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Crystal structures of murine carnitine acetyltransferase in ternary complexes with its substrates.

ABSTRACT: Carnitine acyltransferases catalyze the reversible exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids in the mitochondria for energy production, and are attractive targets for drug discovery against diabetes and obesity. To help define in molecular detail the catalytic mechanism of these enzymes, we report here the high resolution crystal structure of wild-type murine carnitine acetyltransferase (CrAT) in a ternary complex with its substrates acetyl-CoA and carnitine, and the structure of the S554A/M564G double mutant in a ternary complex with the substrates CoA and hexanoylcarnitine. Detailed analyses suggest that these structures may be good mimics for the Michaelis complexes for the forward and reverse reactions of the enzyme, representing the first time that such complexes of CrAT have been studied in molecular detail. The structural information provides significant new insights into the catalytic mechanism of CrAT and possibly carnitine acyltransferases in general.

SUBMITTER: Hsiao YS

PROVIDER: S-EPMC2940834 | biostudies-literature | 2006 Sep

REPOSITORIES: biostudies-literature

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Crystal structures of murine carnitine acetyltransferase in ternary complexes with its substrates.

Hsiao Yu-Shan YS Jogl Gerwald G Tong Liang L

The Journal of biological chemistry 20060726 38

Carnitine acyltransferases catalyze the reversible exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids in the mitochondria for energy production, and are attractive targets for drug discovery against diabetes and obesity. To help define in molecular detail the catalytic mechanism of these enzymes, we report here the high resolution crystal structure of wild-type murine carnitine a ...[more]

PMID: 16870616

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Project description:BackgroundThe carnitine acetyltransferase (CrAT) is a mitochondrial matrix protein that directly influences intramitochondrial acetyl-CoA pools. Murine CrAT is encoded by a single gene located in the opposite orientation head to head to the PPP2R4 gene, sharing a very condensed bi-directional promoter. Since decreased CrAT expression is correlated with metabolic inflexibility and subsequent pathological consequences, our aim was to reveal and define possible activators of CrAT transcription in the normal embryonic murine liver cell line BNL CL. 2 and via which nuclear factors based on key metabolites mainly regulate hepatic expression of CrAT. Here we describe a functional characterization of the CrAT promoter region under conditions of L-carnitine deficiency and supplementation as well as fenofibrate induction in cell culture cells.ResultsThe murine CrAT promoter displays some characteristics of a housekeeping gene: it lacks a TATA-box, is very GC-rich and harbors two Sp1 binding sites. Analysis of the promoter activity of CrAT by luciferase assays uncovered a L-carnitine sensitive region within -342 bp of the transcription start. Electrophoretic mobility shift and supershift assays proved the sequence element (-228/-222) to be an L-carnitine sensitive RXR? binding site, which also showed sensitivity to application of anti-PPAR? and anti-PPARbp antibodies. In addition we analysed this specific RXR?/PPAR? site by Southwestern Blotting technique and could pin down three protein factors binding to this promoter element. By qPCR we could quantify the nutrigenomic effect of L-carnitine itself and fenofibrate.ConclusionsOur results indicate a cooperative interplay of L-carnitine and PPAR? in transcriptional regulation of murine CrAT, which is of nutrigenomical relevance. We created experimental proof that the muCrAT gene clearly is a PPAR? target. Both L-carnitine and fenofibrate are inducers of CrAT transcripts, but the important hyperlipidemic drug fenofibrate being a more potent one, as a consequence of its pharmacological interaction.

Dataset Information

Crystal structures of murine carnitine acetyltransferase in ternary complexes with its substrates.

Publications

Crystal structures of murine carnitine acetyltransferase in ternary complexes with its substrates.

Similar Datasets

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