title: Functional consequences and structural interpretation of mutations of human choline acetyltransferase.
abstract: Choline acetyltransferase (ChAT; EC 2.3.1.6) catalyzes synthesis of acetylcholine from acetyl-CoA (AcCoA) and choline in cholinergic neurons. Mutations in CHAT cause potentially lethal congenital myasthenic syndromes associated with episodic apnea (ChAT-CMS). Here, we analyze the functional consequences of 12 missense and one nonsense mutations of CHAT in 11 patients. Nine of the mutations are novel. We examine expression of the recombinant missense mutants in Bosc 23 cells, determine their kinetic properties and thermal stability, and interpret the functional effects of 11 mutations in the context of the atomic structural model of human ChAT. Five mutations (p.Trp421Ser, p.Ser498Pro, p.Thr553Asn, p.Ala557Thr, and p.Ser572Trp) reduce enzyme expression to less than 50% of wild-type. Mutations with severe kinetic effects are located in the active-site tunnel (p.Met202Arg, p.Thr553Asn, and p.Ala557Thr) or adjacent to the substrate binding site (p.Ser572Trp), or exert their effect allosterically (p.Trp421Ser and p.Ile689Ser). Two mutations with milder kinetic effects (p.Val136Met and p.Ala235Thr) are also predicted to act allosterically. One mutation (p.Thr608Asn) below the nucleotide binding site of CoA enhances dissociation of AcCoA from the enzyme-substrate complex. Two mutations introducing a proline residue into an α-helix (p.Ser498Pro and p.Ser704Pro) impair the thermal stability of ChAT.
authors.affiliation: Department of Neurology and Muscle Research Laboratory, Mayo Clinic, Rochester, Minnesota 55905, USA.
authors.forename: Xin-Ming
authors.initials: XM
authors.lastname: Shen
authors.forename: Thomas O
authors.initials: TO
authors.lastname: Crawford
authors.forename: Joan
authors.initials: J
authors.lastname: Brengman
authors.forename: Gyula
authors.initials: G
authors.lastname: Acsadi
authors.forename: Susan
authors.initials: S
authors.lastname: Iannaconne
authors.forename: Emin
authors.initials: E
authors.lastname: Karaca
authors.forename: Chaouky
authors.initials: C
authors.lastname: Khoury
authors.forename: Jean K
authors.initials: JK
authors.lastname: Mah
authors.forename: Shimon
authors.initials: S
authors.lastname: Edvardson
authors.forename: Zeljko
authors.initials: Z
authors.lastname: Bajzer
authors.forename: David
authors.initials: D
authors.lastname: Rodgers
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Engel
mesh_terms: D001665:Binding Sites; D002795:Choline O-Acetyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D059329:Cholinergic Neurons / Q000201:enzymology / Q000378:metabolism; D056726:Genetic Association Studies; D006801:Humans; D007700:Kinetics; D009154:Mutation; D020294:Myasthenic Syndromes, Congenital / Q000235:genetics; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D002795:Choline O-Acetyltransferase