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• W3022103780 abstract "Mammalian acetylcholinesterase (AChE) is well-studied, being important in both cholinergic brain synapses and the peripheral nervous systems and also a key drug target for many diseases. In contrast, little is known about the structures and molecular mechanism of prokaryotic acetylcholinesterases. We report here the structural and biochemical characterization of ChoE, a putative bacterial acetylcholinesterase from Pseudomonas aeruginosa. Analysis of WT and mutant strains indicated that ChoE is indispensable for P. aeruginosa growth with acetylcholine as the sole carbon and nitrogen source. The crystal structure of ChoE at 1.35 Å resolution revealed that this enzyme adopts a typical fold of the SGNH hydrolase family. Although ChoE and eukaryotic AChEs catalyze the same reaction, their overall structures bear no similarities constituting an interesting example of convergent evolution. Among Ser-38, Asp-285, and His-288 of the catalytic triad residues, only Asp-285 was not essential for ChoE activity. Combined with kinetic analyses of WT and mutant proteins, multiple crystal structures of ChoE complexed with substrates, products, or reaction intermediate revealed the structural determinants for substrate recognition, snapshots of the various catalytic steps, and the molecular basis of substrate inhibition at high substrate concentrations. Our results indicate that substrate inhibition in ChoE is due to acetate release being blocked by the binding of a substrate molecule in a nonproductive mode. Because of the distinct overall folds and significant differences of the active site between ChoE and eukaryotic AChEs, these structures will serve as a prototype for other prokaryotic acetylcholinesterases. Mammalian acetylcholinesterase (AChE) is well-studied, being important in both cholinergic brain synapses and the peripheral nervous systems and also a key drug target for many diseases. In contrast, little is known about the structures and molecular mechanism of prokaryotic acetylcholinesterases. We report here the structural and biochemical characterization of ChoE, a putative bacterial acetylcholinesterase from Pseudomonas aeruginosa. Analysis of WT and mutant strains indicated that ChoE is indispensable for P. aeruginosa growth with acetylcholine as the sole carbon and nitrogen source. The crystal structure of ChoE at 1.35 Å resolution revealed that this enzyme adopts a typical fold of the SGNH hydrolase family. Although ChoE and eukaryotic AChEs catalyze the same reaction, their overall structures bear no similarities constituting an interesting example of convergent evolution. Among Ser-38, Asp-285, and His-288 of the catalytic triad residues, only Asp-285 was not essential for ChoE activity. Combined with kinetic analyses of WT and mutant proteins, multiple crystal structures of ChoE complexed with substrates, products, or reaction intermediate revealed the structural determinants for substrate recognition, snapshots of the various catalytic steps, and the molecular basis of substrate inhibition at high substrate concentrations. Our results indicate that substrate inhibition in ChoE is due to acetate release being blocked by the binding of a substrate molecule in a nonproductive mode. Because of the distinct overall folds and significant differences of the active site between ChoE and eukaryotic AChEs, these structures will serve as a prototype for other prokaryotic acetylcholinesterases. In mammals, acetylcholinesterase (AChE) plays a pivotal role in cholinergic brain synapses and neuromuscular junctions through the termination of impulse transmission via rapid hydrolysis of acetylcholine (ACh), an important cationic neurotransmitter in the nervous system (1Tougu V. Acetylcholinesterase: mechanism of catalysis and inhibition.Curr. Med. Chem. Central Nervous Syst. Agents. 2001; 1: 155-17010.2174/1568015013358536Crossref Google Scholar). The rapid hydrolysis of ACh into acetic acid and choline by AChE is achieved at a rate close to that of a diffusion-controlled reaction and, therefore, AChE is recognized as one of the most efficient enzymes in nature (2Quinn D.M. Acetylcholinesterase: enzyme structure, reaction dynamics, and virtual transition states.Chem. Rev. 1987; 87: 955-97910.1021/cr00081a005Crossref Scopus (884) Google Scholar). Having a catalytic triad consisting of Ser, His, and Glu residues, AchE follows the classical esterase mechanism (3Rauwerdink A. Kazlauskas R.J. How the same core catalytic machinery catalyzes 17 different reactions: the serine-histidine-aspartate catalytic triad of α/β-hydrolase fold enzymes.ACS Catal. 2015; 5 (28580193): 6153-617610.1021/acscatal.5b01539Crossref PubMed Scopus (116) Google Scholar) and two half-reactions are required to complete the full catalytic cycle. In recent decades, the structure and function study of the AChEs from metazoans has been particularly important in the context of the development of inhibitors to block the activity of AChE for various purposes, e.g. the treatment of Alzheimer's disease (4Colović M.B. Krstić D.Z. Lazarević-Pašti T.D. Bondžić A.M. Vasić V.M. Acetylcholinesterase inhibitors: pharmacology and toxicology.Curr. Neuropharmacol. 2013; 11 (24179466): 315-33510.2174/1570159X11311030006Crossref PubMed Scopus (1071) Google Scholar). The first crystal structure of an acetylcholinesterase from Torpedo californica (TcAChE) was published nearly 30 years ago (5Sussman J. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein.Science. 1991; 253: 872-87910.1126/science.1678899Crossref PubMed Scopus (2319) Google Scholar). This was followed by the determination of ∼250 structures of AChEs of eukaryotic sources, many of which are in complex with various inhibitors (6Dvir H. Silman I. Harel M. Rosenberry T.L. Sussman J.L. Acetylcholinesterase: from 3D structure to function.Chem. Biol. Interact. 2010; 187 (20138030): 10-2210.1016/j.cbi.2010.01.042Crossref PubMed Scopus (392) Google Scholar). Despite the long history of research on structure and inhibition of eukaryotic AChEs (7Silman I. Sussman J.L. Recent developments in structural studies on acetylcholinesterase.J. Neurochem. 2017; 142 (28503857): 19-2510.1111/jnc.13992Crossref PubMed Scopus (13) Google Scholar, 8Cavalcante S.F.A. Simas A.B.C. Barcellos M.C. de Oliveira V.G.M. Sousa R.B. Cabral P.A.M. Kuca K. Franca T.C.C. Acetylcholinesterase: the “Hub” for neurodegenerative diseases and chemical weapons convention.Biomolecules. 2020; 10 (32155996): e41410.3390/biom10030414Crossref PubMed Scopus (6) Google Scholar), novel insights have been gained into these pivotal enzymes in recent years, e.g. the dynamics of back door opening (9Bourne Y. Renault L. Marchot P. Crystal structure of snake venom acetylcholinesterase in complex with inhibitory antibody fragment Fab410 bound at the peripheral site: evidence for open and closed states of a back door channel.J. Biol. Chem. 2015; 290 (25411244): 1522-153510.1074/jbc.M114.603902Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), the steric and dynamic parameters implicated in the reaction within the active-site gorge (10Bourne Y. Sharpless K.B. Taylor P. Marchot P. Steric and dynamic parameters influencing in situ cycloadditions to form triazole inhibitors with crystalline acetylcholinesterase.J. Am. Chem. Soc. 2016; 138 (26731630): 1611-162110.1021/jacs.5b11384Crossref PubMed Scopus (22) Google Scholar), and the possible repurposing of AChE inhibitors as anti-cancer agents (11Lazarevic-Pasti T. Leskovac A. Momic T. Petrovic S. Vasic V. Modulators of acetylcholinesterase activity: from Alzheimer's disease to anti-cancer drugs.Curr. Med. Chem. 2017; 24 (28685687): 3283-330910.2174/0929867324666170705123509Crossref PubMed Scopus (40) Google Scholar). ACh hydrolyzing activity has also been recognized in many prokaryotic species from various bacterial classes (12Sánchez D.G. Otero L.H. Hernández C.M. Serra A.L. Encarnación S. Domenech C.E. Lisa A.T. A Pseudomonas aeruginosa PAO1 acetylcholinesterase is encoded by the PA4921 gene and belongs to the SGNH hydrolase family.Microbiol. Res. 2012; 167 (22192836): 317-32510.1016/j.micres.2011.11.005Crossref PubMed Scopus (14) Google Scholar) although they lack a nervous system. This was especially the case for the bacteria from the Pseudomonas genus, which are able to inhabit a large range of environments with many species such as Pseudomonas aeruginosa being pathogens for many different hosts (13Azam M.W. Khan A.U. Updates on the pathogenicity status of Pseudomonas aeruginosa.Drug. Discov. Today. 2019; 24 (30036575): 350-35910.1016/j.drudis.2018.07.003Crossref PubMed Scopus (58) Google Scholar). Acetylcholinesterase activity has been observed in Pseudomonas fluorescens after growth with ACh as the sole source of carbon (14Goldstein D.B. Goldstein A. An adaptive bacterial cholinesterase from Pseudomonas species.J. Gen. Microbiol. 1953; 8 (13035026): 8-1710.1099/00221287-8-1-8Crossref PubMed Scopus (18) Google Scholar, 15Rochu D. Rothlisberger C. Taupin C. Renault F. Gagnon J. Masson P. Purification, molecular characterization and catalytic properties of a Pseudomonas fluorescens enzyme having cholinesterase-like activity.Biochim. Biophys. Acta. 1998; 1385 (9630567): 126-13810.1016/S0167-4838(98)00042-9Crossref PubMed Scopus (11) Google Scholar). The putative acetylcholinesterase from P. aeruginosa strain PAO1, named ChoE, has also been characterized (12Sánchez D.G. Otero L.H. Hernández C.M. Serra A.L. Encarnación S. Domenech C.E. Lisa A.T. A Pseudomonas aeruginosa PAO1 acetylcholinesterase is encoded by the PA4921 gene and belongs to the SGNH hydrolase family.Microbiol. Res. 2012; 167 (22192836): 317-32510.1016/j.micres.2011.11.005Crossref PubMed Scopus (14) Google Scholar, 16Garber N. Nachshon I. Localization of cholinesterase in Pseudomonas aeruginosa strain K.J. Gen. Microbiol. 1980; 117 (6771368): 279-28310.1099/00221287-117-1-279PubMed Google Scholar). Despite its small size (287 residues) relative to that of eukaryotic AChEs (∼560 residues), ChoE is able to catalyze the hydrolysis of both acetylthiocholine (ATCh) and its analog propionylthiocholine (PTCh). The purified ChoE enzyme of P. aeruginosa is inhibited by substrate at high substrate concentrations, exhibiting enzymatic properties similar to eukaryotic AChEs (12Sánchez D.G. Otero L.H. Hernández C.M. Serra A.L. Encarnación S. Domenech C.E. Lisa A.T. A Pseudomonas aeruginosa PAO1 acetylcholinesterase is encoded by the PA4921 gene and belongs to the SGNH hydrolase family.Microbiol. Res. 2012; 167 (22192836): 317-32510.1016/j.micres.2011.11.005Crossref PubMed Scopus (14) Google Scholar). In view of the enzymatic activity of ChoE as well as the abundance of ACh present in the corneal epithelium (17Pesin S.R. Candia O.A. Acetylcholine concentration and its role in ionic transport by the corneal epithelium.Invest. Ophthalmol. Vis. Sci. 1982; 22 (6978868): 651-659PubMed Google Scholar), it was proposed that ChoE could be a virulence factor involved in corneal infection by P. aeruginosa (18Domenech C.E. Garrido M.N. Lisa T.A. Pseudomonas aeruginosa cholinesterase and phosphorylcholine phosphatase: two enzymes contributing to corneal infection.FEMS Microbiol. Lett. 1991; 82: 131-13510.1111/j.1574-6968.1991.tb04853.xCrossref Scopus (19) Google Scholar). Production of choline by ChoE for subsequent catabolic processes by other metabolic pathways was also suggested to be linked to the pathogenicity of P. aeruginosa and its survival during murine lung infection (19Lisa T.A. Lucchesi G.I. Domenech C.E. Pathogenicity of Pseudomonas aeruginosa and its relationship to the choline metabolism through the action of cholinesterase, acid phosphatase, and phospholipase C.Curr. Microbiol. 1994; 29: 193-19910.1007/BF01570153Crossref Scopus (20) Google Scholar, 20Wargo M.J. Choline catabolism to glycine betaine contributes to Pseudomonas aeruginosa survival during murine lung infection.PLoS ONE. 2013; 8 (23457628): e5685010.1371/journal.pone.0056850Crossref PubMed Scopus (29) Google Scholar). In contrast to the eukaryotic AChEs, little is known about the structure and molecular mechanism of prokaryotic acetylcholinesterases. Due to the low sequence identity (<26%) between ChoE and the proteins whose structures are available in the Protein Data Bank (PDB), it remains elusive how this enzyme works at the molecular level. In this study, we have performed structural and biochemical characterization of ChoE. Multiple structures were determined at a resolution of 1.35-1.85 Å in the presence of substrates, products, or reaction intermediate using both WT and the catalytic triad mutants. These results have uncovered the first atomic details of a putative bacterial acetylcholinesterase and provided insights into the mechanism of substrate inhibition. Choline can serve as the sole source of carbon and nitrogen to support P. aeruginosa growth and can be acquired through an acetylcholinesterase activity of ChoE, encoded by the choE (PA4921) gene (12Sánchez D.G. Otero L.H. Hernández C.M. Serra A.L. Encarnación S. Domenech C.E. Lisa A.T. A Pseudomonas aeruginosa PAO1 acetylcholinesterase is encoded by the PA4921 gene and belongs to the SGNH hydrolase family.Microbiol. Res. 2012; 167 (22192836): 317-32510.1016/j.micres.2011.11.005Crossref PubMed Scopus (14) Google Scholar). Growth of the WT PAO1 strain and the deletion mutant strain PAO1::ΔPA4921 (PW9287) was assessed in a microplate reader at 37 °C and both strains were unable to grow in salt solution containing 0.66% KH2PO4, 0.3% K2HPO4, and 0.3% MgSO4, unless choline was added as a source of carbon and nitrogen. The WT strain was able to use ACh as a substrate, but the mutant strain could not (Table 1). These results confirmed that ChoE is required for the bacteria to grow when ACh is the sole source of carbon and nitrogen.Table 1P. aeruginosa growth in salt solution supplemented or not with choline or acetylcholineSalt solutionSalt solution with 20 mm cholineSalt solution with 20 mm acetylcholineWT strain (PAO1)−aGrowth is shown by a plus (+) or a double plus (++), according to the extent of growth, and no growth by a minus (−). The results were obtained from two technical replicates of two biological replicates.+++Mutant strain (PAO1::ΔPA4921)−++−a Growth is shown by a plus (+) or a double plus (++), according to the extent of growth, and no growth by a minus (−). The results were obtained from two technical replicates of two biological replicates. Open table in a new tab Using both ATCh and PTCh substrates, we studied the enzyme kinetics of WT ChoE and mutants of the catalytic triad and substrate-binding residues. Our results showed that WT ChoE has similar activity on ATCh and PTCh (Table 2 and Fig. 1A). Based on the apparent catalytic efficiency (kcat/Km), ChoE exhibits slightly higher specificity on PTCh, consistent with the previous report (12Sánchez D.G. Otero L.H. Hernández C.M. Serra A.L. Encarnación S. Domenech C.E. Lisa A.T. A Pseudomonas aeruginosa PAO1 acetylcholinesterase is encoded by the PA4921 gene and belongs to the SGNH hydrolase family.Microbiol. Res. 2012; 167 (22192836): 317-32510.1016/j.micres.2011.11.005Crossref PubMed Scopus (14) Google Scholar). Using an elevated concentration (30 nm) of ChoE, we also detected its activity toward butyrylthiocholine (BTCh) (Table 2 and Fig. 1A) in contrast to the previous report in which a lower concentration of enzyme was used (12Sánchez D.G. Otero L.H. Hernández C.M. Serra A.L. Encarnación S. Domenech C.E. Lisa A.T. A Pseudomonas aeruginosa PAO1 acetylcholinesterase is encoded by the PA4921 gene and belongs to the SGNH hydrolase family.Microbiol. Res. 2012; 167 (22192836): 317-32510.1016/j.micres.2011.11.005Crossref PubMed Scopus (14) Google Scholar). In our assays, substrate inhibition was observed at high concentrations of the substrate with a KSI(app) of 0.9 mm for ATCh. Substrate inhibition was modeled upon the mechanism first described by Krupka (21Krupka R.M. The mechanism of action of acetylcholinesterase: substrate inhibition and the binding of inhibitors.Biochemistry. 1963; 2 (13927118): 76-8210.1021/bi00901a015Crossref PubMed Scopus (20) Google Scholar) that considered substrate inhibition of AChE as arising from trapping an EIS (acyl-enzyme intermediate substrate complex) at high substrate concentrations. Such EIS complex of ChoE would be kinetically undistinguishable from an EPS complex (enzyme product-substrate ternary complex) and is thus fully consistent with the EPS observed in the crystal structures of ChoE (see below). Although the kinetic data could be equally well fitted with a model describing inhibition as arising from the formation of an enzyme-substrate-substrate complex (ESS, Table 2), as assumed by Sánchez et al. (12Sánchez D.G. Otero L.H. Hernández C.M. Serra A.L. Encarnación S. Domenech C.E. Lisa A.T. A Pseudomonas aeruginosa PAO1 acetylcholinesterase is encoded by the PA4921 gene and belongs to the SGNH hydrolase family.Microbiol. Res. 2012; 167 (22192836): 317-32510.1016/j.micres.2011.11.005Crossref PubMed Scopus (14) Google Scholar), an ESS inhibitory complex of ChoE appears unlikely given the lack of evidence of a second substrate-binding site by crystallography.Table 2Kinetic data of WT ChoE and mutantsEnzymeSubstratekcat(app)Km(app)KSI(app)kcat/Kms−1mmM−1 s−1WTaFit to Equation 1 (Krupka) (21) and “Materials and methods.” Fit to Equation 2 gave identical values.ATCh200 ± 250.10 ± 0.020.9 ± 0.22.0 × 106S38AATCh-bUnmeasurable.---H288NATCh----D285NcFit to Michaelis-Menten equation (Equation 3).ATCh5.0 ± 0.31.6 ± 0.4-3.1 × 103Y106AcFit to Michaelis-Menten equation (Equation 3).ATCh99 ± 42.15 ± 0.25-4.6 × 104W287AcFit to Michaelis-Menten equation (Equation 3).ATCh214 ± 91.64 ± 0.18-1.3 × 105WTaFit to Equation 1 (Krupka) (21) and “Materials and methods.” Fit to Equation 2 gave identical values.PTCh316 ± 430.07 ± 0.021.0 ± 0.34.5 × 106S38APTCh----H288NPTCh----D285NcFit to Michaelis-Menten equation (Equation 3).PTCh6.2 ± 0.20.28 ± 0.05-2.2 × 104Y106AcFit to Michaelis-Menten equation (Equation 3).PTCh179 ± 51.37 ± 0.11-1.3 × 105W287AcFit to Michaelis-Menten equation (Equation 3).PTCh190 ± 51.60 ± 0.13-1.2 × 105WTaFit to Equation 1 (Krupka) (21) and “Materials and methods.” Fit to Equation 2 gave identical values.BTCh6.2 ± 0.20.024 ± 0.0046.2 ± 0.92.6 × 105WTcFit to Michaelis-Menten equation (Equation 3).pNPA83 ± 130.8 ± 0.3-1.0 × 105WTcFit to Michaelis-Menten equation (Equation 3).pNPP439 ± 450.35 ± 0.10-1.2 × 106WTcFit to Michaelis-Menten equation (Equation 3).pNPB5.8 ± 0.30.12 ± 0.01-4.8 × 104WTpNPO----WTaFit to Equation 1 (Krupka) (21) and “Materials and methods.” Fit to Equation 2 gave identical values.ATCh + PI10.2 ± 0.70.1 ± 0.0211.9 ± 4.31.0 × 105WTaFit to Equation 1 (Krupka) (21) and “Materials and methods.” Fit to Equation 2 gave identical values.ATCh + TEAC57.6 ± 1.80.06 ± 0.01175 ± 1049.6 × 105a Fit to Equation 1 (Krupka) (21Krupka R.M. The mechanism of action of acetylcholinesterase: substrate inhibition and the binding of inhibitors.Biochemistry. 1963; 2 (13927118): 76-8210.1021/bi00901a015Crossref PubMed Scopus (20) Google Scholar) and “Materials and methods.” Fit to Equation 2 gave identical values.b Unmeasurable.c Fit to Michaelis-Menten equation (Equation 3). Open table in a new tab The enzymatic assays carried out with the catalytic triad mutants (S38A, D285N, and H288N) showed that the catalytic activity was abolished in both S38A and H288N mutants, whereas the D285N mutation diminished the enzymatic activity severely, indicating that both Ser-38 and His-288 are indispensable for ChoE to fulfill its hydrolase function (Table 2 and Fig. 1B). Notably, replacement of Asp-285 by an asparagine has led to a 40-fold reduction of kcat(app) together with an increase of Km(app) by 16-fold, reflecting a 640-fold drop of catalytic efficiency when ATCh is the substrate. The large impact on kcat(app) upon mutation indicates that Asp-285 is, albeit not essential, an important component of catalytic triad in ChoE. The kinetic analysis of the mutants of substrate-binding residues (Y106A and W287A) (Table 2 and Fig. S1) using ATCh and PTCh as substrates indicated that the Y106A mutation has led to less than ∼2-fold drop of kcat(app) but to a 14-20-fold increase of Km(app), whereas the W287A mutation has resulted in a small or no change of kcat(app) accompanied by a 16-fold increase of Km(app). Intriguingly, substrate inhibition is abolished for all three mutants D285N, Y106A, and W287A. The kinetic analysis of ChoE with the substrates 4-nitrophenyl acetate (pNPA), 4-nitrophenyl propionate (pNPP), 4-nitrophenyl butyrate (pNPB), and 4-nitrophenyl octanoate (pNPO) demonstrated that ChoE exhibits a lower specificity on these p-nitrophenyl esters as compared with the ATCh and PTCh substrates (Table 2 and Fig. 1C). Importantly, the esters bearing extra C-C bonds beyond the propionyl group, such as pNPB and pNPO, are either not substrates or very poor substrates, consistent with our molecular docking in which the esters with a long tail could not be accommodated in the active site pocket with a proper geometry required for catalysis (Fig. S2). Taken together with the data on ATCh, PTCh, and BTCh, these results suggest that the substrates of ChoE with high specificity are limited to the esters having an acetyl or a propionyl group. Notably, no substrate inhibition was observed with two of the p-nitrophenyl esters (pNPA and pNPP). This may well be the case of the third substrate (pNPB) but because it was not soluble at more than 1 mm in our assay condition, the absence of substrate inhibition could not be verified with this substrate. Prompted by the docking results (Fig. S2), we have also investigated the inhibitory effects of tetraethylammonium chloride (TEAC) and propidium iodide (PI), the latter of which is an inhibitor that can bind to either the peripheral anionic site (PAS) of mouse AChE (22Bourne Y. Taylor P. Radic Z. Marchot P. Structural insights into ligand interactions at the acetylcholinesterase peripheral anionic site.EMBO J. 2003; 22 (12505979): 1-1210.1093/emboj/cdg005Crossref PubMed Scopus (300) Google Scholar) or the A-site of human butyrylcholinesterase (23Rosenberry T.L. Brazzolotto X. Macdonald I.R. Wandhammer M. Trovaslet-Leroy M. Darvesh S. Nachon F. Comparison of the binding of reversible inhibitors to human butyrylcholinesterase and acetylcholinesterase: a crystallographic, kinetic and calorimetric study.Molecules. 2017; 22: 209810.3390/molecules22122098Crossref PubMed Scopus (67) Google Scholar). These two molecules contain a quaternary ammonium group, similar to ATCh and PTCh. Kinetic analysis has confirmed that the presence of either molecule at 1 mm could indeed inhibit the activity of ChoE leading to a 3.5-20-fold decrease of kcat(app) when ATCh was used as the substrate, whereas essentially not affecting Km(app). Interestingly, the KSI(app) values for substrate inhibition by ATCh were much higher (13-194–fold) in the presence of TEAC or PI (Table 2 and Fig. 1D). The DNA sequence encoding the signal peptide (residues 1-20) at the N-terminal was not included in our construct. All the residues (21-307) in the mature protein are built into the model because of the excellent quality of electron density maps. Having five β-strands with an order of β2-β1-β3-β4-β5 and 10 α-helices, ChoE has the characteristic α/β/α-globular fold of the SGNH hydrolase family (24Mølgaard A. Kauppinen S. Larsen S. Rhamnogalacturonan acetylesterase elucidates the structure and function of a new family of hydrolases.Structure. 2000; 8 (10801485): 373-38310.1016/S0969-2126(00)00118-0Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 25Akoh C.C. Lee G.-C. Liaw Y.-C. Huang T.-H. Shaw J.-F. GDSL family of serine esterases/lipases.Prog. Lipid Res. 2004; 43 (15522763): 534-55210.1016/j.plipres.2004.09.002Crossref PubMed Scopus (403) Google Scholar) (Fig. 2A). The most similar structures of ChoE, obtained through the DALI search (26Holm L. Rosenström P. Dali server: conservation mapping in 3D.Nucleic Acids Res. 2010; 38 (20457744): W545-W54910.1093/nar/gkq366Crossref PubMed Scopus (2905) Google Scholar), include the esterase EstJ15 (PDB 5XTU) (27Mazlan S. Ali M.S.M. Rahman R. Sabri S. Jonet M.A. Leow T.C. Crystallization and structure elucidation of GDSL esterase of photobacterium sp. J15.Int. J. Biol. Macromol. 2018; 119 (30102982): 1188-119410.1016/j.ijbiomac.2018.08.022Crossref PubMed Scopus (6) Google Scholar), the phospholipase A2 VvPlpA (PDB 6JKZ) (28Wan Y. Liu C. Ma Q. Structural analysis of a Vibrio phospholipase reveals an unusual Ser-His-chloride catalytic triad.J. Biol. Chem. 2019; 294 (31073025): 11391-1140110.1074/jbc.RA119.008280Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) (Fig. 2B), and the esterase EstA (29van den Berg B. Crystal structure of a full-length autotransporter.J. Mol. Biol. 2010; 396 (20060837): 627-63310.1016/j.jmb.2009.12.061Crossref PubMed Scopus (119) Google Scholar). However, these enzymes share a relatively low sequence identity of 23–26% with ChoE. Notably, EstJ15 has the same Ser-His-Asp catalytic triad as that in ChoE, whereas VvPlpA lacks the Asp residue in the catalytic triad of ChoE and EstJ15 and instead utilizes an unusual Ser-His-chloride catalytic triad to fulfill the catalysis (28Wan Y. Liu C. Ma Q. Structural analysis of a Vibrio phospholipase reveals an unusual Ser-His-chloride catalytic triad.J. Biol. Chem. 2019; 294 (31073025): 11391-1140110.1074/jbc.RA119.008280Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Similar to EstJ15 and VvPlpA, ChoE is monomeric based on size exclusion chromatography and the crystal structures. It is worth mentioning that, for the 10 most similar proteins retrieved from the DALI search, none contained a substrate or a product molecule in the active site. Quite remarkably, despite the same reaction being catalyzed by ChoE and eukaryotic AChEs, their overall structures bear no similarities. All the structures reported here were obtained from the crystallization conditions containing 100 mm MES buffer. The native ChoE structure determined at a resolution of 1.35 Å shows unambiguously the presence of a MES molecule in the substrate-binding pocket located at the center of ChoE (Fig. 3A). Calculation of electrostatic potential indicated that this pocket is negatively charged (Fig. 3B), a structural feature facilitating the entrapment of the positively charged quaternary ammonium moiety of the ACh substrate. As shown in Fig. 3C, the MES molecule is bound to the substrate-binding pocket lined by the aromatic residues Trp-73, Tyr-106, Tyr-107, Phe-150, and Trp-287. At the bottom of the pocket are the catalytic residues Ser-38 and His-288 as well as the oxyanion hole composed of Gly-98 and Asn-147. Asp-285, the third residue forming the catalytic triad, is next to His-288, such that His-288 is within the hydrogen bonding distance (2.6-2.9 Å) of both Asp-285 and Ser-38, the pattern of which is similar to that of eukaryotic AChEs (5Sussman J. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein.Science. 1991; 253: 872-87910.1126/science.1678899Crossref PubMed Scopus (2319) Google Scholar) and resembles that of serine proteases as well (30Hedstrom L. Serine protease mechanism and specificity.Chem. Rev. 2002; 102 (12475199): 4501-452410.1021/cr000033xCrossref PubMed Scopus (1214) Google Scholar, 31Lo Y.C. Lin S.C. Shaw J.F. Liaw Y.C. Crystal structure of Escherichia coli thioesterase I/protease I/lysophospholipase L1: consensus sequence blocks constitute the catalytic center of SGNH-hydrolases through a conserved hydrogen bond network.J. Mol. Biol. 2003; 330 (12842470): 539-55110.1016/S0022-2836(03)00637-5Crossref PubMed Scopus (90) Google Scholar). In this structure, the ethanesulfonic moiety of MES is exposed to the solvent and interacts with Tyr-107 and Asp-154 via water-mediated hydrogen bonds, whereas its morpholine ring is buried inside the pocket with its oxygen atom close to a well-defined water molecule (Wat-459) (3.2 Å) at the bottom of the pocket. Located at the center of the oxyanion hole, this water molecule is stabilized by the hydrogen bonds with the main chain amide groups of Ser-38 and Gly-98, the hydroxyl group of Ser-38 as well as the side chain amide group of Asn-147. Both Asp-285 and His-288 are located at a U-turn loop where Asp-285 forms hydrogen bonds with the hydroxyl of Tyr-68 and the main chain amides of Glu-286, Trp-287, and His-288 at this loop. These interactions contribute significantly to the stabilization of the local structure as evidenced by the fact that the D285A mutant is insoluble, presumably due to the perturbation or disruption of the overall folding of the protein. Similarly, the imidazole ring of His-288 is sandwiched between Trp-73 and Trp-287 and also establishes van der Waals contacts with Tyr-39, suggesting a structural role of the His-288 side chain. To minimize the detrimental effects on the local structure and increase our chance to obtain structures of relevant protein-ligand complexes as exemplified in our previous work (32Shi R. Lamb S.S. Bhat S. Sulea T. Wright G.D. Matte A. Cygler M. Crystal structure of StaL, a glycopeptide antibiotic sulfotransferase from Streptomyces toyocaensis.J. Biol. Chem. 2007; 282 (17329243): 13073-1308610.1074/jbc.M611912200Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 33Shi R. McDonald L. Cui Q. Matte A. Cygler M. Ekiel I. Structural and mechanistic insight into covalent substrate binding by Escherichia coli" @default.
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• W3022103780 date "2020-06-01" @default.
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• W3022103780 title "Structural insights into the putative bacterial acetylcholinesterase ChoE and its substrate inhibition mechanism" @default.
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• W3022103780 doi "https://doi.org/10.1074/jbc.ra119.011809" @default.
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