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

Zhihong Li, Xiaoshuai Zhang, Chunran Li, Andrey Kovalevsky, Qun Wan

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4 Scopus citations

Abstract

XynII is a family 11 glycoside hydrolase that uses the retaining mechanism for catalysis. In the active site, E177 works as the acid/base and E86 works as the nucleophile. Mutating an uncharged residue (N44) to an acidic residue (D) near E177 decreases the enzyme’s optimal pH by ~ 1.0 unit. D44 was previously suggested to be a second proton carrier for catalysis. To test this hypothesis, we abolished the activity of E177 by mutating it to be Q, and mutated N44 to be D or E. These double mutants have dramatically decreased activities. Our high-resolution crystallographic structures and the microscopic pKa calculations show that D44 has similar position and pKa value during catalysis, indicating that D44 changes electrostatics around E177, which makes it prone to rotate as the acid/base in acidic conditions, thus decreases the pH optimum. Our results could be helpful to design enzymes with different pH optimum.

Original languageEnglish
Pages (from-to)671-680
Number of pages10
JournalProtein Journal
Volume39
Issue number6
DOIs
StatePublished - Dec 2020

Funding

Q.W. was supported by the National Natural Science Foundation of China (No. 31670790), the Fundamental Research Funds for the Central Universities (No. KYXK202009), a Chinese Spallation Neutron Source User Special Grant, the Qing Lan Project of Jiangsu Province, and the Six Talent Peaks Project of Jiangsu Province. We thank the staff of the BL17U, BL18U1 and BL19U1 beamlines at Shanghai Synchrotron Radiation Facility, Shanghai, P.R. China, for assistance during X-ray data collection. Research at ORNL’s HFIR and Spallation Neutron Source was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. Q.W. was supported by the National Natural Science Foundation of China (No. 31670790), the Fundamental Research Funds for the Central Universities (No. KYXK202009), a Chinese Spallation Neutron Source User Special Grant, the Qing Lan Project of Jiangsu Province, and the Six Talent Peaks Project of Jiangsu Province. We thank the staff of the BL17U, BL18U1 and BL19U1 beamlines at Shanghai Synchrotron Radiation Facility, Shanghai, P.R. China, for assistance during X-ray data collection. Research at ORNL’s HFIR and Spallation Neutron Source was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy.

Keywords

  • Electrostatics
  • General acid/base catalysis
  • Glycoside hydrolase
  • Mutagenesis
  • X-ray crystallography
  • pH-dependent activity

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