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.
Highlights
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 [1]
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 [12]
Growth of the WT PAO1 strain and the deletion mutant strain PAO1::DPA4921 (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
Summary
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 [1]. The first crystal structure of an acetylcholinesterase from Torpedo californica (TcAChE) was published nearly 30 years ago [5] This was followed by the determination of ;250 structures of AChEs of eukaryotic sources, many of which are in complex with various inhibitors [6]. Despite the long history of research on structure and inhibition of eukaryotic AChEs [7, 8], novel insights have been gained into these pivotal enzymes in recent years, e.g. the dynamics of back door opening [9], the steric and dynamic parameters implicated in the reaction within the active-site gorge [10], and the possible repurposing of AChE inhibitors as anti-cancer agents [11]. Production of choline by ChoE for subsequent catabolic processes by other metabolic pathways was suggested to be linked to the pathogenicity of P. aeruginosa and its survival during murine lung infection [19, 20]
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