Nα-acetyl-L-ornithine deacetylase from Escherichia coli and a ninhydrin-based assay to enable inhibitor identification

Kelley, Emma H.; Osipiuk, Jerzy; Korbas, Malgorzata; Endres, Michael; Bland, Alayna; Ehrman, Victoria; Joachimiak, Andrzej; Olsen, Kenneth W.; Becker, Daniel P.

doi:10.3389/fchem.2024.1415644

N^α-acetyl-L-ornithine deacetylase from Escherichia coli and a ninhydrin-based assay to enable inhibitor identification

Journal Article · Thu Jul 11 00:00:00 UTC 2024 · Frontiers in Chemistry

DOI:https://doi.org/10.3389/fchem.2024.1415644· OSTI ID:2470259

Kelley, Emma H. ^[1]; Osipiuk, Jerzy ^[2]; Korbas, Malgorzata ^[3]; Endres, Michael ^[4]; Bland, Alayna ^[1]; Ehrman, Victoria ^[1]; Joachimiak, Andrzej ^[2]; Olsen, Kenneth W. ^[1]; Becker, Daniel P. ^[1]

Loyola University Chicago, IL (United States)
Argonne National Laboratory (ANL), Argonne, IL (United States); Univ. of Chicago, IL (United States)
Univ. of Saskatchewan, Saskatoon, SK (Canada). Canadian Light Source, Inc.
Univ. of Chicago, IL (United States)

Bacteria are becoming increasingly resistant to antibiotics, therefore there is an urgent need for new classes of antibiotics to fight antibiotic resistance. Mammals do not express N^α -acetyl-L-ornithine deacetylase (ArgE), an enzyme that is critical for bacterial survival and growth, thus ArgE represents a promising new antibiotic drug target, as inhibitors would not suffer from mechanism-based toxicity. A new ninhydrin-based assay was designed and validated that included the synthesis of the substrate analog N⁵, N⁵-di-methyl N^α-acetyl-L-ornithine (k_cat/K_m = 7.32 ± 0.94 × 10⁴ M^-1s^-1). This new assay enabled the screening of potential inhibitors that absorb in the UV region, and thus is superior to the established 214 nm assay. Using this new ninhydrin-based assay, captopril was confirmed as an ArgE inhibitor (IC₅₀ = 58.7 μM; K_i = 37.1 ± 0.85 μM), and a number of phenylboronic acid derivatives were identified as inhibitors, including 4-(diethylamino)phenylboronic acid (IC₅₀ = 50.1 μM). Selected inhibitors were also tested in a thermal shift assay with ArgE using SYPRO Orange dye against Escherichia coli ArgE to observe the stability of the enzyme in the presence of inhibitors (captopril K_i = 35.9 ± 5.1 μM). The active site structure of di-Zn EcArgE was confirmed using X-ray absorption spectroscopy, and we reported two X-ray crystal structures of E. coli ArgE. In summary, we describe the development of a new ninhydrin-based assay for ArgE, the identification of captopril and phenylboronic acids as ArgE inhibitors, thermal shift studies with ArgE + captopril, and the first two published crystal structures of ArgE (mono-Zn and di-Zn).

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS); Brookhaven National Laboratory (BNL), Upton, NY (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF); USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-06CH11357; SC0012704

OSTI ID:: 2470259

Journal Information:: Frontiers in Chemistry, Journal Name: Frontiers in Chemistry Vol. 12; ISSN 2296-2646

Publisher:: Frontiers Research FoundationCopyright Statement

Country of Publication:: United States

Language:: English

References (61)

Scalable molecular dynamics with NAMD Phillips, James C.; Braun, Rosemary; Wang, Wei Journal of Computational Chemistry, Vol. 26, Issue 16, p. 1781-1802 https://doi.org/10.1002/jcc.20289	journal	January 2005
Basic Local Alignment Search Tool Altschul, S. Journal of Molecular Biology, Vol. 215, Issue 3 https://doi.org/10.1006/jmbi.1990.9999	journal	October 1990
The Crystal Structure of Tris-inhibited Phospholipase C from Bacillus cereus at 1·9 Å Resolution Hansen, Sissel; Hansen, Lars Kristian; Hough, Edward Journal of Molecular Biology, Vol. 231, Issue 3 https://doi.org/10.1006/jmbi.1993.1333	journal	June 1993
Inhibitors of N α-acetyl-l-ornithine deacetylase: synthesis, characterization and analysis of their inhibitory potency Hlaváček, J.; Pícha, J.; Vaněk, V. Amino Acids, Vol. 38, Issue 4 https://doi.org/10.1007/s00726-009-0326-8	journal	August 2009
Regulation of arginine biosynthesis, catabolism and transport in Escherichia coli Charlier, Daniel; Bervoets, Indra Amino Acids, Vol. 51, Issue 8 https://doi.org/10.1007/s00726-019-02757-8	journal	July 2019
Characterization of the catalytically active Mn(II)-loaded argE-encoded N-acetyl-l-ornithine deacetylase from Escherichia coli McGregor, Wade C.; Swierczek, Sabina I.; Bennett, Brian JBIC Journal of Biological Inorganic Chemistry, Vol. 12, Issue 5 https://doi.org/10.1007/s00775-007-0211-4	journal	February 2007
Metal–ion promiscuity of microbial enzyme DapE at its second metal-binding site Paul, Atanuka; Mishra, Sabyashachi JBIC Journal of Biological Inorganic Chemistry, Vol. 26, Issue 5 https://doi.org/10.1007/s00775-021-01875-7	journal	July 2021
X-ray absorption spectroscopy Yano, Junko; Yachandra, Vittal K. Photosynthesis Research, Vol. 102, Issue 2-3 https://doi.org/10.1007/s11120-009-9473-8	journal	August 2009
VMD: Visual molecular dynamics Humphrey, William; Dalke, Andrew; Schulten, Klaus Journal of Molecular Graphics, Vol. 14, Issue 1 https://doi.org/10.1016/0263-7855(96)00018-5	journal	February 1996
Acetylornithine deacetylase, succinyldiaminopimelate desuccinylase and carboxypeptidase G2 are evolutionarily related Boyen, Anne; Charlier, Daniel; Charlier, Josée Gene, Vol. 116, Issue 1 https://doi.org/10.1016/0378-1119(92)90621-u	journal	July 1992
Mono-N-acyl-2,6-diaminopimelic acid derivatives: Analysis by electromigration and spectroscopic methods and examination of enzyme inhibitory activity Hlaváček, Jan; Vítovcová, Miloslava; Sázelová, Petra Analytical Biochemistry, Vol. 467 https://doi.org/10.1016/j.ab.2014.08.032	journal	December 2014
Determination of dissociation constants of protein ligands by thermal shift assay Bhayani, Jaina A.; Ballicora, Miguel A. Biochemical and Biophysical Research Communications, Vol. 590 https://doi.org/10.1016/j.bbrc.2021.12.041	journal	January 2022
Synthesis and characterization of the N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) alternate substrate analog N,N-dimethyl-l,l-SDAP Liveris, Zachary J.; Kelley, Emma H.; Simmons, Emma Bioorganic & Medicinal Chemistry, Vol. 91 https://doi.org/10.1016/j.bmc.2023.117415	journal	August 2023
Inhibitors of bacterial N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) and demonstration of in vitro antimicrobial activity Gillner, Danuta; Armoush, Nicola; Holz, Richard C. Bioorganic & Medicinal Chemistry Letters, Vol. 19, Issue 22 https://doi.org/10.1016/j.bmcl.2009.09.077	journal	November 2009
Tetrazole-based inhibitors of the bacterial enzyme N-succinyl-l,l-2,6-diaminopimelic acid desuccinylase as potential antibiotics DiPuma, Thomas; Thabthimthong, Teerana; Kelley, Emma H. Bioorganic & Medicinal Chemistry Letters, Vol. 83 https://doi.org/10.1016/j.bmcl.2023.129177	journal	March 2023
Structure of a novel N-acetyl-l-citrulline deacetylase from Xanthomonas campestris Shi, Dashuang; Yu, Xiaolin; Roth, Lauren Biophysical Chemistry, Vol. 126, Issue 1-3 https://doi.org/10.1016/j.bpc.2006.05.013	journal	March 2007
Structural characterization of Zn(II)-, Co(II)-, and Mn(II)-loaded forms of the argE-encoded N-acetyl-L-ornithine deacetylase from Escherichia coli Tao, Ye; Shokes, Jacob E.; McGregor, Wade C. Journal of Inorganic Biochemistry, Vol. 111 https://doi.org/10.1016/j.jinorgbio.2012.02.005	journal	June 2012
Structural Basis for Catalysis by the Mono- and Dimetalated Forms of the dapE-Encoded N-succinyl-l,l-Diaminopimelic Acid Desuccinylase Nocek, Boguslaw P.; Gillner, Danuta M.; Fan, Yao Journal of Molecular Biology, Vol. 397, Issue 3 https://doi.org/10.1016/j.jmb.2010.01.062	journal	April 2010
Improvement of acid resistance of Zn-doped dentin by newly generated chemical bonds Naito, Katsuaki; Kuwahara, Yasutaka; Yamamoto, Hiroko Materials & Design, Vol. 215 https://doi.org/10.1016/j.matdes.2022.110412	journal	March 2022
An efficient method for the preparation of N,N-disubstituted 1,2-diamines Mitchell, Judith M.; Finney, Nathaniel S. Tetrahedron Letters, Vol. 41, Issue 44 https://doi.org/10.1016/s0040-4039(00)01501-x	journal	October 2000
Atomic-Resolution 1.3 Å Crystal Structure, Inhibition by Sulfate, and Molecular Dynamics of the Bacterial Enzyme DapE Kochert, Matthew; Nocek, Boguslaw P.; Habeeb Mohammad, Thahani S. Biochemistry, Vol. 60, Issue 12 https://doi.org/10.1021/acs.biochem.0c00926	journal	March 2021
Inhibition of the dapE -Encoded N -Succinyl- l , l -diaminopimelic Acid Desuccinylase from Neisseria meningitidis by l -Captopril Starus, Anna; Nocek, Boguslaw; Bennett, Brian Biochemistry, Vol. 54, Issue 31 https://doi.org/10.1021/acs.biochem.5b00475	journal	July 2015
Structural Evidence of a Major Conformational Change Triggered by Substrate Binding in DapE Enzymes: Impact on the Catalytic Mechanism Nocek, Boguslaw; Reidl, Cory; Starus, Anna Biochemistry, Vol. 57, Issue 5 https://doi.org/10.1021/acs.biochem.7b01151	journal	January 2018
Biochemical and Structural Analysis of the Bacterial Enzyme Succinyl-Diaminopimelate Desuccinylase (DapE) from Acinetobacter baumannii Kelley, Emma H.; Minasov, George; Konczak, Katherine ACS Omega https://doi.org/10.1021/acsomega.3c08231	journal	January 2024
Acetylation of human serum albumin by p-nitrophenyl acetate Means, Gary E.; Bender, Myron L. Biochemistry, Vol. 14, Issue 22 https://doi.org/10.1021/bi00693a031	journal	November 1975
X-ray Absorption Spectroscopy of the Zinc-Binding Sites in the Class B2 Metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria Costello, Alison L.; Sharma, Narayan P.; Yang, Ke-Wu Biochemistry, Vol. 45, Issue 45 https://doi.org/10.1021/bi061547e	journal	October 2006
Enzyme Recruitment and Its Role in Metabolic Expansion Schulenburg, Cindy; Miller, Brian G. Biochemistry, Vol. 53, Issue 5 https://doi.org/10.1021/bi401667f	journal	January 2014
The Dual Biosynthetic Capability of N -Acetylornithine Aminotransferase in Arginine and Lysine Biosynthesis Ledwidge, Richard; Blanchard, John S. Biochemistry, Vol. 38, Issue 10 https://doi.org/10.1021/bi982574a	journal	February 1999
1-Butaneboronic Acid Binding to Aeromonas proteolytica Aminopeptidase: A Case of Arrested Development^, De Paola, Carin C.; Bennett, Brian; Holz, Richard C. Biochemistry, Vol. 38, Issue 28 https://doi.org/10.1021/bi9900572	journal	June 1999
Mechanistic Analysis of the argE -Encoded N -Acetylornithine Deacetylase ^† Javid-Majd, Farah; Blanchard, John S. Biochemistry, Vol. 39, Issue 6 https://doi.org/10.1021/bi992177f	journal	February 2000
argE -Encoded N -Acetyl- l -Ornithine Deacetylase from Escherichia c oli Contains a Dinuclear Metalloactive Site McGregor, Wade C.; Swierczek, Sabina I.; Bennett, Brian Journal of the American Chemical Society, Vol. 127, Issue 40 https://doi.org/10.1021/ja054081g	journal	October 2005
CHARMM36m: an improved force field for folded and intrinsically disordered proteins Huang, Jing; Rauscher, Sarah; Nawrocki, Grzegorz Nature Methods, Vol. 14, Issue 1 https://doi.org/10.1038/nmeth.4067	journal	November 2016
Characterizing metal-binding sites in proteins with X-ray crystallography Handing, Katarzyna B.; Niedzialkowska, Ewa; Shabalin, Ivan G. Nature Protocols, Vol. 13, Issue 5 https://doi.org/10.1038/nprot.2018.018	journal	April 2018
Horizontal gene transfer in eukaryotic evolution Keeling, Patrick J.; Palmer, Jeffrey D. Nature Reviews Genetics, Vol. 9, Issue 8 https://doi.org/10.1038/nrg2386	journal	August 2008
Development of NMR and thermal shift assays for the evaluation ofMycobacterium tuberculosisisocitrate lyase inhibitors Bhusal, Ram Prasad; Patel, Krunal; Kwai, Brooke X. C. MedChemComm, Vol. 8, Issue 11 https://doi.org/10.1039/c7md00456g	journal	January 2017
Larch: An Analysis Package for XAFS and Related Spectroscopies Newville, Matthew Journal of Physics: Conference Series, Vol. 430 https://doi.org/10.1088/1742-6596/430/1/012007	journal	April 2013
ESPript/ENDscript: extracting and rendering sequence and 3D information from atomic structures of proteins Gouet, P. Nucleic Acids Research, Vol. 31, Issue 13 https://doi.org/10.1093/nar/gkg556	journal	July 2003
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
Substructure solution with SHELXD Schneider, Thomas R.; Sheldrick, George M. Acta Crystallographica Section D Biological Crystallography, Vol. 58, Issue 10 https://doi.org/10.1107/s0907444902011678	journal	September 2002
PHENIX : building new software for automated crystallographic structure determination Adams, Paul D.; Grosse-Kunstleve, Ralf W.; Hung, Li-Wei Acta Crystallographica Section D Biological Crystallography, Vol. 58, Issue 11 https://doi.org/10.1107/s0907444902016657	journal	October 2002
Coot model-building tools for molecular graphics Emsley, Paul; Cowtan, Kevin Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132 https://doi.org/10.1107/s0907444904019158	journal	November 2004
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
Molecular replacement with MOLREP Vagin, Alexei; Teplyakov, Alexei Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 1 https://doi.org/10.1107/s0907444909042589	journal	December 2009
Pi sampling: a methodical and flexible approach to initial macromolecular crystallization screening Gorrec, Fabrice; Palmer, Colin M.; Lebon, Guillaume Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 5 https://doi.org/10.1107/s0907444911008754	journal	April 2011
The CCP4 suite programs for protein crystallography No authors listed Acta Crystallographica Section D Biological Crystallography, Vol. 50, Issue 5, p. 760-763 https://doi.org/10.1107/s0907444994003112	journal	September 1994
Refinement of Macromolecular Structures by the Maximum-Likelihood Method Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta Crystallographica Section D Biological Crystallography, Vol. 53, Issue 3 https://doi.org/10.1107/s0907444996012255	journal	May 1997
The Structural Biology Center 19ID undulator beamline: facility specifications and protein crystallographic results Rosenbaum, Gerd; Alkire, Randy W.; Evans, Gwyndaf Journal of Synchrotron Radiation, Vol. 13, Issue 1 https://doi.org/10.1107/s0909049505036721	journal	December 2005
FMX – the Frontier Microfocusing Macromolecular Crystallography Beamline at the National Synchrotron Light Source II Schneider, Dieter K.; Shi, Wuxian; Andi, Babak Journal of Synchrotron Radiation, Vol. 28, Issue 2 https://doi.org/10.1107/s1600577520016173	journal	February 2021
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
Identification and Characterization of the Myxococcus xanthus argE Gene Harris, Baruch Z.; Singer, Mitchell Journal of Bacteriology, Vol. 180, Issue 23 https://doi.org/10.1128/.180.23.6412-6414.1998	journal	January 1998
Arginine Metabolism Powers Salmonella Resistance to Oxidative Stress Margolis, Alyssa; Liu, Lin; Porwollik, Steffen Infection and Immunity, Vol. 91, Issue 6 https://doi.org/10.1128/iai.00120-23	journal	June 2023
Structural and biochemical characterization of the Escherichia coli argE gene product. Meinnel, T.; Schmitt, E.; Mechulam, Y. Journal of Bacteriology, Vol. 174, Issue 7 https://doi.org/10.1128/jb.174.7.2323-2331.1992	journal	January 1992
Antimicrobial Resistance in Escherichia coli Poirel, Laurent; Madec, Jean-Yves; Lupo, Agnese Microbiology Spectrum, Vol. 6, Issue 4 https://doi.org/10.1128/microbiolspec.arba-0026-2017	journal	July 2018
Competitive sorption of Ni and Zn at the aluminum oxide/water interface: an XAFS study Gou, Wenxian; Siebecker, Matthew G.; Wang, Zimeng Geochemical Transactions, Vol. 19, Issue 1 https://doi.org/10.1186/s12932-018-0054-7	journal	March 2018
Metabolic engineering of Escherichia coli for enhanced arginine biosynthesis Ginesy, Mireille; Belotserkovsky, Jaroslav; Enman, Josefine Microbial Cell Factories, Vol. 14, Issue 1 https://doi.org/10.1186/s12934-015-0211-y	journal	March 2015
The Dimerization Domain in DapE Enzymes Is required for Catalysis Nocek, Boguslaw; Starus, Anna; Makowska-Grzyska, Magdalena PLoS ONE, Vol. 9, Issue 5 https://doi.org/10.1371/journal.pone.0093593	journal	May 2014
Practical spectrophotometric assay for the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase, a potential antibiotic target Heath, Tahirah K.; Lutz, Marlon R.; Reidl, Cory T. PLOS ONE, Vol. 13, Issue 4 https://doi.org/10.1371/journal.pone.0196010	journal	April 2018
Uptake and speciation of zinc in edible plants grown in smelter contaminated soils Mishra, Bhoopesh; McDonald, Louis M.; Roy, Mimi PLOS ONE, Vol. 15, Issue 4 https://doi.org/10.1371/journal.pone.0226180	journal	April 2020
A Quantitative Model of Thermal Stabilization and Destabilization of Proteins by Ligands Cimmperman, Piotras; Baranauskienė, Lina; Jachimovičiūtė, Simona Biophysical Journal, Vol. 95, Issue 7 https://doi.org/10.1529/biophysj.108.134973	journal	October 2008
Indoline-6-Sulfonamide Inhibitors of the Bacterial Enzyme DapE Reidl, Cory T.; Heath, Tahirah K.; Darwish, Iman Antibiotics, Vol. 9, Issue 9 https://doi.org/10.3390/antibiotics9090595	journal	September 2020
Characterization of a Gene Encoding Acetylornithine Deacetylase from Rice Shafiqul I., Md.; Kim, Jung-Sup Biotechnology(Faisalabad), Vol. 13, Issue 2 https://doi.org/10.3923/biotech.2014.54.60	journal	February 2014