| Mutagenesis of specific amino acids converts carnitine acetyltransferase into carnitine palmitoyltransferase. | |
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MedLine Citation:
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PMID: 16681386 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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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:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Biochemistry Volume: 45 ISSN: 0006-2960 ISO Abbreviation: Biochemistry Publication Date: 2006 May |
Date Detail:
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Created Date: 2006-05-09 Completed Date: 2006-08-08 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 0370623 Medline TA: Biochemistry Country: United States |
Other Details:
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Languages: eng Pagination: 6133-41 Citation Subset: IM |
Affiliation:
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Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, E-08028 Spain. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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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:
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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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