title: Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificity.
abstract: Bile acid CoA:amino acid N-acyltransferase (BAAT) is the terminal enzyme in the synthesis of bile salts from cholesterol and catalyzes the conjugation of taurine or glycine to bile acid CoA thioesters to form bile acid N-acylamidates. BAAT has a dual localization to the cytosol and peroxisomes, possibly due to an inefficient carboxy-terminal peroxisomal targeting signal (PTS), -serine-glutamine-leucine (-SQL). Mutational analysis was used to define the role of the carboxy terminus in peroxisomal localization and kinetic activity. Amidation activity of BAAT and BAAT lacking the final two amino acids (AAs) (BAAT-S) were similar, whereas the activity of BAAT with a canonical PTS sequence (BAAT-SKL) was increased >2.5-fold. Kinetic analysis of BAAT and BAAT-SKL showed that BAAT-SKL had a lower Km for taurine and glycine as well as a greater Vmax There was no difference in the affinity for cholyl-CoA. In contrast to BAAT, BAAT-SKL forms bile acid N-acylamidates with β-alanine. BAAT-S immunoprecipitated when incubated with peroxisomal biogenesis factor 5 (Pex5) and rabbit anti-Pex5 antibodies; however, deleting the final 12 AAs prevented coimmunoprecipitation with Pex5, indicating the Pex5 interaction involves more than the -SQL sequence. These results indicate that even small changes in the carboxy terminus of BAAT can have significant effects on activity and substrate specificity.
authors.affiliation: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294.
authors.forename: Nathan A
authors.initials: NA
authors.lastname: Styles
authors.affiliation: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294.
authors.forename: Erin M
authors.initials: EM
authors.lastname: Shonsey
authors.affiliation: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294.
authors.forename: Josie L
authors.initials: JL
authors.lastname: Falany
authors.affiliation: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294.
authors.forename: Amber L
authors.initials: AL
authors.lastname: Guidry
authors.affiliation: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294.
authors.forename: Stephen
authors.initials: S
authors.lastname: Barnes
authors.affiliation: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294 cfalany@uab.edu.
authors.forename: Charles N
authors.initials: CN
authors.lastname: Falany
mesh_terms: D001647:Bile Acids and Salts / Q000235:genetics* / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004252:DNA Mutational Analysis; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009154:Mutation; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000201:enzymology; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D051049:Sphingosine N-Acyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001647:Bile Acids and Salts; C000614264:PEX5 protein, human; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D051049:Sphingosine N-Acyltransferase
keywords: N-acyltransferase; bile acid amidation; bile acid conjugation; bile acid ligase; glycine; peroxisomal biogenesis factor 5; peroxisomal targeting signal; taurine