Direct observation of intermediates in the SufS cysteine desulfurase reaction reveals functional roles of conserved active-site residues

Blahut, Matthew; Wise, Courtney E.; Bruno, Michael R.; Dong, Guangchao; Makris, Thomas M.; Frantom, Patrick A.; Dunkle, Jack A.; Outten, F. Wayne

doi:10.1074/jbc.ra119.009471

Title: Direct observation of intermediates in the SufS cysteine desulfurase reaction reveals functional roles of conserved active-site residues

Journal Article · Thu Jun 27 00:00:00 EDT 2019 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.ra119.009471· OSTI ID:1577152

Blahut, Matthew ^[1]; Wise, Courtney E. ^[1]; Bruno, Michael R. ^[2]; Dong, Guangchao ^[1]; Makris, Thomas M. ^[1];

^[2];

^[1]

Univ. of South Carolina, Columbia, SC (United States)
Univ. of Alabama, Tuscaloosa, AL (United States)

Iron-sulfur (Fe-S) clusters are necessary for the proper functioning of numerous metalloproteins. Fe-S cluster (Isc) and sulfur utilization factor (Suf) pathways are the key biosynthetic routes responsible for generating these Fe-S cluster prosthetic groups in Escherichia coli. Although Isc dominates under normal conditions, Suf takes over during periods of iron depletion and oxidative stress. Sulfur acquisition via these systems relies on the ability to remove sulfur from free cysteine using a cysteine desulfurase mechanism. In the Suf pathway, the dimeric SufS protein uses the cofactor pyridoxal 5'-phosphate (PLP) to abstract sulfur from free cysteine, resulting in the production of alanine and persulfide. Despite much progress, the stepwise mechanism by which this PLP-dependent enzyme operates remains unclear. Here, using rapid-mixing kinetics in conjunction with X-ray crystallography, we analyzed the pre-steady-state kinetics of this process while assigning early intermediates of the mechanism. We employed H123A and C364A SufS variants to trap Cys-aldimine and Cys-ketimine intermediates of the cysteine desulfurase reaction, enabling direct observations of these intermediates and associated conformational changes of the SufS active site. Of note, we propose that Cys-364 is essential for positioning the Cys-aldimine for Cα deprotonation, His-123 acts to protonate the Ala-enamine intermediate, and Arg-56 facilitates catalysis by hydrogen bonding with the sulfhydryl of Cys-aldimine. Our results, along with previous SufS structural findings, suggest a detailed model of the SufS-catalyzed reaction from Cys binding to C–S bond cleavage and indicate that Arg-56, His-123, and Cys-364 are critical SufS residues in this C–S bond cleavage pathway.

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Research Organization:: Argonne National Lab. (ANL), Argonne, IL (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES)

Contributing Organization:: Advanced Photon Source (APS), Argonne National Laboratory (ANL), Argonne, IL (US)

Grant/Contract Number:: S10_RR25528; S10_RR028976; W-31-109-Eng-38; GM112919

OSTI ID:: 1577152

Journal Information:: Journal of Biological Chemistry, Vol. 294, Issue 33; 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: 17 works

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References (45)

A nifS -like Gene, csdB , Encodes an Escherichia coli Counterpart of Mammalian Selenocysteine Lyase : GENE CLONING, PURIFICATION, CHARACTERIZATION AND PRELIMINARY X-RAY CRYSTALLOGRAPHIC STUDIES Mihara, Hisaaki; Maeda, Masaki; Fujii, Tomomi Journal of Biological Chemistry, Vol. 274, Issue 21 https://doi.org/10.1074/jbc.274.21.14768	journal	May 1999
The E. coli SufS-SufE sulfur transfer system is more resistant to oxidative stress than IscS-IscU Dai, Yuyuan; Outten, F. Wayne FEBS Letters, Vol. 586, Issue 22 https://doi.org/10.1016/j.febslet.2012.10.001	journal	October 2012
Iron/sulfur proteins biogenesis in prokaryotes: Formation, regulation and diversity Roche, Béatrice; Aussel, Laurent; Ezraty, Benjamin Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1827, Issue 3 https://doi.org/10.1016/j.bbabio.2012.12.010	journal	March 2013
Mechanism-Based Trapping of the Quinonoid Intermediate by Using the K276R Mutant of PLP-Dependent 3-Aminobenzoate Synthase PctV in the Biosynthesis of Pactamycin Hirayama, Akane; Miyanaga, Akimasa; Kudo, Fumitaka ChemBioChem, Vol. 16, Issue 17 https://doi.org/10.1002/cbic.201500426	journal	October 2015
Structure of a NifS Homologue: X-ray Structure Analysis of CsdB, an Escherichia coli Counterpart of Mammalian Selenocysteine Lyase ^† ^, ^‡ Fujii, Tomomi; Maeda, Masaki; Mihara, Hisaaki Biochemistry, Vol. 39, Issue 6 https://doi.org/10.1021/bi991732a	journal	February 2000
Reaction Mechanism and Molecular Basis for Selenium/Sulfur Discrimination of Selenocysteine Lyase Omi, Rie; Kurokawa, Suguru; Mihara, Hisaaki Journal of Biological Chemistry, Vol. 285, Issue 16 https://doi.org/10.1074/jbc.M109.084475	journal	February 2010
Mechanism for the Desulfurization of L-Cysteine Catalyzed by the nifS Gene Product Zheng, Limin; White, Robert H.; Cash, Valerie L. Biochemistry, Vol. 33, Issue 15 https://doi.org/10.1021/bi00181a031	journal	April 1994
Free energy landscapes of prototropic tautomerism in pyridoxal 5′-phosphate schiff bases at the active site of an enzyme in aqueous medium Soniya, Kumari; Chandra, Amalendu Journal of Computational Chemistry, Vol. 39, Issue 21 https://doi.org/10.1002/jcc.25338	journal	May 2018
Analysis of the E. coli NifS CsdB protein at 2.0 Å reveals the structural basis for perselenide and persulfide intermediate formation Lima, Christopher D. Journal of Molecular Biology, Vol. 315, Issue 5 https://doi.org/10.1006/jmbi.2001.5308	journal	February 2002
Controlling reaction specificity in pyridoxal phosphate enzymes Toney, Michael D. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol. 1814, Issue 11 https://doi.org/10.1016/j.bbapap.2011.05.019	journal	November 2011
Structural Evidence for Dimer-Interface-Driven Regulation of the Type II Cysteine Desulfurase, SufS Dunkle, Jack A.; Bruno, Michael R.; Outten, F. Wayne Biochemistry, Vol. 58, Issue 6 https://doi.org/10.1021/acs.biochem.8b01122	journal	December 2018
Iron-sulfur clusters: ever-expanding roles Fontecave, Marc Nature Chemical Biology, Vol. 2, Issue 4 https://doi.org/10.1038/nchembio0406-171	journal	April 2006
SufC: an unorthodox cytoplasmic ABC/ATPase required for [Fe-S] biogenesis under oxidative stress Nachin, L. The EMBO Journal, Vol. 22, Issue 3 https://doi.org/10.1093/emboj/cdg061	journal	February 2003
Sulfur Mobilization in Cyanobacteria: THE CATALYTIC MECHANISM OF L-CYSTINE C-S LYASE (C-DES) FROM SYNECHOCYSTIS Campanini, Barbara; Schiaretti, Francesca; Abbruzzetti, Stefania Journal of Biological Chemistry, Vol. 281, Issue 50 https://doi.org/10.1074/jbc.M607098200	journal	October 2006
Cysteine Sulfinate Desulfinase, a NIFS-like Protein of Escherichia coli with Selenocysteine Lyase and Cysteine Desulfurase Activities : GENE CLONING, PURIFICATION, AND CHARACTERIZATION OF A NOVEL PYRIDOXAL ENZYME Mihara, Hisaaki; Kurihara, Tatsuo; Yoshimura, Tohru Journal of Biological Chemistry, Vol. 272, Issue 36 https://doi.org/10.1074/jbc.272.36.22417	journal	September 1997
Kinetic and Structural Characterization of Slr0077/SufS, the Essential Cysteine Desulfurase from Synechocystis sp. PCC 6803 ^† ^, ^‡ Tirupati, Bhramara; Vey, Jessica Lynn; Drennan, Catherine L. Biochemistry, Vol. 43, Issue 38 https://doi.org/10.1021/bi0491447	journal	September 2004
Snapshots of the Cystine Lyase C-DES during Catalysis: STUDIES IN SOLUTION AND IN THE CRYSTALLINE STATE Kaiser, Jens T.; Bruno, Stefano; Clausen, Tim Journal of Biological Chemistry, Vol. 278, Issue 1 https://doi.org/10.1074/jbc.M209862200	journal	October 2002
Genetic Analysis of the isc Operon in Escherichia coli Involved in the Biogenesis of Cellular Iron-Sulfur Proteins Tokumoto, U.; Takahashi, Y. Journal of Biochemistry, Vol. 130, Issue 1 https://doi.org/10.1093/oxfordjournals.jbchem.a002963	journal	July 2001
SufE Transfers Sulfur from SufS to SufB for Iron-Sulfur Cluster Assembly Layer, Gunhild; Gaddam, S. Aparna; Ayala-Castro, Carla N. Journal of Biological Chemistry, Vol. 282, Issue 18 https://doi.org/10.1074/jbc.M608555200	journal	March 2007
Structure of External Aldimine of Escherichia coli CsdB, an IscS/Nifs Homolog: Implications for Its Specificity toward Selenocysteine Mihara, H.; Fujii, T.; Kato, S. -i. Journal of Biochemistry, Vol. 131, Issue 5 https://doi.org/10.1093/oxfordjournals.jbchem.a003151	journal	May 2002
Kinetic Analysis of Cysteine Desulfurase CD0387 from Synechocystis sp. PCC 6803: Formation of the Persulfide Intermediate Behshad, Elham; Bollinger, J. Martin Biochemistry, Vol. 48, Issue 50 https://doi.org/10.1021/bi802161u	journal	December 2009
Pyridoxal Phosphate Enzymes: Mechanistic, Structural, and Evolutionary Considerations Eliot, Andrew C.; Kirsch, Jack F. Annual Review of Biochemistry, Vol. 73, Issue 1 https://doi.org/10.1146/annurev.biochem.73.011303.074021	journal	June 2004
Biogenesis of Fe-S Cluster by the Bacterial Suf System: SufS AND SufE FORM A NEW TYPE OF CYSTEINE DESULFURASE Loiseau, Laurent; Ollagnier-de-Choudens, Sandrine; Nachin, Laurence Journal of Biological Chemistry, Vol. 278, Issue 40 https://doi.org/10.1074/jbc.M305953200	journal	July 2003
A suf operon requirement for Fe-S cluster assembly during iron starvation in Escherichia coli: suf operon role during iron starvation Outten, F. Wayne; Djaman, Ouliana; Storz, Gisela Molecular Microbiology, Vol. 52, Issue 3 https://doi.org/10.1111/j.1365-2958.2004.04025.x	journal	April 2004
Crystal structure of the nickel–iron hydrogenase from Desulfovibrio gigas Volbeda, Anne; Charon, Marie-Hélène; Piras, Claudine Nature, Vol. 373, Issue 6515 https://doi.org/10.1038/373580a0	journal	February 1995
Evidence for a Two-Base Mechanism Involving Tyrosine-265 from Arginine-219 Mutants of Alanine Racemase ^† Sun, Shaoxian; Toney, Michael D. Biochemistry, Vol. 38, Issue 13 https://doi.org/10.1021/bi982924t	journal	March 1999
Structure, mechanism, and substrate specificity of kynureninase Phillips, Robert S. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol. 1814, Issue 11 https://doi.org/10.1016/j.bbapap.2010.12.003	journal	November 2011
Chloroplast iron-sulfur cluster protein maturation requires the essential cysteine desulfurase CpNifS Van Hoewyk, D.; Abdel-Ghany, S. E.; Cohu, C. M. Proceedings of the National Academy of Sciences, Vol. 104, Issue 13 https://doi.org/10.1073/pnas.0700774104	journal	March 2007
MUSCLE: multiple sequence alignment with high accuracy and high throughput Edgar, R. C. Nucleic Acids Research, Vol. 32, Issue 5, p. 1792-1797 https://doi.org/10.1093/nar/gkh340	journal	March 2004
Current Advances on Structure-Function Relationships of Pyridoxal 5′-Phosphate-Dependent Enzymes Liang, Jing; Han, Qian; Tan, Yang Frontiers in Molecular Biosciences, Vol. 6 https://doi.org/10.3389/fmolb.2019.00004	journal	March 2019
Mechanistic characterization of sulfur transfer from cysteine desulfurase SufS to the iron-sulfur scaffold SufU in Bacillus subtilis Albrecht, Alexander G.; Peuckert, Florian; Landmann, Hannes FEBS Letters, Vol. 585, Issue 3 https://doi.org/10.1016/j.febslet.2011.01.005	journal	January 2011
The SufE Protein and the SufBCD Complex Enhance SufS Cysteine Desulfurase Activity as Part of a Sulfur Transfer Pathway for Fe-S Cluster Assembly in Escherichia coli Outten, F. Wayne; Wood, Matthew J.; Muñoz, F. Michael Journal of Biological Chemistry, Vol. 278, Issue 46 https://doi.org/10.1074/jbc.M308004200	journal	August 2003
Protected Sulfur Transfer Reactions by the Escherichia coli Suf System Selbach, Bruna P.; Pradhan, Pradyumna K.; Dos Santos, Patricia C. Biochemistry, Vol. 52, Issue 23 https://doi.org/10.1021/bi4001479	journal	May 2013
SufE D74R Substitution Alters Active Site Loop Dynamics To Further Enhance SufE Interaction with the SufS Cysteine Desulfurase Dai, Yuyuan; Kim, Dokyong; Dong, Guangchao Biochemistry, Vol. 54, Issue 31 https://doi.org/10.1021/acs.biochem.5b00663	journal	July 2015
pH Studies on the Mechanism of the Pyridoxal Phosphate-Dependent Dialkylglycine Decarboxylase ^† Zhou, Xianzhi; Toney, Michael D. Biochemistry, Vol. 38, Issue 1 https://doi.org/10.1021/bi981455s	journal	January 1999
Crystal Structure of Bacillus subtilis Cysteine Desulfurase SufS and Its Dynamic Interaction with Frataxin and Scaffold Protein SufU Blauenburg, Bastian; Mielcarek, Andreas; Altegoer, Florian PLOS ONE, Vol. 11, Issue 7 https://doi.org/10.1371/journal.pone.0158749	journal	July 2016
Escherichia coli SufE Sulfur Transfer Protein Modulates the SufS Cysteine Desulfurase through Allosteric Conformational Dynamics Singh, Harsimran; Dai, Yuyuan; Outten, F. Wayne Journal of Biological Chemistry, Vol. 288, Issue 51 https://doi.org/10.1074/jbc.M113.525709	journal	November 2013
Changes in Protein Dynamics in Escherichia coli SufS Reveal a Possible Conserved Regulatory Mechanism in Type II Cysteine Desulfurase Systems Kim, Dokyong; Singh, Harsimran; Dai, Yuyuan Biochemistry, Vol. 57, Issue 35 https://doi.org/10.1021/acs.biochem.7b01275	journal	March 2018
Shared-intermediates in the biosynthesis of thio-cofactors: Mechanism and functions of cysteine desulfurases and sulfur acceptors Black, Katherine A.; Dos Santos, Patricia C. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol. 1853, Issue 6 https://doi.org/10.1016/j.bbamcr.2014.10.018	journal	June 2015
The CsdA cysteine desulphurase promotes Fe/S biogenesis by recruiting Suf components and participates to a new sulphur transfer pathway by recruiting CsdL (ex-YgdL), a ubiquitin-modifying-like protein: The E.âcoli CsdA cysteine desulphurase Trotter, Valentine; Vinella, Daniel; Loiseau, Laurent Molecular Microbiology, Vol. 74, Issue 6 https://doi.org/10.1111/j.1365-2958.2009.06954.x	journal	December 2009
Detection of a gem-diamine and a stable quinonoid intermediate in the reaction catalyzed by serine–glyoxylate aminotransferase from Hyphomicrobium methylovorum Karsten, William E.; Cook, Paul F. Biochimica et Biophysica Acta (BBA) - General Subjects, Vol. 1790, Issue 6 https://doi.org/10.1016/j.bbagen.2009.02.012	journal	June 2009
Kinetic and Mutational Studies of Three NifS Homologs from Escherichia coli: Mechanistic Difference between L-Cysteine Desulfurase and L-Selenocysteine Lyase Reactions Mihara, H.; Kurihara, T.; Yoshimura, T. Journal of Biochemistry, Vol. 127, Issue 4 https://doi.org/10.1093/oxfordjournals.jbchem.a022641	journal	April 2000
Structural Changes during Cysteine Desulfurase CsdA and Sulfur Acceptor CsdE Interactions Provide Insight into the trans -Persulfuration Kim, Sunmin; Park, SangYoun Journal of Biological Chemistry, Vol. 288, Issue 38 https://doi.org/10.1074/jbc.M113.480277	journal	August 2013
Mechanisms of iron–sulfur cluster assembly: the SUF machinery Fontecave, M.; Choudens, S. Ollagnier de; Py, B. JBIC Journal of Biological Inorganic Chemistry, Vol. 10, Issue 7 https://doi.org/10.1007/s00775-005-0025-1	journal	October 2005
Mechanisms of iron–sulfur cluster assembly: the SUF machinery Fontecave, M.; Ollagnier de Choudens, S.; Py, B. JBIC Journal of Biological Inorganic Chemistry, Vol. 11, Issue 1 https://doi.org/10.1007/s00775-005-0051-z	journal	November 2005

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59 BASIC BIOLOGICAL SCIENCES
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
X-ray crystallography
iron-sulfur protein
pre-steady-state kinetics
enzyme catalysis
enzyme mechanism
cysteine desulfurase
cysteine sulfur bond cleavage
PLP
PLP-dependent sulfur abstraction
SufS

Title: Direct observation of intermediates in the SufS cysteine desulfurase reaction reveals functional roles of conserved active-site residues

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