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

Abstract

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 ismore » 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.« less

Authors:
 [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
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
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)
OSTI Identifier:
1570236
Grant/Contract Number:  
AC02-06CH11357
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 294; Journal Issue: 37; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; ApbE; NQR; RNF; flavin transferase; random sequential kinetic mechanism; pKa; general base catalysis; Histidine

Citation Formats

Fang, Xuan, Osipiuk, Jerzy, Chakravarthy, Srinivas, Yuan, Ming, Menzer, William M., Nissen, Devin, Liang, Pingdong, Raba, Daniel A., Tuz, Karina, Howard, Andrew J., Joachimiak, Andrzej, Minh, David D. L., and Juarez, Oscar. Conserved residue His-257 of Vibrio cholerae flavin transferase ApbE plays a critical role in substrate binding and catalysis. United States: N. p., 2019. Web. doi:10.1074/jbc.RA119.008261.
Fang, Xuan, Osipiuk, Jerzy, Chakravarthy, Srinivas, Yuan, Ming, Menzer, William M., Nissen, Devin, Liang, Pingdong, Raba, Daniel A., Tuz, Karina, Howard, Andrew J., Joachimiak, Andrzej, Minh, David D. L., & Juarez, Oscar. Conserved residue His-257 of Vibrio cholerae flavin transferase ApbE plays a critical role in substrate binding and catalysis. United States. doi:10.1074/jbc.RA119.008261.
Fang, Xuan, Osipiuk, Jerzy, Chakravarthy, Srinivas, Yuan, Ming, Menzer, William M., Nissen, Devin, Liang, Pingdong, Raba, Daniel A., Tuz, Karina, Howard, Andrew J., Joachimiak, Andrzej, Minh, David D. L., and Juarez, Oscar. Fri . "Conserved residue His-257 of Vibrio cholerae flavin transferase ApbE plays a critical role in substrate binding and catalysis". United States. doi:10.1074/jbc.RA119.008261. https://www.osti.gov/servlets/purl/1570236.
@article{osti_1570236,
title = {Conserved residue His-257 of Vibrio cholerae flavin transferase ApbE plays a critical role in substrate binding and catalysis},
author = {Fang, Xuan and Osipiuk, Jerzy and Chakravarthy, Srinivas and Yuan, Ming and Menzer, William M. and Nissen, Devin and Liang, Pingdong and Raba, Daniel A. and Tuz, Karina and Howard, Andrew J. and Joachimiak, Andrzej and Minh, David D. L. and Juarez, Oscar},
abstractNote = {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.},
doi = {10.1074/jbc.RA119.008261},
journal = {Journal of Biological Chemistry},
number = 37,
volume = 294,
place = {United States},
year = {2019},
month = {7}
}

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Works referenced in this record:

The Chemical and Biological Versatility of Riboflavin
journal, August 2000


Improved Treatment of Ligands and Coupling Effects in Empirical Calculation and Rationalization of p K a Values
journal, June 2011

  • Søndergaard, Chresten R.; Olsson, Mats H. M.; Rostkowski, Michał
  • Journal of Chemical Theory and Computation, Vol. 7, Issue 7
  • DOI: 10.1021/ct200133y

Three-dimensional structure of p-cresol methylhydroxylase (flavocytochrome c) from Pseudomonas putida at 3.0-.ANG. resolution
journal, January 1991

  • Mathews, F. Scott; Chen, Zhi Wei; Bellamy, Henry D.
  • Biochemistry, Vol. 30, Issue 1
  • DOI: 10.1021/bi00215a034

MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes
journal, July 2004

  • Davis, I. W.; Murray, L. W.; Richardson, J. S.
  • Nucleic Acids Research, Vol. 32, Issue Web Server
  • DOI: 10.1093/nar/gkh398

Covalent Flavinylation Is Essential for Efficient Redox Catalysis in Vanillyl-alcohol Oxidase
journal, December 1999

  • Fraaije, Marco W.; van den Heuvel, Robert H. H.; van Berkel, Willem J. H.
  • Journal of Biological Chemistry, Vol. 274, Issue 50
  • DOI: 10.1074/jbc.274.50.35514

Refinement of Macromolecular Structures by the Maximum-Likelihood Method
journal, May 1997

  • Murshudov, G. N.; Vagin, A. A.; Dodson, E. J.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 53, Issue 3
  • DOI: 10.1107/S0907444996012255

FAD Binding by ApbE Protein from Salmonella enterica: a New Class of FAD-Binding Proteins
journal, December 2010

  • Boyd, J. M.; Endrizzi, J. A.; Hamilton, T. L.
  • Journal of Bacteriology, Vol. 193, Issue 4
  • DOI: 10.1128/JB.00730-10

Purification and Characterization of the Recombinant Na + -Translocating NADH:Quinone Oxidoreductase from Vibrio cholerae
journal, March 2002

  • Barquera, Blanca; Hellwig, Petra; Zhou, Weidong
  • Biochemistry, Vol. 41, Issue 11
  • DOI: 10.1021/bi011873o

Urocanate reductase: identification of a novel anaerobic respiratory pathway in Shewanella oneidensis MR-1 : Urocanate reductase of
journal, November 2012

  • Bogachev, Alexander V.; Bertsova, Yulia V.; Bloch, Dmitry A.
  • Molecular Microbiology, Vol. 86, Issue 6
  • DOI: 10.1111/mmi.12067

Molecular determinants for FMN-binding in Desulfovibrio gigas flavoredoxin
journal, August 2007


TcpP protein is a positive regulator of virulence gene expression in Vibrio cholerae
journal, January 1998

  • Hase, C. C.; Mekalanos, J. J.
  • Proceedings of the National Academy of Sciences, Vol. 95, Issue 2
  • DOI: 10.1073/pnas.95.2.730

The Structural Biology Center 19ID undulator beamline: facility specifications and protein crystallographic results
journal, December 2005

  • Rosenbaum, Gerd; Alkire, Randy W.; Evans, Gwyndaf
  • Journal of Synchrotron Radiation, Vol. 13, Issue 1
  • DOI: 10.1107/S0909049505036721

The flavinyl transferase ApbE of Pseudomonas stutzeri matures the NosR protein required for nitrous oxide reduction
journal, February 2017

  • Zhang, Lin; Trncik, Christian; Andrade, Susana L. A.
  • Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1858, Issue 2
  • DOI: 10.1016/j.bbabio.2016.11.008

Crystal Structure of Glucooligosaccharide Oxidase from Acremonium strictum : A NOVEL FLAVINYLATION OF 6-S-CYSTEINYL, 8α-N1-HISTIDYL FAD
journal, September 2005

  • Huang, Chun-Hsiang; Lai, Wen-Lin; Lee, Meng-Hwan
  • Journal of Biological Chemistry, Vol. 280, Issue 46
  • DOI: 10.1074/jbc.M506078200

PHENIX : building new software for automated crystallographic structure determination
journal, October 2002

  • Adams, Paul D.; Grosse-Kunstleve, Ralf W.; Hung, Li-Wei
  • Acta Crystallographica Section D Biological Crystallography, Vol. 58, Issue 11
  • DOI: 10.1107/S0907444902016657

Generating disulfides enzymatically: Reaction products and electron acceptors of the endoplasmic reticulum thiol oxidase Ero1p
journal, January 2006

  • Gross, E.; Sevier, C. S.; Heldman, N.
  • Proceedings of the National Academy of Sciences, Vol. 103, Issue 2
  • DOI: 10.1073/pnas.0506448103

Expression and mutagenesis of the NqrC subunit of the NQR respiratory Na + pump from Vibrio cholerae with covalently attached FMN
journal, March 2001


Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: The current state of affairs
journal, January 1998

  • Mewies, Martin; McIntire, William S.; Scrutton, Nigel S.
  • Protein Science, Vol. 7, Issue 1
  • DOI: 10.1002/pro.5560070102

The Na + /e stoichiometry of the Na + -motive NADH : quinone oxidoreductase in Vibrio alginolyticus
journal, June 1997


The origin of the sodium-dependent NADH oxidation by the respiratory chain of Klebsiella pneumoniae
journal, April 2004


Covalently bound flavin in the NqrB and NqrC subunits of Na + -translocating NADH-quinone reductase from Vibrio alginolyticus
journal, May 2000


The CCP4 suite programs for protein crystallography
journal, September 1994


AMPylation of Rho GTPases by Vibrio VopS Disrupts Effector Binding and Downstream Signaling
journal, January 2009


UCSF Chimera?A visualization system for exploratory research and analysis
journal, January 2004

  • Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.
  • Journal of Computational Chemistry, Vol. 25, Issue 13
  • DOI: 10.1002/jcc.20084

Kinetic and Structural Insights into the Mechanism of AMPylation by VopS Fic Domain
journal, April 2010

  • Luong, Phi; Kinch, Lisa N.; Brautigam, Chad A.
  • Journal of Biological Chemistry, Vol. 285, Issue 26
  • DOI: 10.1074/jbc.M110.114884

Serine Protease Mechanism and Specificity
journal, December 2002


Origin and Evolution of the Sodium -Pumping NADH: Ubiquinone Oxidoreductase
journal, May 2014


Sodium Ion Cycle in Bacterial Pathogens: Evidence from Cross-Genome Comparisons
journal, September 2001


Characterization of the Pseudomonas aeruginosa NQR complex, a bacterial proton pump with roles in autopoisoning resistance
journal, August 2018

  • Raba, Daniel A.; Rosas-Lemus, Monica; Menzer, William M.
  • Journal of Biological Chemistry, Vol. 293, Issue 40
  • DOI: 10.1074/jbc.RA118.003194

Dissecting the Energetics of the Apoflavodoxin-FMN Complex
journal, March 2000

  • Lostao, Anabel; El Harrous, Mohamed; Daoudi, Fatna
  • Journal of Biological Chemistry, Vol. 275, Issue 13
  • DOI: 10.1074/jbc.275.13.9518

PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical p K a Predictions
journal, December 2010

  • Olsson, Mats H. M.; Søndergaard, Chresten R.; Rostkowski, Michal
  • Journal of Chemical Theory and Computation, Vol. 7, Issue 2
  • DOI: 10.1021/ct100578z

The TP0796 Lipoprotein of Treponema pallidum Is a Bimetal-dependent FAD Pyrophosphatase with a Potential Role in Flavin Homeostasis
journal, February 2013

  • Deka, Ranjit K.; Brautigam, Chad A.; Liu, Wei Z.
  • Journal of Biological Chemistry, Vol. 288, Issue 16
  • DOI: 10.1074/jbc.M113.449975

Covalent Binding of Flavins to RnfG and RnfD in the Rnf Complex from Vibrio cholerae
journal, October 2008

  • Backiel, Julianne; Zagorevski, Dmitri V.; Wang, Zhenyu
  • Biochemistry, Vol. 47, Issue 43
  • DOI: 10.1021/bi800920j

Flavin transferase: the maturation factor of flavin-containing oxidoreductases
journal, August 2018

  • Bogachev, Alexander V.; Baykov, Alexander A.; Bertsova, Yulia V.
  • Biochemical Society Transactions, Vol. 46, Issue 5
  • DOI: 10.1042/BST20180524

HKL -3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes
journal, July 2006

  • Minor, Wladek; Cymborowski, Marcin; Otwinowski, Zbyszek
  • Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 8
  • DOI: 10.1107/S0907444906019949

Structure of the Flavocoenzyme of Two Homologous Amine Oxidases:  Monomeric Sarcosine Oxidase and N -Methyltryptophan Oxidase
journal, April 1999

  • Wagner, Mary Ann; Khanna, Peeyush; Jorns, Marilyn Schuman
  • Biochemistry, Vol. 38, Issue 17
  • DOI: 10.1021/bi982955o

Enzyme Kinetics. Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Irwin H. Segel
journal, March 1976

  • Segal, Harold L.
  • The Quarterly Review of Biology, Vol. 51, Issue 1
  • DOI: 10.1086/409080

Coot model-building tools for molecular graphics
journal, November 2004

  • Emsley, Paul; Cowtan, Kevin
  • Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132
  • DOI: 10.1107/S0907444904019158

Mechanisms of flavoprotein-catalyzed reactions
journal, April 1989


Crystal structure of the ApbE protein (TM1553) from Thermotoga maritima at 1.58 Å resolution
journal, June 2006

  • Han, Gye Won; Sri Krishna, S.; Schwarzenbacher, Robert
  • Proteins: Structure, Function, and Bioinformatics, Vol. 64, Issue 4
  • DOI: 10.1002/prot.20950

The Fic Domain: Regulation of Cell Signaling by Adenylylation
journal, April 2009


Catalytically important flavin linked through a phosphoester bond in a eukaryotic fumarate reductase
journal, June 2018


Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration
journal, March 2005


Biochemical Evidence That Berberine Bridge Enzyme Belongs to a Novel Family of Flavoproteins Containing a Bi-covalently Attached FAD Cofactor
journal, May 2006

  • Winkler, Andreas; Hartner, Franz; Kutchan, Toni M.
  • Journal of Biological Chemistry, Vol. 281, Issue 30
  • DOI: 10.1074/jbc.M603267200

Alternative Pyrimidine Biosynthesis Protein ApbE Is a Flavin Transferase Catalyzing Covalent Attachment of FMN to a Threonine Residue in Bacterial Flavoproteins
journal, April 2013

  • Bertsova, Yulia V.; Fadeeva, Maria S.; Kostyrko, Vitaly A.
  • Journal of Biological Chemistry, Vol. 288, Issue 20
  • DOI: 10.1074/jbc.M113.455402

Molecular replacement with MOLREP
journal, December 2009

  • Vagin, Alexei; Teplyakov, Alexei
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 1
  • DOI: 10.1107/S0907444909042589

What’s in a covalent bond?: On the role and formation of covalently bound flavin cofactors
journal, June 2009


Studies on Succinate Dehydrogenase. 8alpha-Histidyl-FAD as the Active Center of Succinate Dehydrogenase
journal, March 1972


Genome Sequence of an Obligate Intracellular Pathogen of Humans: Chlamydia trachomatis
journal, October 1998


Dynamic energy dependency of Chlamydia trachomatis on host cell metabolism during intracellular growth: Role of sodium-based energetics in chlamydial ATP generation
journal, November 2017

  • Liang, Pingdong; Rosas-Lemus, Mónica; Patel, Dhwani
  • Journal of Biological Chemistry, Vol. 293, Issue 2
  • DOI: 10.1074/jbc.M117.797209

Structural basis of Fic-mediated adenylylation
journal, July 2010

  • Xiao, Junyu; Worby, Carolyn A.; Mattoo, Seema
  • Nature Structural & Molecular Biology, Vol. 17, Issue 8
  • DOI: 10.1038/nsmb.1867

The Binding of Riboflavin-5'-Phosphate in a Flavoprotein: Flavodoxin at 2.0-A Resolution
journal, December 1973

  • Watenpaugh, K. D.; Sieker, L. C.; Jensen, L. H.
  • Proceedings of the National Academy of Sciences, Vol. 70, Issue 12
  • DOI: 10.1073/pnas.70.12.3857

Two-Dimensional NMR-Studies of the Flavin Binding Site of Desulfovibrio vulgaris Flavodoxin in Its Threex States
journal, November 1994

  • Peelen, S.; Vervoort, J.
  • Archives of Biochemistry and Biophysics, Vol. 314, Issue 2
  • DOI: 10.1006/abbi.1994.1445

Molecular insights into the enzymatic diversity of flavin-trafficking protein (Ftp; formerly ApbE) in flavoprotein biogenesis in the bacterial periplasm
journal, December 2015

  • Deka, Ranjit K.; Brautigam, Chad A.; Liu, Wei Z.
  • MicrobiologyOpen, Vol. 5, Issue 1
  • DOI: 10.1002/mbo3.306

Kinetic characterization of Vibrio cholerae ApbE: Substrate specificity and regulatory mechanisms
journal, October 2017


Role of sodium bioenergetics in Vibrio cholerae
journal, May 2001


The concerted action of a positive charge and hydrogen bonds dynamically regulates the p K a of the nucleophilic cysteine in the NrdH-redoxin family : p
journal, December 2013

  • Van Laer, Koen; Oliveira, Margarida; Wahni, Khadija
  • Protein Science, Vol. 23, Issue 2
  • DOI: 10.1002/pro.2397

    Works referencing / citing this record:

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

    • Bertsova, Yulia V.; Serebryakova, Marina V.; Anashkin, Victor A.
    • FEMS Microbiology Letters, Vol. 366, Issue 22
    • DOI: 10.1093/femsle/fnz252