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The Lysozyme Inhibitor Thionine Acetate Is Also an Inhibitor of the Soluble Lytic Transglycosylase Slt35 from Escherichia Coli

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The Lysozyme Inhibitor Thionine Acetate Is Also an Inhibitor of the Soluble Lytic Transglycosylase Slt35 from Escherichia Coli molecules Article The Lysozyme Inhibitor Thionine Acetate Is Also an Inhibitor of the Soluble Lytic Transglycosylase Slt35 from Escherichia coli Aysha B. Mezoughi 1 , Chiara M. Costanzo 2, Gregor M. Parker 1, Enas M. Behiry 1, Alan Scott 1, Andrew C. Wood 1, Sarah E. Adams 1, Richard B. Sessions 3 and E. Joel Loveridge 1,2,* 1 School of Chemistry, Cardiff University, Park Place, Cardiff CF10 3AT, UK; [email protected] (A.B.M.); [email protected] (G.M.P.); [email protected] (E.M.B.); [email protected] (A.S.); [email protected] (A.C.W.); [email protected] (S.E.A.) 2 Department of Chemistry, Swansea University, Singleton Park, Swansea SA2 8PP, UK; [email protected] 3 School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK; [email protected] * Correspondence: [email protected] Abstract: Lytic transglycosylases such as Slt35 from E. coli are enzymes involved in bacterial cell wall remodelling and recycling, which represent potential targets for novel antibacterial agents. Here, we investigated a series of known glycosidase inhibitors for their ability to inhibit Slt35. While glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, thiamet G and miglitol had no effect, the phenothiazinium dye thionine acetate was found to be a weak inhibitor. IC50 values and binding constants for thionine acetate were similar for Slt35 and the hen egg white lysozyme. Molecular docking simulations suggest that thionine binds to the active site of both Slt35 and Citation: Mezoughi, A.B.; Costanzo, lysozyme, although it does not make direct interactions with the side-chain of the catalytic Asp and C.M.; Parker, G.M.; Behiry, E.M.; Glu residues as might be expected based on other inhibitors. Thionine acetate also increased the Scott, A.; Wood, A.C.; Adams, S.E.; potency of the beta-lactam antibiotic ampicillin against a laboratory strain of E. coli. Sessions, R.B.; Loveridge, E.J. The Lysozyme Inhibitor Thionine Acetate Keywords: Is Also an Inhibitor of the Soluble lytic transglycosylase; thionine acetate; enzyme inhibition; antibacterial Lytic Transglycosylase Slt35 from Escherichia coli. Molecules 2021, 26, 4189. https://doi.org/ 10.3390/molecules26144189 1. Introduction Bacterial cells are surrounded by a peptidoglycan sacculus on the outside of the cy- Academic Editor: F. J. (Frank) Dekker toplasmic membrane, which is essential for maintaining cell strength and integrity [1]. Peptidoglycan is composed of glycan strands of repeating N-acetylglucosamine (NAG) Received: 14 June 2021 and N-acetylmuramic acid (NAM) residues linked by β-1,4-glycosidic bonds, cross-linked Accepted: 6 July 2021 via peptide side-chains [2]. Escherichia coli turns over about 50% of its sacculus per gen- Published: 9 July 2021 eration, to allow cell growth and division, insertion of proteins into the cell wall and other processes [3]. The muropeptides released from the sacculus are recycled by the cell. Publisher’s Note: MDPI stays neutral The sacculus degradation and remodelling activities are catalysed by enzymes including with regard to jurisdictional claims in glycosidases, amidases, endopeptidases and carboxypeptidases [4,5]. published maps and institutional affil- Lytic transglycosylases are important peptidoglycan-degrading glycosidases [6–8], iations. that catalyse cleavage of the β-1,4-glycosidic bond between NAM and NAG residues. Un- like lysozymes, they do so by catalysing an intramolecular transglycosylation reaction that forms 1,6-anhydromuropeptides [9], which can be recycled to form fresh peptidoglycan but which may also act as inducers of β-lactamase production [10] and as virulence fac- Copyright: © 2021 by the authors. tors [11–13]. Many bacterial species, including human pathogens such as Bacillus anthracis, Licensee MDPI, Basel, Switzerland. Staphylococcus aureus, Neisseria meningitides and Neisseria gonorrhoeae, inhibit the activity of This article is an open access article both lysozyme and lytic transglycosylases by acetylation of C-6 hydroxyl moieties of NAM distributed under the terms and residues in their peptidoglycan [14,15]. This is used to control lytic transglycosylase activity conditions of the Creative Commons and so prevent autolysis [7]. Vertebrate lysozymes are also inhibited by the proteinaceous Attribution (CC BY) license (https:// inhibitor Ivy, which is produced by certain Gram-negative bacteria [16]. Inhibition of the creativecommons.org/licenses/by/ 4.0/). membrane-bound lytic transglycosylase MltB from P. aeruginosa by Ivy was suggested to Molecules 2021, 26, 4189. https://doi.org/10.3390/molecules26144189 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 4189 2 of 13 Molecules 2021, 26, 4189 eties of NAM residues in their peptidoglycan [14,15]. This is used to control lytic transgly-2 of 13 cosylase activity and so prevent autolysis [7]. Vertebrate lysozymes are also inhibited by the proteinaceous inhibitor Ivy, which is produced by certain Gram-negative bacteria [16]. Inhibitionregulate the of autolyticthe membrane-bound activity of lytic lytic transglycosylases transglycosylase in Gram-negativeMltB from P. aeruginosa bacteria thatby Ivy are wasunable suggested to O-acetylate to regulate their the peptidoglycan autolytic activi [17ty]. of However, lytic transglycosylases despite preventing in Gram-nega- autolysis, tivespecific bacteria inhibition that are of LTsunable may to also O-acetylat increasee their the potency peptidoglycan of β-lactam [17]. antibioticsHowever, despite against preventingbacteria, as autolysis, exemplified specifi by thec inhibition action of of the LTs bulgecins may also [18 increase–25]. the potency of β-lactam antibioticsIminosugars, against alkaloidsbacteria, as and exemplifie their syntheticd by the analogues action of (Figure the bulgecins1) can act [18–25]. as glycosidase inhibitorsIminosugars, by mimicking alkaloids the and glycosyloxocarbenium their synthetic analogues ion (Figure intermediate 1) can act and as related glycosidase tran- inhibitorssition states by inmimicking enzymatic the glycoside glycosyloxocarbe hydrolysisnium [26 ].ion 1-Deoxynojirimycin, intermediate and related a D-glucose transi- tionanalogue, states in is enzymatic the best known glycoside iminosugar. hydrolysis The[26].alkaloid 1-Deoxynojirimycin, castanospermine a D-glucose is also ana- a β- logue,glucosidase is the best inhibitor known [27 iminosugar.]. The NAG-thiazoline The alkaloid derivative castanospermine thiamet is G also is a potenta β-glucosidase inhibitor inhibitorof β-acetylglucosaminidases [27]. The NAG-thiazoline [28],while derivative miglitol thiamet (Glyset) G is is a usedpotent as inhibitor a drug for of treatment β-acetyl- glucosaminidasesof type II diabetes [28], [29 ].while Thionine miglitol acetate, (Glyset) a planar is used cationic as a drug phenothiazinium for treatment of dye, type has II diabetesbeen shown [29]. toThionine bind to acetate, lysozyme a planar [30]. cati Thisonic compound phenothiazinium is also used dye, inhas electrochemical been shown to bindand photochemicalto lysozyme [30]. biosensors This compound [31,32] is and also has used antibacterial in electrochemical activity towardand photochemical pathogenic biosensorsbacteria [33 [31,32]]. and has antibacterial activity toward pathogenic bacteria [33]. FigureFigure 1. StructuresStructures of of some some reported glycosid glycosidasease inhibitors and thionine acetate. InIn this this study, study, we evaluated thionine acetate and some known glycosidase inhibitors againstagainst hen hen egg egg white white lysozyme lysozyme and and Slt35, Slt35, using using a a combination combination of of enzyme enzyme activity activity assays, assays, dissociationdissociation constant constant determination determination and molecular docking analysis. 2.2. Results Results and and Discussion Discussion 2.1. Enzyme Activity Assay 2.1. Enzyme Activity Assay 2.1.1. Effect of the Buffer 2.1.1. Effect of the Buffer A turbidimetric assay [34] was used to determine the activity of hen egg white lysozymeA turbidimetric and Slt35, assay by monitoring [34] was used peptidoglycan to determine solubilisation the activity of when hen egg the white enzyme lyso- is zymeadded and to Micrococcus Slt35, by monitoring lysodeikticus peptidoglycell suspension.can solubilisation Although this when assay the has enzyme previously is added been toperformed Micrococcus in sodiumlysodeikticus phosphate cell suspension. buffer for theAlthough same enzymes this assay [17 has], we previously found Tris-maleate been per- formedto give in superior sodium enzyme phosphate activity: buffer maximum for the same 0.018 enzymes A min −[17],1 for we phosphate found Tris-maleate (pH 6.4) and to give0.068 superior A min− enzyme1 for Tris-maleate activity: maximum (pH 5.8), 0.018 both containingA min−1 for 100phosphate mM NaCl. (pH 6.4) Acetate, and 0.068 MES Aand min HEPES−1 for buffersTris-maleate gave very(pH poor5.8), activity:both containing maximum 100 0.020 mM A NaCl. min−1 Acetate,for sodium MES acetate and HEPES(pH 5.5 ),buffers 0.003 gave A min very−1 forpoor MES activity: (pH 6.0)maximum and 0.003 0.020 A A min min−1−1for for HEPESsodium (pHacetate 7.0), (pH all 5.5),containing 0.003 A 100 min mM−1 for NaCl. MES (pH 6.0) and 0.003 A min−1 for HEPES (pH 7.0), all containing 100 mM NaCl. 2.1.2. Effect of Salts 2.1.2. TheEffect effect of Salts of NaCl, KCl, MgCl2 and CaCl2 on enzyme activity was determined. The activityThe of effect Slt35 of was NaCl, not KCl, determined
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