Comparative functional and structural analysis of Pseudomonas aeruginosa d-alanine-d-alanine ligase isoforms as prospective antibiotic targets.
ATP-grasp
Ddl
X-ray crystallography
cell wall biosynthesis
cycloserine
Journal
The FEBS journal
ISSN: 1742-4658
Titre abrégé: FEBS J
Pays: England
ID NLM: 101229646
Informations de publication
Date de publication:
Dec 2023
Dec 2023
Historique:
revised:
02
07
2023
received:
04
05
2023
accepted:
14
08
2023
pubmed:
15
8
2023
medline:
15
8
2023
entrez:
15
8
2023
Statut:
ppublish
Résumé
Pseudomonas aeruginosa is a major human pathogen in the healthcare setting. The emergence of multi-drug-resistant and extensive drug-resistant P. aeruginosa is of great concern, and clearly indicates that new alternatives to current first-line antibiotics are required in the future. Inhibition of d-alanine-d-alanine production presents as a promising avenue as it is a key component in the essential process of cell wall biosynthesis. In P. aeruginosa, d-alanine-d-alanine production is facilitated by two isoforms, d-alanine-d-alanine ligase A (PaDdlA) and d-alanine-d-alanine ligase B (PaDdlA), but neither enzyme has been individually characterised to date. Here, we present the functional and structural characterisation of PaDdlA and PaDdlB, and assess their potential as antibiotic targets. This was achieved using a combination of in vitro enzyme-activity assays and X-ray crystallography. The former revealed that both isoforms effectively catalyse d-alanine-d-alanine production with near identical efficiency, and that this is effectively disrupted by the model d-alanine-d-alanine ligase inhibitor, d-cycloserine. Next, each isoform was co-crystallised with ATP and either d-alanine-d-alanine or d-cycloserine, allowing direct comparison of the key structural features. Both isoforms possess the same structural architecture and share a high level of conservation within the active site. Although residues forming the d-alanine pocket are completely conserved, the ATP-binding pocket possesses several amino acid substitutions resulting in a differing chemical environment around the ATP adenine base. Together, these findings support that the discovery of dual PaDdlA/PaDdlB competitive inhibitors is a viable approach for developing new antibiotics against P. aeruginosa.
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
5536-5553Informations de copyright
© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.
Références
Murray CJ, Ikuta KS, Sharara F, Swetschinski L, Aguilar GR, Gray A, Han C, Bisignano C, Rao P, Wool E et al. (2022) Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 399, 629-655.
Sarkar P, Yarlagadda V, Ghosh C & Haldar J (2017) A review on cell wall synthesis inhibitors with an emphasis on glycopeptide antibiotics. Medchemcomm 8, 516-533.
Anderson EM, Shaji Saji N, Anderson AC, Brewer D, Clarke AJ & Khursigara CM (2022) Pseudomonas aeruginosa alters peptidoglycan composition under nutrient conditions resembling cystic fibrosis lung infections. mSystems 7, e0015622.
Pederick JL, Thompson AP, Bell SG & Bruning JB (2020) d-Alanine-d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations. J Biol Chem 295, 7894-7904.
Fujihira T, Kanematsu S, Umino A, Yamamoto N & Nishikawa T (2007) Selective increase in the extracellular d-serine contents by d-cycloserine in the rat medial frontal cortex. Neurochem Int 51, 233-236.
Zawadzke LE, Bugg TDH & Walsh CT (1991) Existence of two D-alanine:D-alanine ligases in Escherichia coli: cloning and sequencing of the ddlA gene and purification and characterization of the DdlA and DdlB enzymes. Biochemistry 30, 1673-1682.
Lee SA, Gallagher LA, Thongdee M, Staudinger BJ, Lippman S, Singh PK & Manoil C (2015) General and condition-specific essential functions of Pseudomonas aeruginosa. Proc Natl Acad Sci USA 112, 5189-5194.
Batson S, de Chiara C, Majce V, Lloyd AJ, Gobec S, Rea D, Fülöp V, Thoroughgood CW, Simmons KJ, Dowson CG et al. (2017) Inhibition of D-ala:D-ala ligase through a phosphorylated form of the antibiotic D-cycloserine. Nat Commun 8, 1939.
Prosser GA & de Carvalho LPS (2013) Kinetic mechanism and inhibition of Mycobacterium tuberculosis d-alanine:d-alanine ligase by the antibiotic d-cycloserine. FEBS J 280, 1150-1166.
Liu S, Chang JS, Herberg JT, Horng M-M, Tomich PK, Lin AH & Marotti KR (2006) Allosteric inhibition of Staphylococcus aureus d-alanine:d-alanine ligase revealed by crystallographic studies. Proc Natl Acad Sci USA 103, 15178-15183.
Pederick JL & Bruning JB (2021) An antimony-phosphomolybdate microassay of ATPase activity through the detection of inorganic phosphate. Anal Biochem 623, 114170.
Neuhaus FC (1962) The enzymatic synthesis of D-alanyl-D-alanine I. Purification and properties of D-alanyl-D-alanine synthetase. J Biol Chem 237, 778-786.
Bruning JB, Murillo AC, Chacon O, Barletta RG & Sacchettini JC (2011) Structure of the Mycobacterium tuberculosis d-alanine:d-alanine ligase, a target of the antituberculosis drug d-cycloserine. Antimicrob Agents Chemother 55, 291-301.
Fan C, Moews PC, Shi Y, Walsh CT & Knox JR (1995) A common fold for peptide synthetases cleaving ATP to ADP: glutathione synthetase and D-alanine:d-alanine ligase of Escherichia coli. Proc Natl Acad Sci USA 92, 1172-1176.
Fan C, Moews PC, Walsh CT & Knox JR (1994) Vancomycin resistance: structure of d-alanine:d-alanine ligase at 2.3 A resolution. Science 266, 439-443.
Parang K & Cole PA (2002) Designing bisubstrate analog inhibitors for protein kinases. Pharmacol Ther 93, 145-157.
Lavogina D, Enkvist E & Uri A (2010) Bisubstrate inhibitors of protein kinases: from principle to practical applications. ChemMedChem 5, 23-34.
Gower CM, Chang MEK & Maly DJ (2014) Bivalent inhibitors of protein kinases. Crit Rev Biochem Mol Biol 49, 102-115.
Elliott TS, Slowey A, Ye Y & Conway SJ (2012) The use of phosphate bioisosteres in medicinal chemistry and chemical biology. Med Chem Commun 3, 735-751.
Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD & Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In The Proteomics Protocols Handbook (Walker JM, ed.), pp. 571-607. Humana Press, Totowa, NJ.
Cowieson NP, Aragao D, Clift M, Ericsson DJ, Gee C, Harrop SJ, Mudie N, Panjikar S, Price JR, Riboldi-Tunnicliffe A et al. (2015) MX1: a bending-magnet crystallography beamline serving both chemical and macromolecular crystallography communities at the Australian Synchrotron. J Synchrotron Radiat 22, 187-190.
Aragão D, Aishima J, Cherukuvada H, Clarken R, Clift M, Cowieson NP, Ericsson DJ, Gee CL, Macedo S, Mudie N et al. (2018) MX2: a high-flux undulator microfocus beamline serving both the chemical and macromolecular crystallography communities at the Australian Synchrotron. J Synchrotron Radiat 25, 885-891.
Kabsch W (2010) XDS. Acta Crystallogr D 66, 125-132.
Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AGW, McCoy A et al. (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr D 67, 235-242.
McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC & Read RJ (2007) Phaser crystallographic software. J Appl Cryst 40, 658-674.
Stein N (2008) CHAINSAW: a program for mutating pdb files used as templates in molecular replacement. J Appl Cryst 41, 641-643.
Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung L-W, Kapral GJ, Grosse-Kunstleve RW et al. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D 66, 213-221.
Emsley P & Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D 60, 2126-2132.
Neuhaus FC (1962) The enzymatic synthesis of D-alanyl-D-alanine II. Kinetic studies on D-alanyl-D-alanine synthetase. J Biol Chem 237, 3128-3135.
The PyMOL Molecular Graphics System. Schrödinger, LLC, New York, NY.
Krissinel E & Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372, 774-797.
Kumar S, Stecher G, Li M, Knyaz C & Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35, 1547-1549.