| Research Focus A snapshot of carnitine acetyltransferase q Rona R. Ramsay and James H. Naismith Centre for Biomolecular Sciences, University of St. Andrews, North Haugh, St. Andrews, UK KY16 9ST Carnitine acetyltransferase (CrAT) is part of the carni- tine system that protects the acylation state of the pools of acetyl-coenzyme A, a key metabolic intermedi- ate, by transferring excess acetate and other short- chain acyl groups to and from carnitine. The homology of CrAT with other carnitine acyltransferases, such as carnitine palmitoyltransferase I (CPT-I) that regulates fatty acid metabolism, make the solving of its structure a landmark in understanding mechanism and ligand binding in this family. Although the first crystals of carnitine acetyltransferase (CrAT) were observed in 1965 [1], its structure has only just been solved [2,3]. It is the first structure of an enzyme from the family of enzymes that transfer activated acyl groups between carnitine and coenzyme A (CoA; Box 1) to modulate acyl-CoA and CoA availability for the wide range of acyl-transfer reactions that are essential for energy production and cell regulation. Acyl-CoA esters, by virtue of their potent effects on gene transcription, enzyme activity, membrane trafficking and secretory processes, are direct effectors of cell function. The carnitine system The carnitine-dependent enzymes and acyl-carnitine carriers transfer acyl groups between the limited and compartmentalized pools of CoA in the cell [4]. CrAT in the mitochondrial matrix catalyzes a freely reversible equili- brium reaction between short-chain acyl-CoA and CoA, and acyl-L-carnitine and L-carnitine (Box 1) so that the fraction acylated is the same for both CoA and L-carnitine. Acetyl-L-carnitine can then be exported into the much larger cytosolic carnitine pool via the carnitine carrier (carnitine/acylcarnitine translocase). For example, under conditions in which high fatty-acid oxidation flux produces large amounts of acetyl-CoA in the mitochondrial matrix, acetyl-L-carnitine levels increase in the cytosol and even increase in the plasma [5]. Similarly, non-metabolized short-chain acyl groups are excreted as their L-carnitine esters [6]. In the reverse direction, acetyl-L-carnitine provides a large reservoir of activated acetyl groups for transfer to CoA for the citric-acid cycle in response to the surge in energy demand when, for example, an insect or pigeon takes flight. CrAT is an abundant, soluble and active enzyme. Its kinetic and chemical mechanism has been thoroughly characterized [7] and sequences have been reported from yeast, mouse, rat and human [8]. The gene encoding CrAT contains the leader sequence for delivery to the mitochon- drial matrix where , 50% of CrAT activity in liver cells is found. When a second start-codon omitting the leader sequence is used, the C-terminal peroxisomal targetting signal (Ala-Lys-Leu) results in its import into the peroxisomal lumen (30% of the activity). The remaining CrAT activity is found in the lumen of the endoplasmic reticulum. No CrAT activity in mammalian cytoplasm means that activated acetyl groups are transferred only between the intra-organelle pools. The structure of CrAT CrAT from mouse [2] and man [3] is a monomer composed of two domains. The chemical reaction takes place in a tunnel that is formed by the interface of the two domains (Fig. 1). The C-terminal domain binds most of the CoA, whereas carnitine is bound in the tunnel. Notably, the N-terminal domain is structurally related to dihydrolipoyl transferase (E2pCD of pyruvate dehydrogenase), which transfers the acetyl group between lipoamide and CoA, and to chloramphenicol acetyltransferase, which acetyl- ates chloramphenicol. The degree of structural conser- vation observed at domain 1 is perhaps unsurprising given that the reaction catalyzed (transfer of an acetyl group) is the same, but it was not apparent from sequence analysis. Structural superposition of CrAT and dihydrolipoyl transferase suggests that the position of the substrate atom to which the acetyl group is transferred from the CoA (3-hydroxyl group of carnitine in CrAT) is conserved relative to the structure. Most importantly, a histidine (His343 in CrAT) – identified as the active site base by site- directed mutagenesis and structural data – is also structurally conserved in an unusual conformation. His343, which is held in position by a hydrogen bond to the backbone carbonyl residue, activates the nucleophile on the acceptor by deprotonation. In the structure of CrAT in the absence of ligands [2], His343 is hydrogen bonded to Glu347 (which is either conserved or conservatively substituted to Asp), which suggests a charge-relay system. However, in the structure with CoA bound, this interaction is not found and, instead, a main-chain carbonyl makes the histidine residue more basic (Fig. 1b). The carboxylate group of carnitine is recognized by Arg518 and a network of hydrogen bonds (Fig. 1b). The positively charged trimethyammonium group has no specific interactions, but stacks against Phe566 and is close to Ser552 (the first conserved serine of the Ser-Thr-Ser motif) – mutation of which increases the K M for L-carnitine in the analogous enzyme, carnitine octanoyltransferase. Corresponding author: Rona R. Ramsay (rrr@st-and.ac.uk). Update TRENDS in Biochemical Sciences Vol.28 No.7 July 2003 343 http://tibs.trends.com