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

Zhihong Li; Xiaoshuai Zhang; Qingqing Wang; Chunran Li; Nianying Zhang; Xinkai Zhang; Birui Xu; Baoliang Ma; Tobias E. Schrader; Leighton Coates; Andrey Kovalevsky; Yandong Huang; Qun Wan

doi:10.1021/acscatal.8b01472

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

Zhihong Li, Xiaoshuai Zhang, Qingqing Wang, Chunran Li, Nianying Zhang, Xinkai Zhang, Birui Xu, Baoliang Ma, Tobias E. Schrader, Leighton Coates, Andrey Kovalevsky, Yandong Huang, Qun Wan

Research output: Contribution to journal › Article › peer-review

19 Scopus citations

Abstract

Glycoside hydrolases (GHs) commonly use the retaining or inverting mechanisms to hydrolyze carbohydrates, and the rates of catalysis are usually pH dependent. Deeper understanding of these pH-dependent reaction mechanisms is of great importance for protein engineering and drug design. We used high-resolution X-ray crystallography to analyze the sugar ring configurations of an oligosaccharide ligand during hydrolysis for the family 11 GH, and the results support the ¹S₃ → ⁴H₃ → ⁴C₁ conformational itinerary. These results indicate that sugar ring flexibility may help to distort and break the glycosidic bond. Constant pH molecular dynamics simulations and neutron crystallography demonstrate that the catalytic glutamate residue (E177) has alternate conformational changes to transfer a proton to cleave the glycosidic bond. Furthermore, a neutron crystallography analysis shows that the H-bond length between E177 and its nearby tyrosine residue (Y88) is shortened when the pH increases, preventing E177 from rotating downward and obtaining a proton from the solvent for catalysis. This result indicates that the H-bond length variation may play a key role in the pH-dependent reaction mechanism. In summary, our results demonstrate that both sugar ring flexibility and protein dynamics are important in the pH-dependent reaction mechanism and may help to engineer GHs with different pH optima.

Original language	English
Pages (from-to)	8058-8069
Number of pages	12
Journal	ACS Catalysis
Volume	8
Issue number	9
DOIs	https://doi.org/10.1021/acscatal.8b01472
State	Published - Sep 7 2018

Funding

Q.W. was supported by the National Natural Science Foundation of China (No. 31670790), the Open Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No.3144060008), the Fundamental Research Funds for the Central Universities (No. KYTZ201604), the Natural Science Foundation of Jiangsu Province of China (No. BK20161443), a China Spallation Neutron Source travel grant, the Qing Lan Project of Jiangsu Province, and the Six Talent Peaks Project of Jiangsu Province. Research at ORNL’s Spallation Neutron Source was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. We thank the SSRF beamlines BL19U1 and BL18U1 for X-ray data collection and the BIODIFF beamline at the FRM II research reactor at the Heinz Maier-Leibnitz Zentrum (MLZ) for neutron data collection. We thank Dr. Lei Wu at Nanjing Agricultural University, Dr. Wenfei Li and Dr. Weitong Ren at Nanjing University, and Dr. Changrui Lu at Donghua University for helpful discussions.

Funders	Funder number
Office of Basic Energy Sciences
Qing Lan Project of Jiangsu Province
Scientific User Facilities Division
State Key Laboratory of Natural Medicines, China Pharmaceutical University	3144060008
U.S. Department of Energy
National Natural Science Foundation of China	31670790
Natural Science Foundation of Jiangsu Province	BK20161443
Six Talent Peaks Project in Jiangsu Province
Fundamental Research Funds for the Central Universities	KYTZ201604

Keywords

H-bond length
constant pH molecular dynamics
glycoside hydrolase
neutron crystallography
pH-dependent reaction mechanism

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10.1021/acscatal.8b01472

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

abstract = "Glycoside hydrolases (GHs) commonly use the retaining or inverting mechanisms to hydrolyze carbohydrates, and the rates of catalysis are usually pH dependent. Deeper understanding of these pH-dependent reaction mechanisms is of great importance for protein engineering and drug design. We used high-resolution X-ray crystallography to analyze the sugar ring configurations of an oligosaccharide ligand during hydrolysis for the family 11 GH, and the results support the 1S3 → 4H3 → 4C1 conformational itinerary. These results indicate that sugar ring flexibility may help to distort and break the glycosidic bond. Constant pH molecular dynamics simulations and neutron crystallography demonstrate that the catalytic glutamate residue (E177) has alternate conformational changes to transfer a proton to cleave the glycosidic bond. Furthermore, a neutron crystallography analysis shows that the H-bond length between E177 and its nearby tyrosine residue (Y88) is shortened when the pH increases, preventing E177 from rotating downward and obtaining a proton from the solvent for catalysis. This result indicates that the H-bond length variation may play a key role in the pH-dependent reaction mechanism. In summary, our results demonstrate that both sugar ring flexibility and protein dynamics are important in the pH-dependent reaction mechanism and may help to engineer GHs with different pH optima.",

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author = "Zhihong Li and Xiaoshuai Zhang and Qingqing Wang and Chunran Li and Nianying Zhang and Xinkai Zhang and Birui Xu and Baoliang Ma and Schrader, {Tobias E.} and Leighton Coates and Andrey Kovalevsky and Yandong Huang and Qun Wan",

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AU - Zhang, Xiaoshuai

AU - Wang, Qingqing

AU - Li, Chunran

AU - Zhang, Nianying

AU - Zhang, Xinkai

AU - Xu, Birui

AU - Ma, Baoliang

AU - Schrader, Tobias E.

AU - Coates, Leighton

AU - Kovalevsky, Andrey

AU - Huang, Yandong

AU - Wan, Qun

PY - 2018/9/7

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N2 - Glycoside hydrolases (GHs) commonly use the retaining or inverting mechanisms to hydrolyze carbohydrates, and the rates of catalysis are usually pH dependent. Deeper understanding of these pH-dependent reaction mechanisms is of great importance for protein engineering and drug design. We used high-resolution X-ray crystallography to analyze the sugar ring configurations of an oligosaccharide ligand during hydrolysis for the family 11 GH, and the results support the 1S3 → 4H3 → 4C1 conformational itinerary. These results indicate that sugar ring flexibility may help to distort and break the glycosidic bond. Constant pH molecular dynamics simulations and neutron crystallography demonstrate that the catalytic glutamate residue (E177) has alternate conformational changes to transfer a proton to cleave the glycosidic bond. Furthermore, a neutron crystallography analysis shows that the H-bond length between E177 and its nearby tyrosine residue (Y88) is shortened when the pH increases, preventing E177 from rotating downward and obtaining a proton from the solvent for catalysis. This result indicates that the H-bond length variation may play a key role in the pH-dependent reaction mechanism. In summary, our results demonstrate that both sugar ring flexibility and protein dynamics are important in the pH-dependent reaction mechanism and may help to engineer GHs with different pH optima.

AB - Glycoside hydrolases (GHs) commonly use the retaining or inverting mechanisms to hydrolyze carbohydrates, and the rates of catalysis are usually pH dependent. Deeper understanding of these pH-dependent reaction mechanisms is of great importance for protein engineering and drug design. We used high-resolution X-ray crystallography to analyze the sugar ring configurations of an oligosaccharide ligand during hydrolysis for the family 11 GH, and the results support the 1S3 → 4H3 → 4C1 conformational itinerary. These results indicate that sugar ring flexibility may help to distort and break the glycosidic bond. Constant pH molecular dynamics simulations and neutron crystallography demonstrate that the catalytic glutamate residue (E177) has alternate conformational changes to transfer a proton to cleave the glycosidic bond. Furthermore, a neutron crystallography analysis shows that the H-bond length between E177 and its nearby tyrosine residue (Y88) is shortened when the pH increases, preventing E177 from rotating downward and obtaining a proton from the solvent for catalysis. This result indicates that the H-bond length variation may play a key role in the pH-dependent reaction mechanism. In summary, our results demonstrate that both sugar ring flexibility and protein dynamics are important in the pH-dependent reaction mechanism and may help to engineer GHs with different pH optima.

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

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