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Title: Conserved residue His-257 of Vibrio cholerae flavin transferase ApbE plays a critical role in substrate binding and catalysis

Journal Article · · Journal of Biological Chemistry
 [1];  [2];  [3];  [1]; ORCiD logo [1];  [1];  [1];  [1];  [1];  [1];  [2]; ORCiD logo [4];  [1]
  1. Illinois Inst. of Technology, Chicago, IL (United States). Dept. of Biological Science
  2. Univ. of Chicago, Chicago, IL (United States). Center for Structural Genomics of Infectious Diseases (CSGID), Consortium for Advanced Science and Engineering; Argonne National Lab. (ANL), Lemont, IL (United States). Biosciences Division, Structural Biology Center
  3. Illinois Inst. of Technology, Chicago, IL (United States). Dept. of Biological Science; Argonne National Lab. (ANL), Lemont, IL (United States). Biophysics Collaborative Access Team, Advanced Proton Source
  4. Illinois Inst. of Technology, Chicago, IL (United States). Dept. of Chemistry
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The flavin transferase ApbE plays essential roles in bacterial physiology, covalently incorporating FMN cofactors into numerous respiratory enzymes that use the integrated cofactors as electron carriers. In this work we performed a detailed kinetic and structural characterization of Vibrio cholerae WT ApbE and mutants of the conserved residue His-257, to understand its role in substrate binding and in the catalytic mechanism of this family. Bi-substrate kinetic experiments revealed that ApbE follows a random Bi Bi sequential kinetic mechanism, in which a ternary complex is formed, indicating that both substrates must be bound to the enzyme for the reaction to proceed. Steady-state kinetic analyses show that the turnover rates of His-257 mutants are significantly smaller than those of WT ApbE, and have increased Km values for both substrates, indicating that the His-257 residue plays important roles in catalysis and in enzyme-substrate complex formation. Analyses of the pH dependence of ApbE activity indicate that the pKa of the catalytic residue (pKES1) increases by 2 pH units in the His-257 mutants, suggesting that this residue plays a role in substrate deprotonation. The crystal structures of WT ApbE and an H257G mutant were determined at 1.61 and 1.92 Å resolutions, revealing that His-257 is located in the catalytic site and that the substitution does not produce major conformational changes. In this paper, we propose a reaction mechanism in which His-257 acts as a general base that deprotonates the acceptor residue, which subsequently performs a nucleophilic attack on FAD for flavin transfer.

Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Organization:
National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID); Illinois Inst. of Technology, Chicago, IL (United States); USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Grant/Contract Number:
AC02-06CH11357
OSTI ID:
1570236
Journal Information:
Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 37 Vol. 294; ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular BiologyCopyright Statement
Country of Publication:
United States
Language:
English

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Cited By (1)

Mutational analysis of the flavinylation and binding motifs in two protein targets of the flavin transferase ApbE journal November 2019

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
ApbE
Histidine
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RNF
flavin transferase
general base catalysis
pKa
random sequential kinetic mechanism