Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Wan, Qun; Parks, Jerry M.; Hanson, B. Leif; Fisher, Suzanne Zoe; Ostermann, Andreas; Schrader, Tobias E.; Graham, David E.; Coates, Leighton; Langan, Paul; Kovalevsky, Andrey

doi:10.1073/pnas.1504986112

Title: Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Journal Article · Mon Sep 21 00:00:00 EDT 2015 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.1504986112· OSTI ID:1265915

Wan, Qun ^[1];

^[2]; Hanson, B. Leif ^[3]; Fisher, Suzanne Zoe ^[4]; Ostermann, Andreas ^[5]; Schrader, Tobias E. ^[6];

^[7]; Coates, Leighton ^[7];

^[7];

^[7]

Nanjing Agricultural Univ., Nanjing (People's Republic of China)
Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Univ. of Toledo, Toledo, OH (United States)
European Spallation Source, Lund (Sweden)
Technische Univ. Munchen, Garching (Germany)
Forschungszentrum Julich GmbH, Garching (Germany)
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pK_a values of key side chains during catalysis. Yet, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. Here, we performed continuum electrostatics calculations to estimate the pK_a values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pK_a of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. Lastly, these findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.

View Accepted Manuscript (DOE)

Cite

Export

Save

Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Laboratory Directed Research and Development (LDRD) Program

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 1265915

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Vol. 112, Issue 40; ISSN 0027-8424

Publisher:: National Academy of Sciences, Washington, DC (United States)Copyright Statement

Country of Publication:: United States

Language:: English

Citation Metrics:

Cited by: 28 works

Citation information provided by
Web of Science

References (47)

Glycosidase inhibition: assessing mimicry of the transition state Gloster, Tracey M.; Davies, Gideon J. Org. Biomol. Chem., Vol. 8, Issue 2 https://doi.org/10.1039/b915870g	journal	January 2010
GH11 xylanases: Structure/function/properties relationships and applications Paës, Gabriel; Berrin, Jean-Guy; Beaugrand, Johnny Biotechnology Advances, Vol. 30, Issue 3 https://doi.org/10.1016/j.biotechadv.2011.10.003	journal	May 2012
Prediction and Rationalization of the pH Dependence of the Activity and Stability of Family 11 Xylanases ^† Kongsted, Jacob; Ryde, Ulf; Wydra, James Biochemistry, Vol. 46, Issue 47 https://doi.org/10.1021/bi7016365	journal	November 2007
Snapshots along an Enzymatic Reaction Coordinate: Analysis of a Retaining β-Glycoside Hydrolase ^† ^, ^‡ Davies, Gideon J.; Mackenzie, Lloyd; Varrot, Annabelle Biochemistry, Vol. 37, Issue 34 https://doi.org/10.1021/bi981315i	journal	August 1998
Cellulases and biofuels Wilson, David B. Current Opinion in Biotechnology, Vol. 20, Issue 3, p. 295-299 https://doi.org/10.1016/j.copbio.2009.05.007	journal	June 2009
The Two Major Xylanases from Trichoderma Reesei: Characterization of Both Enzymes and Genes Törrönen, Anneli; Mach, Robert L.; Messner, Robert Nature Biotechnology, Vol. 10, Issue 11 https://doi.org/10.1038/nbt1192-1461	journal	November 1992
X-ray crystallographic studies of family 11 xylanase Michaelis and product complexes: implications for the catalytic mechanism Wan, Qun; Zhang, Qiu; Hamilton-Brehm, Scott Acta Crystallographica Section D Biological Crystallography, Vol. 70, Issue 1 https://doi.org/10.1107/S1399004713023626	journal	December 2013
Reactions of anionic nucleophiles with .alpha.-D-glucopyranosyl fluoride in aqueous solution through a concerted, ANDN (SN2) mechanism Banait, Narinder S.; Jencks, William P. Journal of the American Chemical Society, Vol. 113, Issue 21 https://doi.org/10.1021/ja00021a021	journal	October 1991
Theory of enzymatic reverse-protonation catalysis Mock, William L. Bioorganic Chemistry, Vol. 20, Issue 4 https://doi.org/10.1016/0045-2068(92)90047-7	journal	December 1992
Spontaneous Hydrolysis of Glycosides Wolfenden, Richard; Lu, Xiangdong; Young, Gregory Journal of the American Chemical Society, Vol. 120, Issue 27 https://doi.org/10.1021/ja9813055	journal	July 1998
Scalable molecular dynamics with NAMD Phillips, James C.; Braun, Rosemary; Wang, Wei Journal of Computational Chemistry, Vol. 26, Issue 16, p. 1781-1802 https://doi.org/10.1002/jcc.20289	journal	January 2005
Preliminary joint X-ray and neutron protein crystallographic studies of endoxylanase II from the fungus Trichoderma longibrachiatum Kovalevsky, Andrey Y.; Hanson, B. Leif; Seaver, Sean Acta Crystallographica Section F Structural Biology and Crystallization Communications, Vol. 67, Issue 2 https://doi.org/10.1107/S174430911005075X	journal	January 2011
Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the ph optimum of a glycosidase 1 1Edited by M. F. Summers Joshi, Manish D.; Sidhu, Gary; Pot, Isabelle Journal of Molecular Biology, Vol. 299, Issue 1 https://doi.org/10.1006/jmbi.2000.3722	journal	May 2000
Dissecting conformational contributions to glycosidase catalysis and inhibition Speciale, Gaetano; Thompson, Andrew J.; Davies, Gideon J. Current Opinion in Structural Biology, Vol. 28 https://doi.org/10.1016/j.sbi.2014.06.003	journal	October 2014
Rapid determination of hydrogen positions and protonation states of diisopropyl fluorophosphatase by joint neutron and X-ray diffraction refinement Blum, Marc-Michael; Mustyakimov, Marat; Rüterjans, Heinz Proceedings of the National Academy of Sciences, Vol. 106, Issue 3 https://doi.org/10.1073/pnas.0807842106	journal	January 2009
[16] SHELXL: High-resolution refinement Sheldrick, George M.; Schneider, Thomas R. Methods in Enzymology https://doi.org/10.1016/S0076-6879(97)77018-6	book	January 1997
Dissecting the Electrostatic Interactions and pH-Dependent Activity of a Family 11 Glycosidase ^† ^, ^‡ Joshi, Manish D.; Sidhu, Gary; Nielsen, Jens E. Biochemistry, Vol. 40, Issue 34 https://doi.org/10.1021/bi0105429	journal	August 2001
CHARMM: The biomolecular simulation program Brooks, B. R.; Brooks, C. L.; Mackerell, A. D. Journal of Computational Chemistry, Vol. 30, Issue 10 https://doi.org/10.1002/jcc.21287	journal	July 2009
The endoxylanases from family 11: computer analysis of protein sequences reveals important structural and phylogenetic relationships Sapag, Amalia; Wouters, Johan; Lambert, Christophe Journal of Biotechnology, Vol. 95, Issue 2 https://doi.org/10.1016/S0168-1656(02)00002-0	journal	May 2002
Stereochemistry and the Mechanism of Enzymatic Reactions Koshland, D. E. Biological Reviews, Vol. 28, Issue 4 https://doi.org/10.1111/j.1469-185X.1953.tb01386.x	journal	November 1953
A Historical Perspective for the Catalytic Reaction Mechanism of Glycosidase; So As to Bring about Breakthrough in Confusing Situation Chiba, Seiya Bioscience, Biotechnology, and Biochemistry, Vol. 76, Issue 2 https://doi.org/10.1271/bbb.110713	journal	February 2012
The p K _a of the General Acid/Base Carboxyl Group of a Glycosidase Cycles during Catalysis: A ¹³ C-NMR Study of Bacillus circulans Xylanase ^† McIntosh, Lawrence P.; Hand, Greg; Johnson, Philip E. Biochemistry, Vol. 35, Issue 31 https://doi.org/10.1021/bi9613234	journal	January 1996
Symposium on Three-Dimensional Structure of Macromolecules of Biological Origin. by Invitation of the Committee on Arrangements for the Autumn Meeting. Presented before the Academy on October 19, 1966. Chairman, Walter Kauzmann: STUDIES ON THE ENZYMIC ACTIVITY OF LYSOZYME, II. THE HYDROLYSIS AND TRANSFER REACTIONS OF N-ACETYLGLUCOSAMINE OLIGOSACCHARIDES Rupley, J. A.; Gates, V. Proceedings of the National Academy of Sciences, Vol. 57, Issue 3 https://doi.org/10.1073/pnas.57.3.496	journal	March 1967
Structural Comparison of Two Major endo-1,4-Xylanases from Trichoderma reesei Torronen, Anneli; Rouvinen, Juha Biochemistry, Vol. 34, Issue 3 https://doi.org/10.1021/bi00003a019	journal	January 1995
Catalytic Reaction Mechanism Based on α-Secondary Deuterium Isotope Effects in Hydrolysis of Trehalose by European Honeybee Trehalase Mori, Haruhide; Lee, Jin-Ha; Okuyama, Masayuki Bioscience, Biotechnology, and Biochemistry, Vol. 73, Issue 11 https://doi.org/10.1271/bbb.90447	journal	November 2009
A solvent-isotope-effect study of proton transfer during catalysis by Escherichia coli (lacZ) β-galactosidase Selwood, T.; Sinnott, M. L. Biochemical Journal, Vol. 268, Issue 2 https://doi.org/10.1042/bj2680317	journal	June 1990
Heterologous expression, purification, crystallization and preliminary X-ray analysis of Trichoderma reesei xylanase II and four variants Wan, Qun; Kovalevsky, Andrey; Zhang, Qiu Acta Crystallographica Section F Structural Biology and Crystallization Communications, Vol. 69, Issue 3 https://doi.org/10.1107/S1744309113001164	journal	February 2013
Crystallographic Snapshots of an Entire Reaction Cycle for a Retaining Xylanase from Streptomyces olivaceoviridis E-86 Suzuki, Ryuichiro; Fujimoto, Zui; Ito, Shigeyasu The Journal of Biochemistry, Vol. 146, Issue 1 https://doi.org/10.1093/jb/mvp047	journal	March 2009
Glycoside hydrolases: Catalytic base/nucleophile diversity Vuong, Thu V.; Wilson, David B. Biotechnology and Bioengineering, Vol. 107, Issue 2, p. 195-205 https://doi.org/10.1002/bit.22838	journal	June 2010
HKL -3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes Minor, Wladek; Cymborowski, Marcin; Otwinowski, Zbyszek Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 8 https://doi.org/10.1107/S0907444906019949	journal	July 2006
The Macromolecular Neutron Diffractometer MaNDi at the Spallation Neutron Source Coates, Leighton; Cuneo, Matthew J.; Frost, Matthew J. Journal of Applied Crystallography, Vol. 48, Issue 4 https://doi.org/10.1107/S1600576715011243	journal	July 2015
THE weighted histogram analysis method for free-energy calculations on biomolecules. I. The method Kumar, Shankar; Rosenberg, John M.; Bouzida, Djamal Journal of Computational Chemistry, Vol. 13, Issue 8 https://doi.org/10.1002/jcc.540130812	journal	October 1992
Enzymatic deconstruction of xylan for biofuel production Dodd, Dylan; Cann, Isaac K. O. GCB Bioenergy, Vol. 1, Issue 1 https://doi.org/10.1111/j.1757-1707.2009.01004.x	journal	February 2009
PHENIX: a comprehensive Python-based system for macromolecular structure solution Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221 https://doi.org/10.1107/S0907444909052925	journal	January 2010
Symposium on Three-Dimensional Structure of Macromolecules of Biological Origin. by Invitation of the Committee on Arrangements for the Autumn Meeting. Presented before the Academy on October 19, 1966. Chairman, Walter Kauzmann: THE HEN EGG-WHITE LYSOZYME MOLECULE Phillips, D. C. Proceedings of the National Academy of Sciences, Vol. 57, Issue 3 https://doi.org/10.1073/pnas.57.3.483	journal	March 1967
Analysis of the dynamic properties of Bacillus circulans xylanase upon formation of a covalent glycosyl-enzyme intermediate Connelly, Gregory P.; Withers, Stephen G.; Mcintosh, Lawrence P. Protein Science, Vol. 9, Issue 3 https://doi.org/10.1110/ps.9.3.512	journal	January 2000
CHARMM Additive All-Atom Force Field for Glycosidic Linkages between Hexopyranoses Guvench, Olgun; Hatcher, Elizabeth; Venable, Richard M. Journal of Chemical Theory and Computation, Vol. 5, Issue 9 https://doi.org/10.1021/ct900242e	journal	August 2009
Insights into transition state stabilization of the β-1,4-glycosidase Cex by covalent intermediate accumulation in active site mutants Notenboom, Valerie; Birsan, Camelia; Nitz, Mark Nature Structural Biology, Vol. 5, Issue 9 https://doi.org/10.1038/1852	journal	September 1998
Nomenclature for sugar-binding subsites in glycosyl hydrolases Davies, Gideon J.; Wilson, Keith S.; Henrissat, Bernard Biochemical Journal, Vol. 321, Issue 2 https://doi.org/10.1042/bj3210557	journal	January 1997
A critical survey of average distances between catalytic carboxyl groups in glycoside hydrolases: Average GH Inter-Carboxyl Distances Mhlongo, Ndumiso N.; Skelton, Adam A.; Kruger, Gert Proteins: Structure, Function, and Bioinformatics, Vol. 82, Issue 9 https://doi.org/10.1002/prot.24528	journal	February 2014
H++ 3.0: automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations Anandakrishnan, R.; Aguilar, B.; Onufriev, A. V. Nucleic Acids Research, Vol. 40, Issue W1 https://doi.org/10.1093/nar/gks375	journal	May 2012
Three-dimensional structure of endo-1,4-beta-xylanase II from Trichoderma reesei: two conformational states in the active site. Törrönen, A.; Harkki, A.; Rouvinen, J. The EMBO Journal, Vol. 13, Issue 11 https://doi.org/10.1002/j.1460-2075.1994.tb06536.x	journal	June 1994
Oxygen-18 leaving group kinetic isotope effects on the hydrolysis of nitrophenyl glycosides. 1. .beta.-Galactosidase-catalyzed hydrolysis Rosenberg, Steven; Kirsch, Jack F. Biochemistry, Vol. 20, Issue 11 https://doi.org/10.1021/bi00514a031	journal	May 1981
Generalized X-ray and neutron crystallographic analysis: more accurate and complete structures for biological macromolecules Adams, Paul D.; Mustyakimov, Marat; Afonine, Pavel V. Acta Crystallographica Section D Biological Crystallography, Vol. 65, Issue 6 https://doi.org/10.1107/S0907444909011548	journal	May 2009
Glucosidase-Catalyzed Hydrolysis of α- d -Glucopyranosyl Pyridinium Salts: Kinetic Evidence for Nucleophilic Involvement at the Glucosidation Transition State Huang, Xicai; Tanaka, Kelly S. E.; Bennet, Andrew J. Journal of the American Chemical Society, Vol. 119, Issue 46 https://doi.org/10.1021/ja963733l	journal	November 1997
Transition state structures for the hydrolysis of alpha-D-glucopyranosyl fluoride by retaining and inverting reactions of glycosylases. Tanaka, Y.; Tao, W.; Blanchard, J. S. Journal of Biological Chemistry, Vol. 269, Issue 51 https://doi.org/10.1016/S0021-9258(18)31636-3	journal	December 1994
[20] Processing of X-ray diffraction data collected in oscillation mode Otwinowski, Zbyszek; Minor, Wladek Macromolecular Crystallography Part A, 307-326 https://doi.org/10.1016/S0076-6879(97)76066-X	book	January 1997

Cited By (5)

Neutron scattering in the biological sciences: progress and prospects Ashkar, Rana; Bilheux, Hassina Z.; Bordallo, Heliosa Acta Crystallographica Section D Structural Biology, Vol. 74, Issue 12 https://doi.org/10.1107/s2059798318017503	journal	December 2018
Neutron and X-ray crystal structures of Lactobacillus brevis alcohol dehydrogenase reveal new insights into hydrogen-bonding pathways Hermann, Johannes; Nowotny, Phillip; Schrader, Tobias E. Acta Crystallographica Section F Structural Biology Communications, Vol. 74, Issue 12 https://doi.org/10.1107/s2053230x18015273	journal	November 2018
Neutron structure of the T26H mutant of T4 phage lysozyme provides insight into the catalytic activity of the mutant enzyme and how it differs from that of wild type: Neutron Structure of the T26H Mutant of T4L Hiromoto, Takeshi; Meilleur, Flora; Shimizu, Rumi Protein Science, Vol. 26, Issue 10 https://doi.org/10.1002/pro.3230	journal	July 2017
Structural and functional characterization of a highly stable endo-β-1,4-xylanase from Fusarium oxysporum and its development as an efficient immobilized biocatalyst Gómez, Sara; Payne, Asia M.; Savko, Martin Biotechnology for Biofuels, Vol. 9, Issue 1 https://doi.org/10.1186/s13068-016-0605-z	journal	September 2016
Titration of ionizable groups in proteins using multiple neutron data sets from a single crystal: application to the small GTPase Ras Knihtila, Ryan; Volmar, Alicia Y.; Meilleur, Flora Acta Crystallographica Section F Structural Biology Communications, Vol. 75, Issue 2 https://doi.org/10.1107/s2053230x18018125	journal	January 2019

Similar Records

Understanding the pH-Dependent Reaction Mechanism of a Glycoside Hydrolase Using High-Resolution X-ray and Neutron Crystallography

Journal Article · Wed Jul 18 00:00:00 EDT 2018 · ACS Catalysis · OSTI ID:1265915

Li, Zhihong; Zhang, Xiaoshuai; Wang, Qingqing; +10 more

Active site and laminarin binding in glycoside hydrolase family 55

Journal Article · Mon Mar 09 00:00:00 EDT 2015 · Journal of Biological Chemistry · OSTI ID:1265915

Bianchetti, Christopher M.; Takasuka, Taichi E.; Deutsch, Sam; +4 more

Studying the Role of a Single Mutation of a Family 11 Glycoside Hydrolase Using High-Resolution X-ray Crystallography

Journal Article · Sat Oct 31 00:00:00 EDT 2020 · The Protein Journal · OSTI ID:1265915

Li, Zhihong; Zhang, Xiaoshuai; Li, Chunran; +2 more

Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
glycoside hydrolase
protonation state
macromolecular neutron
crystallography
xylanase
molecular simulations

Title: Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Citation Formats

References (47)

Cited By (5)

Similar Records

Related Subjects