Document Detail


Mutagenesis of specific amino acids converts carnitine acetyltransferase into carnitine palmitoyltransferase.
MedLine Citation:
PMID:  16681386     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Carnitine acyltransferases catalyze the exchange of acyl groups between carnitine and CoA. The members of the family can be classified on the basis of their acyl-CoA selectivity. Carnitine acetyltransferases (CrATs) are very active toward short-chain acyl-CoAs but not toward medium- or long-chain acyl-CoAs. Previously, we identified an amino acid residue (Met(564) in rat CrAT) that was critical to fatty acyl-chain-length specificity. M564G-mutated CrAT behaved as if its natural substrates were medium-chain acyl-CoAs, similar to that of carnitine octanoyltransferase (COT). To extend the specificity of rat CrAT to other substrates, we have performed new mutations. Using in silico molecular modeling procedures, we have now identified a second putative amino acid involved in acyl-CoA specificity (Asp(356) in rat CrAT). The double CrAT mutant D356A/M564G showed 6-fold higher activity toward palmitoyl-CoA than that of the single CrAT mutant M564G and a new activity toward stearoyl-CoA. We show that by performing two amino acid replacements a CrAT can be converted into a pseudo carnitine palmitoyltransferase (CPT) in terms of substrate specificity. To change CrAT specificity from carnitine to choline, we also prepared a mutant CrAT that incorporates four amino acid substitutions (A106M/T465V/T467N/R518N). The quadruple mutant shifted the catalytic discrimination between l-carnitine and choline in favor of the latter substrate and showed a 9-fold increase in catalytic efficiency toward choline compared with that of the wild-type. Molecular in silico docking supports kinetic data for the positioning of substrates in the catalytic site of CrAT mutants.
Authors:
Antonio G Cordente; Eduardo López-Viñas; María Irene Vázquez; Paulino Gómez-Puertas; Guillermina Asins; Dolors Serra; Fausto G Hegardt
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biochemistry     Volume:  45     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  2006 May 
Date Detail:
Created Date:  2006-05-09     Completed Date:  2006-08-08     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  6133-41     Citation Subset:  IM    
Affiliation:
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, E-08028 Spain.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Amino Acids / chemistry*,  genetics
Animals
Carnitine O-Acetyltransferase / chemistry*,  genetics
Carnitine O-Palmitoyltransferase / chemistry*,  genetics
Fluorometry
Kinetics
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Radiometry
Rats
Sequence Homology, Amino Acid
Substrate Specificity
Chemical
Reg. No./Substance:
0/Amino Acids; EC 2.3.1.21/Carnitine O-Palmitoyltransferase; EC 2.3.1.7/Carnitine O-Acetyltransferase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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