SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2021955903> ?p ?o ?g. }

Showing items 1 to 100 of ±149 with 100 items per page.

W2021955903 endingPage "1441" @default.
W2021955903 startingPage "1432" @default.
W2021955903 abstract "The structure and dynamics of the enzyme-substrate complex of Bacillus 1,3–1,4-β-glucanase, one of the most active glycoside hydrolases, is investigated by means of Car-Parrinello molecular dynamics simulations (CPMD) combined with force field molecular dynamics (QM/MM CPMD). It is found that the substrate sugar ring located at the –1 subsite adopts a distorted 1S3 skew-boat conformation upon binding to the enzyme. With respect to the undistorted 4C1 chair conformation, the 1S3 skew-boat conformation is characterized by: (a) an increase of charge at the anomeric carbon (C1), (b) an increase of the distance between C1 and the leaving group, and (c) a decrease of the intraring O5-C1 distance. Therefore, our results clearly show that the distorted conformation resembles both structurally and electronically the transition state of the reaction in which the substrate acquires oxocarbenium ion character, and the glycosidic bond is partially broken. Together with analysis of the substrate conformational dynamics, it is concluded that the main determinants of substrate distortion have a structural origin. To fit into the binding pocket, it is necessary that the aglycon leaving group is oriented toward the β region, and the skew-boat conformation naturally fulfills this premise. Only when the aglycon is removed from the calculation the substrate recovers the all-chair conformation, in agreement with the recent determination of the enzyme product structure. The QM/MM protocol developed here is able to predict the conformational distortion of substrate binding in glycoside hydrolases because it accounts for polarization and charge reorganization at the –1 sugar ring. It thus provides a powerful tool to model E·S complexes for which experimental information is not yet available. The structure and dynamics of the enzyme-substrate complex of Bacillus 1,3–1,4-β-glucanase, one of the most active glycoside hydrolases, is investigated by means of Car-Parrinello molecular dynamics simulations (CPMD) combined with force field molecular dynamics (QM/MM CPMD). It is found that the substrate sugar ring located at the –1 subsite adopts a distorted 1S3 skew-boat conformation upon binding to the enzyme. With respect to the undistorted 4C1 chair conformation, the 1S3 skew-boat conformation is characterized by: (a) an increase of charge at the anomeric carbon (C1), (b) an increase of the distance between C1 and the leaving group, and (c) a decrease of the intraring O5-C1 distance. Therefore, our results clearly show that the distorted conformation resembles both structurally and electronically the transition state of the reaction in which the substrate acquires oxocarbenium ion character, and the glycosidic bond is partially broken. Together with analysis of the substrate conformational dynamics, it is concluded that the main determinants of substrate distortion have a structural origin. To fit into the binding pocket, it is necessary that the aglycon leaving group is oriented toward the β region, and the skew-boat conformation naturally fulfills this premise. Only when the aglycon is removed from the calculation the substrate recovers the all-chair conformation, in agreement with the recent determination of the enzyme product structure. The QM/MM protocol developed here is able to predict the conformational distortion of substrate binding in glycoside hydrolases because it accounts for polarization and charge reorganization at the –1 sugar ring. It thus provides a powerful tool to model E·S complexes for which experimental information is not yet available. Glycoside hydrolases (GHs) 2The abbreviations used are: GHsglycoside hydrolasesMUmethylumbelliferylCPMDCar-Parrinello molecular dynamicsQMquantum mechanicsMMmolecular mechanicsMDmolecular dynamicsCPCar-ParrinelloDFTdensity functional theoryglyglycosidica.u.atomic units. are the enzymes responsible for the hydrolysis of glycosidic bonds and play important biological functions such as glycan processing in glycoproteins, remodeling the cell walls, and polysaccharide modification and degradation. The reaction mechanism, a classical textbook example of enzymatic reaction, has attracted much interest because genetically inherited disorders of glycoside hydrolysis often occur and because inhibitors of these enzymes can act as new therapeutic agents for the treatment of viral infections (1Davies G. Henrissat B. Structure. 1995; 3: 853-859Abstract Full Text Full Text PDF PubMed Scopus (1628) Google Scholar, 2Hurtley S. Service R. Szuromi P. Science. 291. 2001: 2263-2502Google Scholar). Despite the large number of GHs known, classified into more than 90 families (1Davies G. Henrissat B. Structure. 1995; 3: 853-859Abstract Full Text Full Text PDF PubMed Scopus (1628) Google Scholar), the catalytic mechanism is similar. They typically operate by means of acid/base catalysis with retention or inversion of the anomeric configuration, although a different mechanism has recently been proposed for the GH family 4 (3Yip V.L.Y. Varrot A. Davies G.J. Rajan S.S. Yang X. Thompson J. Anderson W.F. Withers S.G. J. Am. Chem. Soc. 2004; 126: 8354-8355Crossref PubMed Scopus (110) Google Scholar). The acid/base reaction is assisted by two essential residues: a proton donor and a nucleophile or general base residue (4White A. Rose D.R. Curr. Opin. Struct. Biol. 1997; 7: 645-651Crossref PubMed Scopus (130) Google Scholar). Inverting enzymes operate by a single nucleophilic substitution, whereas retaining glycosidases follow a double displacement mechanism via formation and hydrolysis of a covalent glycosyl-enzyme intermediate. Both steps involve oxocarbenium ion-like transition states (Fig. 1). A current issue in the understanding of GH mechanisms is the conformational itinerary that the substrate follows during the reaction (5Davies G.J. Ducros V.M.-A. Varrot A. Zechel D.L. Biochem. Soc. Trans. 2003; 31: 523-527Crossref PubMed Google Scholar, 6Taylor E.J. Goyal A. Guerreiro C.I.P.D. Prates J.A.M. Money V. Ferry N. Morland C. Planas A. Macdonald J.A. Stick R.V. Gilbert H.J. Fontes C.M.G.A. Davies G.J. J. Biol. Chem. 2005; 280: 32761-32767Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), in which substrate distortion is induced upon binding to the enzyme to reach a transition state with sp 2The abbreviations used are: GHsglycoside hydrolasesMUmethylumbelliferylCPMDCar-Parrinello molecular dynamicsQMquantum mechanicsMMmolecular mechanicsMDmolecular dynamicsCPCar-ParrinelloDFTdensity functional theoryglyglycosidica.u.atomic units. geometry at the anomeric carbon. glycoside hydrolases methylumbelliferyl Car-Parrinello molecular dynamics quantum mechanics molecular mechanics molecular dynamics Car-Parrinello density functional theory glycosidic atomic units. glycoside hydrolases methylumbelliferyl Car-Parrinello molecular dynamics quantum mechanics molecular mechanics molecular dynamics Car-Parrinello density functional theory glycosidic atomic units. Bacterial 1,3–1,4-β-glucanases are highly active retaining endoglycosidases (7Planas A. Biochim. Biochim. Acta. 2000; 1543: 361-382Crossref PubMed Scopus (236) Google Scholar) belonging to family 16 retaining glycoside hydrolases (7Planas A. Biochim. Biochim. Acta. 2000; 1543: 361-382Crossref PubMed Scopus (236) Google Scholar, 8Coutinho P.M. Henrissat B. Gilbert H.J. Davies G.J. Henrissat B. Svensson B. Recent Advances in Carbohydrate Bioengineering. The Royal Society of Chemistry, Cambridge1999Google Scholar). These enzymes act on linear β-glucans containing β-1,3 and β-1,4 linkages such as cereal β-glucans and lichenan, with a strict cleavage specificity for β-1,4 glycosidic bonds on 3-O-substituted glucosyl residues. Two Glu residues act as nucleophile and general acid/base catalyst, respectively (9Viladot J.L. de Ramon E. Durany O. Planas A. Biochemistry. 1998; 37: 11332-11342Crossref PubMed Scopus (92) Google Scholar). The Michaelis complex of neither 1,3–1,4-β-glucanases nor another member of the GH 16 enzymes has yet to be characterized. However, during the last decade a growing number of crystallographic studies on retaining β-glycoside hydrolases have shown that the substrate binds to the enzyme in a distorted conformation (5Davies G.J. Ducros V.M.-A. Varrot A. Zechel D.L. Biochem. Soc. Trans. 2003; 31: 523-527Crossref PubMed Google Scholar). In particular, the saccharide unit binding at subsite 3Subsite is defined as the group of amino acid residues in the enzyme binding site that interacts with a single monosaccharyl unit of the oligo or polysaccharide substrate. The scissile glycosidic bond is in-between subsites –1 and +1, and subsites are numbered –1, –2, ···, –n towards the non-reducing end of the bound saccharide, and +1, +2, ···, +n on the reducing end site (20Davies G.J. Wilson K.S. Henrissat B. Biochem. J. 1997; 321: 557-559Crossref PubMed Scopus (852) Google Scholar). We will use the abbreviation “–1 sugar ring” to refer to the saccharide unit binding at subsite –1. –1 is found to adopt a boat (1,4B) or skew-boat (1S3 or 1S5) type conformation instead of the relaxed 4C1 chair conformation (Fig. 2a). Ring-distorted conformations have been observed in the complex of endoglucanase I from Fusarium oxysporum with a non-hydrolyzable inhibitor (10Sulzenbacher G. Driguez H. Henrissat B. Schulein M. Davies G.J. Biochemistry. 1996; 35: 15280-15287Crossref PubMed Scopus (228) Google Scholar), as well as cellulase Cel5A from Bacillus agaradhaerens (11Davies G.J. Mackenzie L. Varrot A. Dauter M.m Brzozowski A.M. Schülein M. Withers S.G. Biochemistry. 1998; 37: 11707-11713Crossref PubMed Scopus (240) Google Scholar), chitobiase from Serratia marcescens (12Tews I. Perrakis A. Oppenheim A. Dauter Z. Wilson K.S. Vorgias C.E. Nat. Struct. Biol. 1996; 3: 638-648Crossref PubMed Scopus (328) Google Scholar), chitinases (13Tews I. Vanscheltinga A.C. Perrakis A. Wilson K.S. Dijkstra B.W. J. Am. Chem. Soc. 1997; 119: 7954-7959Crossref Scopus (275) Google Scholar, 14Van Aalten D.M.F. Komander D. Synstad B. Gåseidnes S. Peter M.G. Eijsink V.G.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8979-8984Crossref PubMed Scopus (399) Google Scholar), β-galactosidase (15Espinosa J.F. Montero E. Vian A. García J.L. Dietrich H. Schmidt R.R. Martín-Lomas M. Imberty A. Canñada F.J. Jiménez-Barbero J. J. Am. Chem. Soc. 1998; 120: 1309-1318Crossref Scopus (96) Google Scholar), and β-mannanase. Similar distorted structures have been characterized for inverting glycosidases and carbohydrate-bound biological receptors (16Ducros V.M.-A. Zechel D.L. Murshudov G.N. Gilbert H.J. Szabó L. Stoll D. Withers S.G. Davies G.J. Angew. Chem. Int. Ed. 2001; 41: 2824-2827Crossref Scopus (121) Google Scholar, 17Zou J.-Y. Kleywegt G.J. Ståhlberg J. Driguez H. Nerinckx W. Claeyssens M. Koivula A. Teeri T.T. Jones T.A. Structure. 1999; 7: 1035-1045Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 18Varrot A. Frandsen T.P. Driguez H. Davies G.J. Acta. Crystallogr. Sect. D. 2002; 58: 2201-2204Crossref PubMed Scopus (29) Google Scholar, 19Guérin D.M.A. Lascombe M.-B. Costabel M. Souchon H. Lamzin V. Béguin P. Alzari P.M. J. Mol. Biol. 2002; 316: 1061-1069Crossref PubMed Scopus (123) Google Scholar, 21Varrot A. Macdonald J. Stick R.V. Pell G. Gilbert H.J. Davies G.J. Chem. Commun. 2003; : 946-947Crossref PubMed Scopus (44) Google Scholar, 22Golan G. Shallom D. Teplitsky A. Zaide G. Shulami S. Baasov T. Stojanoff V. Thompson A. Shoham Y. Shoham G. J. Biol. Chem. 2004; 279: 3014-3024Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 23Sabini E. Sulzenbacher G. Dauter M. Dauter Z. Jorgensen P.L. Schülein M. Dupont C. Davies G.J. Wilson K.S. Chem. Biol. 1999; 7: 483-492Abstract Full Text PDF Scopus (128) Google Scholar). Recent crystallographic studies on Thermobifida fusca endoglucanase Cel6A show a significant decrease in activity upon mutation of the closest Tyr residue, a feature, which has been attributed to a conformation change of the substrate in subsite –1 from skew-boat to chair (24Zhou W.L. Irwin D.C. Escovar-Kousen J. Wilson D.B. Biochemistry. 2004; 43: 9655-9663Crossref PubMed Scopus (99) Google Scholar, 25Larsson, A., Bergfors, T., Dultz, E., Irwin, D., Roos, A., Driguez, H., Wilson, D., Jones, A. (2005), 6th Carbohydrate Bioengineering Meeting, Barcelona, Spain, AbstractsGoogle Scholar). It is also worth noting that a 4C1-2S0 equilibrium has been recently detected in the binding of glycosylaminoglycans to polypeptides by NMR techniques (26Canales A. Angulo J. Ojeda R. Bruix M. Fayos R. Lozano R. Giménez-Gallego G. Martín-Lomas M. Nieto P.M. Jiménez-Barbero J. J. Am. Chem. Soc. 2005; 127: 5778-5779Crossref PubMed Scopus (67) Google Scholar). This kind of saccharide ring distortion has favorable mechanistic consequences in glycoside hydrolysis. As shown in Fig. 2b, the 1S3 distortion places the glycosidic oxygen near the acid/base residue. It also reduces the steric interaction between the hydrogen at the anomeric carbon and the nucleophile, and places the aglycon (i.e. the leaving group) in a pseudo-axial position that facilitates nucleophile attack on the anomeric carbon (27Kirby A.J. Acc. Chem. Res. 1984; 17: 305-311Crossref Scopus (97) Google Scholar). These distortions in the Michaelis complex are therefore in the pathway to reach the transition state of the reaction. The fact that all β-glycoside hydrolase enzyme-substrate complexes so far characterized by x-ray crystallography bear a distorted substrate suggests that substrate distortion is a general feature of β-glycoside hydrolases. However, in most cases these distortions are encountered in inhibitor-enzyme complexes (i.e. modified forms of the substrate) or in complexes with inactive enzyme mutants, which also raises the question whether the inhibitor or the mutant could affect the substrate conformation (28García-Herrero A. Montero E. Munñoz J.J. Espinosa J.F. Vián A. García J.L. Asensio J.L. Canñada F.J. Jiménez-Barbero J. J. Am. Chem. Soc. 2002; 124: 4804-4810Crossref PubMed Scopus (80) Google Scholar). Theoretical calculations can be very helpful to solve these issues, as they can be performed directly on the “native” substrate-bound enzyme. Classical molecular dynamics simulations demonstrated that the boat conformation at subsite –1 is critical in the mechanism of family 18 chitinases (29Brameld K.A. Goddard W.A. J. Am. Chem. Soc. 1998; 120: 3571-3580Crossref Scopus (87) Google Scholar). Recent studies confirmed that the –1 sugar moiety of the substrate in cellulase Cel6A from Trichoderma reesei, an inverting glycosidase, adopts a skew-boat conformation (2S0) (30André G. Kanchanawong P. Palma R. Cho H. Deng X. Irwin D. Himmel M.E. Wilson D.B. Brady J.W. Protein Eng. 1. 2003; 6: 125-134Crossref Scopus (31) Google Scholar). Similarly, modeling studies of β-galactosidase and xylanases provided evidence of substrate distortion (15Espinosa J.F. Montero E. Vian A. García J.L. Dietrich H. Schmidt R.R. Martín-Lomas M. Imberty A. Canñada F.J. Jiménez-Barbero J. J. Am. Chem. Soc. 1998; 120: 1309-1318Crossref Scopus (96) Google Scholar, 31Kankainen M. Laitinen T. Peräkylä Phys. Chem. Chem. Phys. 2004; 6: 5074-5080Crossref Google Scholar). All these studies rely on parametrized expressions (force fields) to describe the interaction among atoms, and thus the interplay of electronic/structural factors on the substrate conformation cannot be analyzed. To overcome these limitations, we have taken a step forward in accuracy and predictive power by using first principles methods, thus taking into account electronic effects and charge rearrangements in the active site. In the framework of our structure/function studies of bacterial 1,3–1,4-β-glucanases (7Planas A. Biochim. Biochim. Acta. 2000; 1543: 361-382Crossref PubMed Scopus (236) Google Scholar, 32Faijes M. Pérez X. Pérez O. Planas A. Biochemistry. 2003; 42: 13304-13318Crossref PubMed Scopus (45) Google Scholar, 33Planas A. Nieto J. Abel M. Segade A. Biocatal. Biotransfor. 2003; 21: 223-231Crossref Scopus (2) Google Scholar, 34Piotukh K. Serra V. Borriss R. Planas A. Biochemistry. 1999; 38: 16092-16104Crossref PubMed Scopus (27) Google Scholar), we investigate here the conformation of the substrate in the Michaelis complex of Bacillus 1,3–1,4-β-glucanase by means of hybrid quantum mechanics/molecular mechanics (QM/MM) simulations (35Wharshel A. Levitt M. J. Mol. Biol. 1976; 103: 227-249Crossref PubMed Scopus (3695) Google Scholar). In this approach, the atoms of the QM region evolve in time under the effect of the quantum mechanical forces, computed using density functional theory (DFT), and the electronic cloud adapts instantaneously to the chemical environment, whereas the forces on the MM region are ruled by a force field. The effect of the enzyme on the properties of the substrate is analyzed, providing insight on the factors leading to the substrate conformation we predict for this enzyme. The substrate chosen for the analysis is the 4-methylumbelliferyl tetrasaccharide shown in Fig. 3, which is a good substrate extensively used in enzyme kinetics (32Faijes M. Pérez X. Pérez O. Planas A. Biochemistry. 2003; 42: 13304-13318Crossref PubMed Scopus (45) Google Scholar, 33Planas A. Nieto J. Abel M. Segade A. Biocatal. Biotransfor. 2003; 21: 223-231Crossref Scopus (2) Google Scholar, 34Piotukh K. Serra V. Borriss R. Planas A. Biochemistry. 1999; 38: 16092-16104Crossref PubMed Scopus (27) Google Scholar). To the best of our knowledge, the conformational itinerary of the substrate in GHs has not yet been investigated by the first principles methods. Enzyme-Substrate Initial Structure—The only structures available for 1,3–1,4-β-glucanase, a family GH16 enzyme, are that of the native enzyme (36Keitel T. Simon O. Borriss R. Heinemann U. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5287-5291Crossref PubMed Scopus (194) Google Scholar, 37Hahn M. Pons J. Planas A. Querol E. Heinemann U. FEBS Lett. 1995; 374: 221-224Crossref PubMed Scopus (77) Google Scholar) and that of the covalent enzyme-inhibitor complex with an epoxybutyl saccharide (36Keitel T. Simon O. Borriss R. Heinemann U. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5287-5291Crossref PubMed Scopus (194) Google Scholar). In addition, the structure of the enzyme product complex has been recently characterized (PDB accession code 1U0A) (69Gaiser O.J. Piotukh K. Ponnuswamy M.N. Planas A. Borriss R. Heinemann U. J. Mol. Biol. 2005; (in press)Google Scholar). This structure was used to build the initial structure of the Michaelis complex. For this purpose, the missing methylumbelliferyl aglycon (MU in Fig. 3) was inserted manually to generate the enzyme-substrate complex. This structure was submitted to a preliminary molecular dynamics simulation (33Planas A. Nieto J. Abel M. Segade A. Biocatal. Biotransfor. 2003; 21: 223-231Crossref Scopus (2) Google Scholar) using the Cornell et al. force field (38Cornell W.D. Cieplak P. Bayly C.I. Gould I.R. Merz Jr., K.M. Ferguson D.M. Spellmeyer D. Fox T. Caldwell J.W. Kollman P.A. J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11640) Google Scholar), as implemented in the HYPERCHEM package, 4www.hyper.com. with the restraint that only the atoms 6 Å from the anomeric carbon were allowed to move. During this procedure the substrate maintained the original chair conformation (4C1) in the four sugar rings. To force a structure similar to that of an oxocarbenium ion, the charge on the anomeric carbon was increased by 0.6 electrons. After this replacement, the sugar ring adopted a distorted 1S3 conformation. For the sake of simplification, hereafter we will use the notations 4C1-substrate and 1S3-substrate to refer to the substrate isomer in which the sugar ring of the –1 subsite adopts either 4C1 or 1S3 conformation, respectively. These two structures were used for the subsequent calculations. First Principles Molecular Dynamics Simulations—First principles molecular dynamics simulations were performed to analyze the dynamics of the isolated substrate and that of the substrate in the presence of the catalytic residues (Glu109 and Glu105). The calculations were performed using the Car-Parrinello method (CP) (40Car R. Parrinello M. Phys. Rev. Lett. 1985; 55: 2471-2474Crossref PubMed Scopus (9385) Google Scholar, 41Marx D. Hutter J. Grotendorst J. Methods and Algorithms of Quantum Chemistry. John von Neumann Institute for Computing, Julich, Germany2000: 301-409Google Scholar), which is based on DFT. Both DFT and the CP method have already been employed with success in a number of investigations of biological processes (see for instance Refs. 42Rovira C. Schulze B. Eichinger M. Evanseck J.D. Parrinello M. Biophys. J. 2001; 81: 435-445Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 43Mordasini T. Curioni A. Andreoni W. J. Biol. Chem. 2003; 278: 4381-4384Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 44Carloni P. Röthlisberger U. Parrinello M. Acc. Chem. Res. 2002; 35: 455-464Crossref PubMed Scopus (269) Google Scholar, 45Magistrato A. DeGrado W.F. Laio A. Röthlisberger U. VandeVondele J. Klein M.L. J. Phys. Chem. B. 2003; 107: 4182-4188Crossref Scopus (37) Google Scholar, 46Piana S. Bucher D. Carloni P. Röthlisberger U. J. Phys. Chem. B. 2004; 108: 11139-11149Crossref Scopus (103) Google Scholar, 47Klein C.D.P. Schiffmann R. Folkers G. Piana S. Röthlisberger U. J. Biol. Chem. 2003; 278: 47862-47867Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 48Rousseau R. Kleinschmidt U.W. Marx D. Ang. Chem. Int. Ed. Eng. 2004; 43: 4804-4807Crossref PubMed Scopus (82) Google Scholar) including carbohydrate structure and dynamics (49Molteni C. Parrinello M. J. Am. Chem. Soc. 1998; 120: 2168-2171Crossref Scopus (145) Google Scholar, 50Suzuki T. Sota T. J. Phys. Chem. B. 2005; 109: 12603-12611Crossref PubMed Scopus (9) Google Scholar, 51Suzuki T. Kawashima H. Kotoku H. Sota T. J. Phys. Chem. B. 2005; 109: 12997-13005Crossref PubMed Scopus (6) Google Scholar, 52Ballone P. Marchi M. Branca C. Magazu S. J. Phys. Chem. B. 2000; 104: 6313-6317Crossref Scopus (55) Google Scholar) and carbohydrate reactivity (see for instance Refs. 53Bérces A. Enright G. Nukada T. Whitfield D.M. J. Am. Chem. Soc. 2001; 123: 5460-5464Crossref PubMed Scopus (57) Google Scholar, 54Stubbs J.M. Marx D. J. Am. Chem. Soc. 2003; 125: 10960-10962Crossref PubMed Scopus (32) Google Scholar, 55Corchado J.C. Sánchez M.L. Aguilar M.A. J. Am. Chem. Soc. 2004; 126: 7311-7319Crossref PubMed Scopus (75) Google Scholar, 56Appell M. Strati G. Willet J.L. Momany F.A. Carbohyd. Res. 2004; 339: 537-551Crossref PubMed Scopus (120) Google Scholar, 57Tvaroska I. André I. Carver J.P. J. Am. Chem. Soc. 2000; 122: 8762-8776Crossref Scopus (57) Google Scholar, 58Fernández-Alonso M.C. Canñada J. Jiménez-Barbero J. Cuevas G. Chem. Phys. Chem. 2005; 6: 671-680Crossref Scopus (28) Google Scholar). The generalized gradient-corrected approximation of DFT, following the prescription of Perdew, Burke, and Ernzerhoff (59Perdew J.P. Burke K. Ernzerhof M. Phys. Rev. Lett. 1996; 77: 3865-3868Crossref PubMed Scopus (139895) Google ScholarErratum Phys. Rev. Lett. 1997; 78: 1396Google Scholar), was used. The choice of this functional is based on its reliability in the description of hydrogen bonds (60Ireta J. Neugebauer J. Sheffler M. J. Phys. Chem. A. 2004; 108: 5692-5698Crossref Scopus (343) Google Scholar). We employed ab initio pseudopotentials, generated within the Troullier-Martins scheme (61Troullier M. Martins J.L. Phys. Rev. B. 1991; 43: 1993-2006Crossref PubMed Scopus (14770) Google Scholar). The Kohn-Sham orbitals (62Koch W. Holthausen M.C. A Chemist's Guide to Density Functional Theory. Wiley-VCH, Weinheim, Germany2000: 41-64Google Scholar) are expanded in a plane wave basis set with the kinetic energy cutoff of 70 Ry. Structural optimizations were performed by means of molecular dynamics with annealing of the atomic velocities, using a time step of 0.12 fs, and the fictitious mass of the electrons was set at 1200 a.u. With this setup the total energy and the fictitious kinetic energy of the electrons were conserved within 1.01 · 10–6 a.u./ps·atom and 3.6 · 10–5 a.u./ps·atom, respectively. The Nosé-Hoover thermostat for the nuclear degrees of freedom was used to maintain the temperature as constant as possible (63Nosé S. Mol. Phys. 1984; 52: 255-268Crossref Scopus (7356) Google Scholar). The systems were enclosed in super-cells of size 17.5 × 12.5 × 12.5 Å3 (isolated substrate) and 15.8 × 13.2 × 17.0 Å3 (substrate + catalytic residues). The calculations were performed with the CPMD program, 5CPMD program, Copyright IBM Corp. 1990–2003, Copyright MPI für Festkörperforschung, Stuttgart 1997–2001. URL: www.cpmd.org. and structure analysis was performed with VMD (65Humphrey W. Dalke A. Schulten K. J. Mol. Graph. 1996; 14: 33Crossref PubMed Scopus (39882) Google Scholar). Atomic charges were computed from the electrostatic potential (ESP). Interaction energies between the substrate and each of the catalytic residues were obtained by subtracting the energy of the complex from the energy of the isolated fragments in their corresponding optimized structure. The energy of the 4H3 transition conformation (the transition state for a conversion between the 1S3 and 4C1 conformers) was obtained by fixing the dihedral angle defined by the C2, C1, O5, and C5 ring atoms (hereafter referred as ϕ angle, shown in Fig. 3a) to zero degrees and optimizing all other degrees of freedom. Similarly, the 1S3 conformation (which is not a local minimum for the isolated substrate) was optimized by fixing the C2-O5-C5-C4 dihedral angle. Hybrid QM/MM Molecular Dynamics Simulations—Hybrid QM/MM simulations on the complete protein were performed for each of the two substrate conformations described above (4C1-substrate and 1S3-substrate). Before starting the QM/MM simulations, a classical molecular dynamics simulation was performed to equilibrate the protein and allow the substrate to accommodate in the binding cavity. The following parameters were used in the classical simulations. The Cornell et al. force field (38Cornell W.D. Cieplak P. Bayly C.I. Gould I.R. Merz Jr., K.M. Ferguson D.M. Spellmeyer D. Fox T. Caldwell J.W. Kollman P.A. J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11640) Google Scholar), as implemented in the AMBER 7.0 program (66Pearlman D.A. Case D.A. Caldwell J.W. Ross W.S. Cheatham T.E. Debolt S. Ferguson D. Seibel G. Kollman P. Comput. Phys. Commun. 1995; 91: 1-41Crossref Scopus (2689) Google Scholar), and the GLYCAM parameter set (67Woods R.J. Dwek R.A. Fraserreid B. J. Phys. Chem. 1995; 99: 3832-3846Crossref Scopus (417) Google Scholar) were used for the protein residues and for the substrate, respectively. The MU aglycon was parameterized using the antechamber module. The atomic charges of the substrate were obtained from a first principles (Car-Parrinello) calculation of the isolated substrate. All His residues (located in the protein surface) were taken as protonated (i.e. positive charge), and all Asp and Glu residues were taken as deprotonated (i.e. negative charge) except Glu109 (the acid-base residue) and Asp107, which is hydrogen-bonded to the nucleophile Glu105. Six chlorine atoms were added to achieve neutrality of the protein structure. The system was enveloped in a 52 Å × 40 Å × 66 Å box of equilibrated TIP3P water molecules and was equilibrated in several steps. First, all water molecules were relaxed with a gradient minimizer and then equilibrated for 20 ps at 150 K (protein constrained). Next, the whole system was minimized and subsequently equilibrated for 20 ps at 300 K. During equilibration, the system was coupled to a heat bath to achieve the desired temperature of 300 K. The simulation was continued for 20 ps at constant pressure, allowing the cell volume to evolve until equilibration. Analysis of the trajectories was carried out by using standard tools of AMBER. Several scenarios were tested for the initial protonation state of the acid/base (Glu109) and the Asp107 residues. The configuration that better maintained the interaction of the catalytic residues with the substrate upon a short test MD run was chosen for the production runs. In this configuration, the OH group of Glu109 interacts with Ogly and the OH group of Asp107 interacts with Glu105. Two separate classical simulations were performed, one with the substrate in the chair conformation (4C1-substrate) and another one with the substrate in the distorted skew-boat conformation (1S3-substrate). While the chair conformer was found to be stable, the skew-boat conformer evolved toward the chair one during the optimization process, unless a larger atomic charge for the anomeric carbon is used. For this reason, the simulation of the skew-boat conformer was performed by using a different charge in C1 (see above). Once the system was equilibrated and the relative position of the substrate and the enzyme did not change (i.e. the root mean-square deviation variations were stabilized), QM/MM simulations were initiated. The method developed by Laio et al. (68Laio A. VandeVondele J. Rothlisberger U. J. Chem. Phys. 2002; 116: 6941-6947Crossref Scopus (557) Google Scholar) was used. This method combines the first principles molecular dynamics method of Car and Parrinello (CPMD) (40Car R. Parrinello M. Phys. Rev. Lett. 1985; 55: 2471-2474Crossref PubMed Scopus (9385) Google Scholar) with a force field molecular dynamics methodology (i.e QM/MM CPMD). In this approach, the system is partitioned into a QM fragment and an MM fragment. The dynamics of the atoms on the QM fragment depends on the electronic density, ρ(r), computed with DFT, whereas that of the atoms on the MM fragment is ruled by an empirical force field. The QM-MM interface is modeled" @default.
W2021955903 created "2016-06-24" @default.
W2021955903 creator A5038087076 @default.
W2021955903 creator A5079082841 @default.
W2021955903 creator A5081831378 @default.
W2021955903 creator A5085600610 @default.
W2021955903 date "2006-01-01" @default.
W2021955903 modified "2023-09-30" @default.
W2021955903 title "Substrate Distortion in the Michaelis Complex of Bacillus 1,3–1,4-β-Glucanase" @default.
W2021955903 cites W1849088874 @default.
W2021955903 cites W1935712031 @default.
W2021955903 cites W1965761289 @default.
W2021955903 cites W1969758693 @default.
W2021955903 cites W1974579245 @default.
W2021955903 cites W1975505188 @default.
W2021955903 cites W1980179156 @default.
W2021955903 cites W1981368803 @default.
W2021955903 cites W1987250042 @default.
W2021955903 cites W1992856312 @default.
W2021955903 cites W2000717347 @default.
W2021955903 cites W2001574266 @default.
W2021955903 cites W2004303971 @default.
W2021955903 cites W2006145970 @default.
W2021955903 cites W2007953948 @default.
W2021955903 cites W2010672871 @default.
W2021955903 cites W2011029745 @default.
W2021955903 cites W2014430192 @default.
W2021955903 cites W2016166635 @default.
W2021955903 cites W2018456240 @default.
W2021955903 cites W2021741281 @default.
W2021955903 cites W2026995511 @default.
W2021955903 cites W2029667189 @default.
W2021955903 cites W2038417675 @default.
W2021955903 cites W2041638382 @default.
W2021955903 cites W2045596260 @default.
W2021955903 cites W2045801831 @default.
W2021955903 cites W2046001409 @default.
W2021955903 cites W2048398687 @default.
W2021955903 cites W2049373071 @default.
W2021955903 cites W2049468314 @default.
W2021955903 cites W2055998893 @default.
W2021955903 cites W2056661808 @default.
W2021955903 cites W2057074235 @default.
W2021955903 cites W2058396359 @default.
W2021955903 cites W2061900269 @default.
W2021955903 cites W2065687317 @default.
W2021955903 cites W2065893364 @default.
W2021955903 cites W2070279852 @default.
W2021955903 cites W2073446347 @default.
W2021955903 cites W2074402389 @default.
W2021955903 cites W2078299239 @default.
W2021955903 cites W2081753121 @default.
W2021955903 cites W2082011590 @default.
W2021955903 cites W2083495362 @default.
W2021955903 cites W2087990820 @default.
W2021955903 cites W2091344572 @default.
W2021955903 cites W2093381148 @default.
W2021955903 cites W2098208486 @default.
W2021955903 cites W2098303032 @default.
W2021955903 cites W2103499539 @default.
W2021955903 cites W2103726069 @default.
W2021955903 cites W2106325630 @default.
W2021955903 cites W2112185869 @default.
W2021955903 cites W2122199548 @default.
W2021955903 cites W2124975968 @default.
W2021955903 cites W2129728418 @default.
W2021955903 cites W2136806985 @default.
W2021955903 cites W2141162642 @default.
W2021955903 cites W2146663527 @default.
W2021955903 cites W2147705013 @default.
W2021955903 cites W2159913888 @default.
W2021955903 cites W2950633309 @default.
W2021955903 doi "https://doi.org/10.1074/jbc.m507643200" @default.
W2021955903 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16260784" @default.
W2021955903 hasPublicationYear "2006" @default.
W2021955903 type Work @default.
W2021955903 sameAs 2021955903 @default.
W2021955903 citedByCount "82" @default.
W2021955903 countsByYear W20219559032012 @default.
W2021955903 countsByYear W20219559032013 @default.
W2021955903 countsByYear W20219559032014 @default.
W2021955903 countsByYear W20219559032015 @default.
W2021955903 countsByYear W20219559032016 @default.
W2021955903 countsByYear W20219559032017 @default.
W2021955903 countsByYear W20219559032018 @default.
W2021955903 countsByYear W20219559032019 @default.
W2021955903 countsByYear W20219559032020 @default.
W2021955903 countsByYear W20219559032021 @default.
W2021955903 countsByYear W20219559032022 @default.
W2021955903 countsByYear W20219559032023 @default.
W2021955903 crossrefType "journal-article" @default.
W2021955903 hasAuthorship W2021955903A5038087076 @default.
W2021955903 hasAuthorship W2021955903A5079082841 @default.
W2021955903 hasAuthorship W2021955903A5081831378 @default.
W2021955903 hasAuthorship W2021955903A5085600610 @default.
W2021955903 hasBestOaLocation W20219559031 @default.
W2021955903 hasConcept C126780896 @default.
W2021955903 hasConcept C181199279 @default.