title: Redesign of cosubstrate specificity and identification of important residues for substrate binding to hChAT.
abstract: In eukaryotes, choline acetyltransferase (ChAT) catalyzes the reversible formation of the neurotransmitter acetylcholine from choline and acetyl-CoA. ChAT belongs to a family of CoA-dependent enzymes that also includes the carnitine acyltransferases CrAT, CrOT, and CPTs. In contrast to CrOT and CPTs that are very active toward medium- and long-chain acyl-CoAs, respectively, CrAT and ChAT display activity toward only short-chain acyl-CoAs. We recently demonstrated the substrate and cosubstrate promiscuity of the wild-type human ChAT (hChAT). To extend the flexibility of this enzyme, we have generated a series of single, double, and triple hChAT mutants. Here we report the conversion of hChAT into choline octanoyltransferase (ChOT) and choline palmitoyltransferase (ChPT). The E337 and C550 residues (numbering from hChAT) were previously shown to dictate the acyl-CoA cosubstrate specificity in the carnitine series. Here we identify and demonstrate the importance of C551, in addition to E337 and C550, in contributing to the acyl-CoA specificity of hChAT. We also show that either C550 or C551 needs to be present for the transfer of medium- and long-chain acyl-CoAs by hChAT. By exploring the potential expansion of the tunnel on the substrate side, we demonstrate that residues M84, Y436, and Y552 play a critical role in binding and holding the choline substrate in the ChAT active site.
authors.affiliation: Life Sciences Institute, 210 Washtenaw Avenue, University of Michigan, Ann Arbor, Michigan 48109-2216, USA.
authors.forename: Keith D
authors.initials: KD
authors.lastname: Green
authors.forename: Vanessa R
authors.initials: VR
authors.lastname: Porter
authors.forename: Yaru
authors.initials: Y
authors.lastname: Zhang
authors.forename: Sylvie
authors.initials: S
authors.lastname: Garneau-Tsodikova
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002384:Catalysis; D002794:Choline / Q000737:chemistry*; D002795:Choline O-Acetyltransferase / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D002795:Choline O-Acetyltransferase; D002794:Choline