Functional and Structural Characterization of Diverse NfsB Chloramphenicol Reductase Enzymes from Human Pathogens

Mullowney, Michael W.; Maltseva, Natalia I.; Endres, Michael; Kim, Youngchang; Joachimiak, Andrzej; Crofts, Terence S.

doi:10.1128/spectrum.00139-22

Title: Functional and Structural Characterization of Diverse NfsB Chloramphenicol Reductase Enzymes from Human Pathogens

Journal Article · 23 February 2022 · Microbiology Spectrum

DOI: https://doi.org/10.1128/spectrum.00139-22 · OSTI ID:1846232

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Northwestern Univ., Evanston, IL (United States)
Univ. of Chicago, IL (United States); Argonne National Lab. (ANL), Argonne, IL (United States)
Argonne National Lab. (ANL), Argonne, IL (United States)
Univ. of Chicago, IL (United States); Argonne National Lab. (ANL), Argonne, IL (United States); Univ. of Chicago, IL (United States)

Phylogenetically diverse bacteria can carry out chloramphenicol reduction, but only a single enzyme has been described that efficiently catalyzes this reaction, the NfsB nitroreductase from Haemophilus influenzae strain KW20. Here, we tested the hypothesis that some NfsB homologs function as housekeeping enzymes with the potential to become chloramphenicol resistance enzymes. We found that expression of H. influenzae and Neisseria spp. nfsB genes, but not Pasteurella multocida nfsB, allows Escherichia coli to resist chloramphenicol by nitroreduction. Mass spectrometric analysis confirmed that purified H. influenzae and N. meningitides NfsB enzymes reduce chloramphenicol to amino-chloramphenicol, while kinetics analyses supported the hypothesis that chloramphenicol reduction is a secondary activity. We combined these findings with atomic resolution structures of multiple chloramphenicol-reducing NfsB enzymes to identify potential key substrate-binding pocket residues. Our work expands the chloramphenicol reductase family and provides mechanistic insights into how a housekeeping enzyme might confer antibiotic resistance.

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

Sponsoring Organization:: USDOE Office of Science (SC); National Institutes of Health (NIH); USDOE

Grant/Contract Number:: AC02-06CH11357; F32CA221327; HHSN272201700060C

OSTI ID:: 1846232

Alternate ID(s):: OSTI ID: 1896795

Journal Information:: Microbiology Spectrum, Vol. 10, Issue 2; ISSN 2165-0497

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: English

References (51)

Reduction of polynitroaromatic compounds: the bacterial nitroreductases Roldán, María Dolores; Pérez-Reinado, Eva; Castillo, Francisco FEMS Microbiology Reviews, Vol. 32, Issue 3 https://doi.org/10.1111/j.1574-6976.2008.00107.x	journal	May 2008
New LIC vectors for production of proteins from genes containing rare codons Eschenfeldt, William H.; Makowska-Grzyska, Magdalena; Stols, Lucy Journal of Structural and Functional Genomics, Vol. 14, Issue 4 https://doi.org/10.1007/s10969-013-9163-9	journal	September 2013
Linking Crystallographic Model and Data Quality Karplus, P. A.; Diederichs, K. Science, Vol. 336, Issue 6084 https://doi.org/10.1126/science.1218231	journal	May 2012
MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes Davis, I. W.; Murray, L. W.; Richardson, J. S. Nucleic Acids Research, Vol. 32, Issue Web Server https://doi.org/10.1093/nar/gkh398	journal	July 2004
Investigating the promiscuity of the chloramphenicol nitroreductase from Haemophilus influenzae towards the reduction of 4-nitrobenzene derivatives Green, Keith D.; Fosso, Marina Y.; Mayhoub, Abdelrahman S. Bioorganic & Medicinal Chemistry Letters, Vol. 29, Issue 9 https://doi.org/10.1016/j.bmcl.2019.02.025	journal	May 2019
Plazomicin Retains Antibiotic Activity against Most Aminoglycoside Modifying Enzymes Cox, Georgina; Ejim, Linda; Stogios, Peter J. ACS Infectious Diseases, Vol. 4, Issue 6 https://doi.org/10.1021/acsinfecdis.8b00001	journal	April 2018
Controlled Intracellular Processing of Fusion Proteins by TEV Protease Kapust, Rachel B.; Waugh, David S. Protein Expression and Purification, Vol. 19, Issue 2 https://doi.org/10.1006/prep.2000.1251	journal	July 2000
A new Coupling Component for Sulfanilamide Determination Bratton, A. Calvin; Marshall, E. K. Journal of Biological Chemistry, Vol. 128, Issue 2 https://doi.org/10.1016/S0021-9258(18)73708-3	journal	May 1939
Announcing the worldwide Protein Data Bank Berman, Helen; Henrick, Kim; Nakamura, Haruki Nature Structural & Molecular Biology, Vol. 10, Issue 12 https://doi.org/10.1038/nsb1203-980	journal	December 2003
Molecular basis of bacterial resistance to chloramphenicol and florfenicol Schwarz, Stefan; Kehrenberg, Corinna; Doublet, Benoît FEMS Microbiology Reviews, Vol. 28, Issue 5 https://doi.org/10.1016/j.femsre.2004.04.001	journal	November 2004
The antibiotic resistome: what's new? Perry, Julie Ann; Westman, Erin Louise; Wright, Gerard D. Current Opinion in Microbiology, Vol. 21 https://doi.org/10.1016/j.mib.2014.09.002	journal	October 2014
Chloramphenicol Is a Substrate for a Novel Nitroreductase Pathway in Haemophilus influenzae Smith, Arnold L.; Erwin, Alice L.; Kline, Toni Antimicrobial Agents and Chemotherapy, Vol. 51, Issue 8 https://doi.org/10.1128/AAC.00087-07	journal	August 2007
Methionine in proteins: The Cinderella of the proteinogenic amino acids Aledo, Juan C. Protein Science, Vol. 28, Issue 10 https://doi.org/10.1002/pro.3698	journal	August 2019
PROCHECK: a program to check the stereochemical quality of protein structures Laskowski, R. A.; MacArthur, M. W.; Moss, D. S. Journal of Applied Crystallography, Vol. 26, Issue 2 https://doi.org/10.1107/S0021889892009944	journal	April 1993
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs Altschul, Stephen F.; Madden, Thomas L.; Schäffer, Alejandro A. Nucleic Acids Research, Vol. 25, Issue 17, p. 3389-3402 https://doi.org/10.1093/nar/25.17.3389	journal	September 1997
High-throughput protein purification and quality assessment for crystallization Kim, Youngchang; Babnigg, Gyorgy; Jedrzejczak, Robert Methods, Vol. 55, Issue 1 https://doi.org/10.1016/j.ymeth.2011.07.010	journal	September 2011
Structural and Mechanistic Studies of Escherichia coli Nitroreductase with the Antibiotic Nitrofurazone Race, Paul R.; Lovering, Andrew L.; Green, Richard M. Journal of Biological Chemistry, Vol. 280, Issue 14 https://doi.org/10.1074/jbc.M409652200	journal	April 2005
Experimental Animal Models for Anaerobic Infections Onderdonk, A. B.; Kasper, D. L.; Mansheim, B. J. Clinical Infectious Diseases, Vol. 1, Issue 2 https://doi.org/10.1093/clinids/1.2.291	journal	March 1979
Structure and Function of CinD (YtjD) of Lactococcus lactis , a Copper-Induced Nitroreductase Involved in Defense against Oxidative Stress Mermod, Mélanie; Mourlane, Frédéric; Waltersperger, Sandro Journal of Bacteriology, Vol. 192, Issue 16 https://doi.org/10.1128/JB.00372-10	journal	August 2010
The Ferredoxin-dependent Reduction of Chloramphenicol by Clostridium acetobutylicum O'Brien, R. W.; Morris, J. G. Journal of General Microbiology, Vol. 67, Issue 3 https://doi.org/10.1099/00221287-67-3-265	journal	August 1971
Bacterial resistance to antibiotics: Enzymatic degradation and modification Wright, G. Advanced Drug Delivery Reviews, Vol. 57, Issue 10 https://doi.org/10.1016/j.addr.2005.04.002	journal	July 2005
Chloramphenicol Derivatives as Antibacterial and Anticancer Agents: Historic Problems and Current Solutions Dinos, George; Athanassopoulos, Constantinos; Missiri, Dionissia Antibiotics, Vol. 5, Issue 2 https://doi.org/10.3390/antibiotics5020020	journal	June 2016
Reduction of Chloromycetin and Related Compounds by Escherichia coli Smith, Grant N.; Worrel, C. S. Journal of Bacteriology, Vol. 65, Issue 3 https://doi.org/10.1128/jb.65.3.313-317.1953	journal	March 1953
Building Phylogenetic Trees from Molecular Data with MEGA Hall, B. G. Molecular Biology and Evolution, Vol. 30, Issue 5 https://doi.org/10.1093/molbev/mst012	journal	March 2013
Crossroads of Antibiotic Resistance and Biosynthesis Wencewicz, Timothy A. Journal of Molecular Biology, Vol. 431, Issue 18 https://doi.org/10.1016/j.jmb.2019.06.033	journal	August 2019
The evolution of substrate discrimination in macrolide antibiotic resistance enzymes Pawlowski, Andrew C.; Stogios, Peter J.; Koteva, Kalinka Nature Communications, Vol. 9, Issue 1 https://doi.org/10.1038/s41467-017-02680-0	journal	January 2018
The Buccaneer software for automated model building. 1. Tracing protein chains Cowtan, Kevin Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 9 https://doi.org/10.1107/S0907444906022116	journal	August 2006
Synthesis and Evaluation of Hetero- and Homodimers of Ribosome-Targeting Antibiotics: Antimicrobial Activity, in Vitro Inhibition of Translation, and Drug Resistance Berkov-Zrihen, Yifat; Green, Keith D.; Labby, Kristin J. Journal of Medicinal Chemistry, Vol. 56, Issue 13 https://doi.org/10.1021/jm400707f	journal	July 2013
HKL -3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes Minor, Wladek; Cymborowski, Marcin; Otwinowski, Zbyszek Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 8 https://doi.org/10.1107/S0907444906019949	journal	July 2006
Features and development of Coot Emsley, P.; Lohkamp, B.; Scott, W. G. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/S0907444910007493	journal	March 2010
Mechanism-Informed Refinement Reveals Altered Substrate-Binding Mode for Catalytically Competent Nitroreductase Pitsawong, Warintra; Haynes, Chad A.; Koder, Ronald L. Structure, Vol. 25, Issue 7 https://doi.org/10.1016/j.str.2017.05.002	journal	July 2017
Protein production by auto-induction in high-density shaking cultures Studier, F. William Protein Expression and Purification, Vol. 41, Issue 1, p. 207-234 https://doi.org/10.1016/j.pep.2005.01.016	journal	May 2005
Discovery and Characterization of a Nitroreductase Capable of Conferring Bacterial Resistance to Chloramphenicol Crofts, Terence S.; Sontha, Pratyush; King, Amber O. Cell Chemical Biology, Vol. 26, Issue 4 https://doi.org/10.1016/j.chembiol.2019.01.007	journal	April 2019
The Moderately Efficient Enzyme: Evolutionary and Physicochemical Trends Shaping Enzyme Parameters Bar-Even, Arren; Noor, Elad; Savir, Yonatan Biochemistry, Vol. 50, Issue 21 https://doi.org/10.1021/bi2002289	journal	May 2011
REFMAC 5 for the refinement of macromolecular crystal structures Murshudov, Garib N.; Skubák, Pavol; Lebedev, Andrey A. Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4 https://doi.org/10.1107/S0907444911001314	journal	March 2011
Protein Production for Structural Genomics Using E. coli Expression Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia Structural Genomics and Drug Discovery https://doi.org/10.1007/978-1-4939-0354-2_7	book	January 2014
MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods Tamura, K.; Peterson, D.; Peterson, N. Molecular Biology and Evolution, Vol. 28, Issue 10 https://doi.org/10.1093/molbev/msr121	journal	May 2011
MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets Kumar, Sudhir; Stecher, Glen; Tamura, Koichiro Molecular Biology and Evolution, Vol. 33, Issue 7 https://doi.org/10.1093/molbev/msw054	journal	March 2016
PHENIX: a comprehensive Python-based system for macromolecular structure solution Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221 https://doi.org/10.1107/S0907444909052925	journal	January 2010
Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active-site residues Hall, Kelsi R.; Robins, Katherine J.; Williams, Elsie M. eLife, Vol. 9 https://doi.org/10.7554/eLife.59081	journal	November 2020
Permeability of currently available microtiter plate sealing tapes fail to fulfil the requirements for aerobic microbial cultivation Sieben, Michaela; Giese, Heiner; Grosch, Jan-Hendrik Biotechnology Journal, Vol. 11, Issue 12 https://doi.org/10.1002/biot.201600054	journal	September 2016
Re-estimating annual deaths due to multidrug-resistant organism infections Burnham, Jason P.; Olsen, Margaret A.; Kollef, Marin H. Infection Control & Hospital Epidemiology, Vol. 40, Issue 1 https://doi.org/10.1017/ice.2018.304	journal	November 2018
Experimental phasing with SHELXC / D / E : combining chain tracing with density modification Sheldrick, George M. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/S0907444909038360	journal	March 2010
Reduction of Chloromycetin by a Cell-free Bacterial Extract and its Relation to Nitrite Reduction Egami, Fujio; Ebata, Mituo; Sato, Ryo Nature, Vol. 167, Issue 4238 https://doi.org/10.1038/167118a0	journal	January 1951
Relationship Between “Chloramphenicol Reductase Activity” and Chloramphenicol Resistance in Escherichia Coli, Merkel, Joseph R.; Steers, Edward Journal of Bacteriology, Vol. 66, Issue 4 https://doi.org/10.1128/jb.66.4.389-396.1953	journal	October 1953
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
Structures of Nitroreductase in Three States: EFFECTS OF INHIBITOR BINDING AND REDUCTION Haynes, Chad A.; Koder, Ronald L.; Miller, Anne-Frances Journal of Biological Chemistry, Vol. 277, Issue 13 https://doi.org/10.1074/jbc.M111334200	journal	January 2002
Gene Cloning, Purification, and Characterization of NfsB, a Minor Oxygen-Insensitive Nitroreductase from Escherichia coli, Similar in Biochemical Properties to FRase I, the Major Flavin Reductase in Vibrio fischeri Zenno, S.; Koike, H.; Tanokura, M. Journal of Biochemistry, Vol. 120, Issue 4 https://doi.org/10.1093/oxfordjournals.jbchem.a021473	journal	October 1996
Acquired Antibiotic Resistance Genes: An Overview van Hoek, Angela H. A. M.; Mevius, Dik; Guerra, Beatriz Frontiers in Microbiology, Vol. 2 https://doi.org/10.3389/fmicb.2011.00203	journal	January 2011
Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements Lutz, R. Nucleic Acids Research, Vol. 25, Issue 6, p. 1203-1210 https://doi.org/10.1093/nar/25.6.1203	journal	March 1997
Genetics of Nitrofurazone Resistance in Escherichia coli McCalla, D. R.; Kaiser, Christel; Green, M. H. L. Journal of Bacteriology, Vol. 133, Issue 1 https://doi.org/10.1128/jb.133.1.10-16.1978	journal	January 1978