X-ray crystallographic snapshots of reaction intermediates in the G117H mutant of human butyrylcholinesterase, a nerve agent target engineered into a catalytic bioscavenger

Abstract

OPs (organophosphylates) exert their acute toxicity through inhibition of acetylcholinesterase, by phosphylation of the catalytic serine residue. Engineering of human butyrylcholinesterase, by substitution of a histidine residue for the glycine residue at position 117, led to the creation of OP hydrolase activity. However, the lack of structural information and poor understanding of the hydrolytic mechanism of the G117H mutant has hampered further improvements in the catalytic activity. We have solved the crystallographic structure of the G117H mutant with a variety of ligands in its active site. A sulfate anion bound to the active site suggested the positioning for an OP prior to phosphylation. A fluoride anion was found in the active site when NaF was added to the crystallization buffer. In the fluoride complex, the imidazole ring from the His117 residue was substantially shifted, adopting a relaxed conformation probably close to that of the unliganded mutant enzyme. Additional X-ray structures were obtained from the transient covalent adducts formed upon reaction of the G117H mutant with the OPs echothiophate and VX [ethyl ({2-[bis(propan-2-yl)amino]ethyl}sulfanyl](methyl)phosphinate]. The position of the His117 residue shifted in response to the introduction of these adducts, overlaying the phosphylserine residue. These structural data suggest that the dephosphylation mechanism involves either a substantial conformational change of the His117 residue or an adjacent nucleophilic substitution by water.

Structural Study of the Complex Stereoselectivity of Human Butyrylcholinesterase for the Neurotoxic V-agents

“…The orientation of VX is identical to that observed in the recently solved structure of the VX-G117H mutant of BChE (PDB entry 2XMG) (26). This is the mirror image of the VX-TcAChE adduct which is of configuration P R : in this latter, the methyl substituent is located in the acyl-binding pocket, and the ethoxy group points toward the catalytic histidine inducing a conformational change (11).…”

Development of organophosphate hydrolase activity in a bacterial homolog of human cholinesterase

“…Various forms of hysteresis in AChE and BChE have been observed kinetically (Masson et al, 2005; Badiou et al, 2008; Masson and Lockridge, 2010; Lushchekina et al, 2014), and possibly structurally (Nachon et al, 2011). Non-linear kinetic curves for BChE G117H also were observed with selected substrates (Millard et al, 1995b).…”

Cholinesterase Inhibitors

“…Since then, they were developed as CWAs (e.g., nerve agents such as VX, VE, VG, and VM), but also as agricultural pesticides (e.g., malathion, parathion, diazinon, fenthion, dichlorvos, trichlorfon, chlorpyrifos, dimethoate, triazophos, and ethion), flame retardants (e.g., triphenyl phosphate), therapeutics to elevate ACh levels (e.g., echothiophate and diisopropyl fluorophosphate, DFP), and veterinary medications. Similar to carbamates, OPs react with the active site of ChEs and other serine esterases due to the structural resemblance to their substrates (Nachon et al, 2011). The phosphorylated enzyme can be reactivated at a considerably slower rate than the carbamylated enzyme, and certain OPs can further undergo dealkyl ation following their interaction with the enzyme, which completely blocks its reactivation (known as aging; Taylor, 1996;Wandhammer et al, 2013) (Figure 52.2A).…”

“…By the early 1990s, these definitions were well complemented by theoretical and experimental structuralmolecular observations. Cloning of cholinesterase genes from plants, inverte brates, and vertebrates (including humans) and the con sequent elucidation of the threedimensional structure of cholinesterases (Figure 52.1B and C;Nicolet et al, 2003;Ngamelue et al, 2007;Dvir et al, 2010;Nachon et al, 2011;Nachon et al, 2013) allowed a better understanding of the structurefunction relationship in these enzymes, while opening new and yetunsolved questions.…”