Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificity

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, poss...

Full description

Bibliographic Details
Main Authors: Nathan A. Styles, Erin M. Shonsey, Josie L. Falany, Amber L. Guidry, Stephen Barnes, Charles N. Falany
Format: Article
Language:English
Published: Elsevier 2016-07-01
Series:Journal of Lipid Research
Subjects:
Online Access:http://www.sciencedirect.com/science/article/pii/S0022227520351518
_version_ 1819060741104205824
author Nathan A. Styles
Erin M. Shonsey
Josie L. Falany
Amber L. Guidry
Stephen Barnes
Charles N. Falany
author_facet Nathan A. Styles
Erin M. Shonsey
Josie L. Falany
Amber L. Guidry
Stephen Barnes
Charles N. Falany
author_sort Nathan A. Styles
collection DOAJ
description 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.
first_indexed 2024-12-21T14:31:48Z
format Article
id doaj.art-e977dd560e6d4c779247b3b901b7fbb1
institution Directory Open Access Journal
issn 0022-2275
language English
last_indexed 2024-12-21T14:31:48Z
publishDate 2016-07-01
publisher Elsevier
record_format Article
series Journal of Lipid Research
spelling doaj.art-e977dd560e6d4c779247b3b901b7fbb12022-12-21T19:00:29ZengElsevierJournal of Lipid Research0022-22752016-07-0157711331143Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificityNathan A. Styles0Erin M. Shonsey1Josie L. Falany2Amber L. Guidry3Stephen Barnes4Charles N. Falany5Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294To whom correspondence should be addressed; To whom correspondence should be addressed; Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294Bile 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.http://www.sciencedirect.com/science/article/pii/S0022227520351518bile acid conjugationbile acid amidationtaurineglycineN-acyltransferasebile acid ligase
spellingShingle Nathan A. Styles
Erin M. Shonsey
Josie L. Falany
Amber L. Guidry
Stephen Barnes
Charles N. Falany
Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificity
Journal of Lipid Research
bile acid conjugation
bile acid amidation
taurine
glycine
N-acyltransferase
bile acid ligase
title Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificity
title_full Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificity
title_fullStr Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificity
title_full_unstemmed Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificity
title_short Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificity
title_sort carboxy terminal mutations of bile acid coa n acyltransferase alter activity and substrate specificity
topic bile acid conjugation
bile acid amidation
taurine
glycine
N-acyltransferase
bile acid ligase
url http://www.sciencedirect.com/science/article/pii/S0022227520351518
work_keys_str_mv AT nathanastyles carboxyterminalmutationsofbileacidcoanacyltransferasealteractivityandsubstratespecificity
AT erinmshonsey carboxyterminalmutationsofbileacidcoanacyltransferasealteractivityandsubstratespecificity
AT josielfalany carboxyterminalmutationsofbileacidcoanacyltransferasealteractivityandsubstratespecificity
AT amberlguidry carboxyterminalmutationsofbileacidcoanacyltransferasealteractivityandsubstratespecificity
AT stephenbarnes carboxyterminalmutationsofbileacidcoanacyltransferasealteractivityandsubstratespecificity
AT charlesnfalany carboxyterminalmutationsofbileacidcoanacyltransferasealteractivityandsubstratespecificity