : European Journal of Biochemistry , 228 :863 , 1995
Abstract : 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.
ESTHER : Bertolini_1995_Eur.J.Biochem_228_863
PubMedSearch : Bertolini_1995_Eur.J.Biochem_228_863
PubMedID: 7737187
Gene_locus related to this paper: geoca-1lipa , geoca-2lipa

Title : Polymorphism in the lipase genes of Geotrichum candidum strains - Bertolini_1994_Eur.J.Biochem_219_119
Author(s) : Bertolini MC , Laramee L , Thomas DY , Cygler M , Schrag JD , Vernet T
Ref : European Journal of Biochemistry , 219 :119 , 1994
Abstract : 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.
ESTHER : Bertolini_1994_Eur.J.Biochem_219_119
PubMedSearch : Bertolini_1994_Eur.J.Biochem_219_119
PubMedID: 8306978
Gene_locus related to this paper: geoca-1lipa , geoca-2lipa

Title : Two conformational states of Candida rugosa lipase - Grochulski_1994_Protein.Sci_3_82
Author(s) : Grochulski P , Li Y , Schrag JD , Cygler M
Ref : Protein Science , 3 :82 , 1994
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 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.
ESTHER : Grochulski_1994_Protein.Sci_3_82
PubMedSearch : Grochulski_1994_Protein.Sci_3_82
PubMedID: 8142901
Gene_locus related to this paper: canru-1lipa

Title : Analogs of reaction intermediates identify a unique substrate binding site in Candida rugosa lipase - Grochulski_1994_Biochemistry_33_3494
Author(s) : Grochulski P , Bouthillier F , Kazlauskas RJ , Serreqi AN , Schrag JD , Ziomek E , Cygler M
Ref : Biochemistry , 33 :3494 , 1994
Abstract : 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.
ESTHER : Grochulski_1994_Biochemistry_33_3494
PubMedSearch : Grochulski_1994_Biochemistry_33_3494
PubMedID: 8142346
Gene_locus related to this paper: canru-1lipa

Title : A structural basis for the chiral preferences of lipases -
Author(s) : Cygler M , Grochulski P , Kazlauskas RJ , Schrag JD , Bouthillier F , Rubin B , Serreqi AN , Gupta AK
Ref : Journal of the American Chemical Society , 116 :3180 , 1994
Gene_locus related to this paper: canru-1lipa

Title : Cloning and expression of Geotrichum candidum lipase II gene in yeast. Probing of the enzyme active site by site-directed mutagenesis - Vernet_1993_J.Biol.Chem_268_26212
Author(s) : Vernet T , Ziomek E , Recktenwald A , Schrag JD , de Montigny C , Tessier DC , Thomas DY , Cygler M
Ref : Journal of Biological Chemistry , 268 :26212 , 1993
Abstract : 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.
ESTHER : Vernet_1993_J.Biol.Chem_268_26212
PubMedSearch : Vernet_1993_J.Biol.Chem_268_26212
PubMedID: 7902836

Title : Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins - Cygler_1993_Protein.Sci_2_366
Author(s) : Cygler M , Schrag JD , Sussman JL , Harel M , Silman I , Gentry MK , Doctor BP
Ref : Protein Science , 2 :366 , 1993
Abstract : 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.
ESTHER : Cygler_1993_Protein.Sci_2_366
PubMedSearch : Cygler_1993_Protein.Sci_2_366
PubMedID: 8453375

Title : 1.8 A refined structure of the lipase from Geotrichum candidum - Schrag_1993_J.Mol.Biol_230_575
Author(s) : Schrag JD , Cygler M
Ref : Journal of Molecular Biology , 230 :575 , 1993
Abstract : 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.
ESTHER : Schrag_1993_J.Mol.Biol_230_575
PubMedSearch : Schrag_1993_J.Mol.Biol_230_575
PubMedID: 8464065
Gene_locus related to this paper: geoca-1lipa , geoca-2lipa

Title : Insights into interfacial activation from an open structure of Candida rugosa lipase - Grochulski_1993_J.Biol.Chem_268_12843
Author(s) : Grochulski P , Li Y , Schrag JD , Bouthillier F , Smith P , Harrison D , Rubin B , Cygler M
Ref : Journal of Biological Chemistry , 268 :12843 , 1993
Abstract : 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.
ESTHER : Grochulski_1993_J.Biol.Chem_268_12843
PubMedSearch : Grochulski_1993_J.Biol.Chem_268_12843
PubMedID: 8509417
Gene_locus related to this paper: canru-1lipa , geoca-1lipa

Title : Pancreatic lipases: evolutionary intermediates in a positional change of catalytic carboxylates? - Schrag_1992_J.Biol.Chem_267_4300
Author(s) : Schrag JD , Winkler FK , Cygler M
Ref : Journal of Biological Chemistry , 267 :4300 , 1992
Abstract : 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.
ESTHER : Schrag_1992_J.Biol.Chem_267_4300
PubMedSearch : Schrag_1992_J.Biol.Chem_267_4300
PubMedID: 1537823
Gene_locus related to this paper: geoca-1lipa

Title : The alpha\/beta hydrolase fold - Ollis_1992_Prot.Engin_5_197
Author(s) : Ollis DL , Cheah E , Cygler M , Dijkstra B , Frolow F , Franken SM , Harel M , Remington SJ , Silman I , Schrag JD , Sussman JL , Verschueren KHG , Goldman A
Ref : Protein Engineering , 5 :197 , 1992
Abstract : 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.
ESTHER : Ollis_1992_Prot.Engin_5_197
PubMedSearch : Ollis_1992_Prot.Engin_5_197
PubMedID: 1409539

Title : Sequence Alignment of Esterases and Lipases Based on 3-D Structures of Two Members of This Family -
Author(s) : 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

Title : Advances in structural understanding of lipases -
Author(s) : Cygler M , Schrag JD , Ergan F
Ref : Biotechnol Genet Eng Rev , 10 :143 , 1992
PubMedID: 1301738

Title : Ser-His-Glu triad forms the catalytic site of the lipase from Geotrichum candidum - Schrag_1991_Nature_351_761
Author(s) : Schrag JD , Li YG , Wu S , Cygler M
Ref : Nature , 351 :761 , 1991
Abstract : 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.
ESTHER : Schrag_1991_Nature_351_761
PubMedSearch : Schrag_1991_Nature_351_761
PubMedID: 2062369
Gene_locus related to this paper: geoca-1lipa