Crystal Structure of Carnitine Acetyltransferase and Implications for the Catalytic Mechanism and Fatty Acid Transport

Abstract

Carnitine acyltransferases have crucial roles in the transport of fatty acids for beta-oxidation. Dysregulation of these enzymes can lead to serious diseases in humans, and they are targets for therapeutic development against diabetes. We report the crystal structures of murine carnitine acetyltransferase (CRAT), alone and in complex with its substrate carnitine or CoA. The structure contains two domains. Surprisingly, these two domains share the same backbone fold, which is also similar to that of chloramphenicol acetyltransferase and dihydrolipoyl transacetylase. The active site is located at the interface between the two domains. Carnitine and CoA are bound in deep channels in the enzyme, on opposite sides of the catalytic His343 residue. The structural information provides a molecular basis for understanding the catalysis by carnitine acyltransferases and for designing their inhibitors. Specifically, our structural information suggests that the substrate carnitine may assist the catalysis by stabilizing the oxyanion in the reaction intermediate.

The brain-specific carnitine palmitoyltransferase-1c regulates energy homeostasis

“…We have tested numerous acyl-CoA thioesters of various chain length and saturation as acyl donors, none of which served as substrates (Table 1). It should be noted that the putative carnitine-binding site is conserved at the primary amino acid sequence level, suggesting that carnitine or a carnitine-like molecule act as the acyl acceptor (26). Thus, the reaction catalyzed by CPT1c remains unresolved.…”

C75 is converted to C75-CoA in the hypothalamus, where it inhibits carnitine palmitoyltransferase 1 and decreases food intake and body weight

“…A model of rat CrAT wt enzyme was constructed by homology modelling using as templates the structures deposited in the Protein Data Bank (PDB) corresponding to human (1NM8) [27], and mouse CrAT (1NDB, 1NDF and 1NDI) [28], essentially as Page 12 of 39 A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 12 described elsewhere [14]. CrAT D356A/M564G was modelled by the same procedures using rat CrAT wt as template.…”

Crystal Structure of Vinorine Synthase, the First Representative of the BAHD Superfamily

“…Other proteins that can be aligned to the VS structure include condensation do- All of these aligned proteins are CoA-dependent acyltransferases and contain the conserved HXXXD motif in the active site. In all these acyltransferases except VibH, His of this motif plays a critical role in the CoA-dependent acyltransfer reaction mechanism (35)(36)(37)(38)(39). VibH also contains the HXXXD motif, and the conserved His is favorably positioned in the active site, but mutation of this His to Ala or Glu has little effect on catalysis, indicating that the HXXXD motif in VibH is not used for an equivalent role in acyltransfer catalysis (40).…”

“…Besides, the N␦1 of His 160 is also hydrogen-bonded with carbonyl oxygen of Ala 163 (2.9 Å) and with the side chain of Asn 293 (3.0 Å). Structures of several related CoA-dependent acyltransferases have been solved in complex with co-factor and substrate, such as Azotobacter vinelandii dihydrolipoyl transacetylase (Protein Data Bank codes 1EAD and 1EAB) with CoA and substrate lipoamide and mouse carnitine acetyltransferase (Protein Data Bank codes 1NDB and 1NDI) with substrate carnitine and CoA (35,36). By superimposing the dihydrolipoyl transacetylase monomer on domain 1 of VS, we could map the CoA and lipoamide binding sites onto VS.…”