Carnitine O-Acetyltransferase as a Central Player in Lipid and Branched-Chain Amino Acid Metabolism, Epigenetics, Cell Plasticity, and Organelle Function

Abstract

Carnitine O-acetyltransferase (CRAT) is a key mitochondrial enzyme involved in maintaining metabolic homeostasis by mediating the reversible transfer of acetyl groups between acetyl-CoA and carnitine. This enzymatic activity ensures the optimal functioning of mitochondrial carbon flux by preventing acetyl-CoA accumulation, buffering metabolic flexibility, and regulating the balance between fatty acid and glucose oxidation. CRAT's interplay with the mitochondrial carnitine shuttle, involving carnitine palmitoyltransferases (CPT1 and CPT2) and the carnitine carrier (SLC25A20), underscores its critical role in energy metabolism. Emerging evidence highlights the structural and functional diversity of CRAT and structurally related acetyltransferases across cellular compartments, illustrating their coordinated role in lipid metabolism, amino acid catabolism, and mitochondrial bioenergetics. Moreover, the structural insights into CRAT have paved the way for understanding its regulation and identifying potential modulators with therapeutic applications for diseases such as diabetes, mitochondrial disorders, and cancer. This review examines CRAT's structural and functional aspects, its relationships with carnitine shuttle members and other carnitine acyltransferases, and its broader role in metabolic health and disease. The potential for targeting CRAT and its associated pathways offers promising avenues for therapeutic interventions aimed at restoring metabolic equilibrium and addressing metabolic dysfunction in disease states.

Keywords: CPT; CRAT; CROT; ChAT; artemisinin; carnitine palmitoyltransferases; choline acyltransferase; mitochondrial carnitine acetyltransferase; molecular modeling; peroxisomal carnitine octanoyltransferase; suramin.

Figure 2

Multiple sequence alignment of the human carnitine acyltransferases and choline acyltransferase. An extract from a multiple sequence alignment of carnitine acyltransferases and choline acetyltransferase sampled by blastp through the indicated metazoan is reported. The represented blocks highlight the conservation of residues crucial for the function of the compared enzymes, such as H343 (important for the catalytic activity), K419 and K23 (important for CoA binding), and D430 and E454 (important for protein stability). All the mentioned residues are labeled and indicated by arrows. Amino acid replacement Y110C and V569M recently associated with an early onset case of Leigh syndrome are also reported for comparative purposes and indicated by labels and arrows. Residue numbering in the amino acid labels corresponds to the residues numbering in the human CRAT sequence with accession number NP_000746.3. The “>” symbols indicate the crystallized carnitine acyltransferases or the choline acetyl transferase, reported in this alignment for comparative purposes, with reference to residues numbering that can vary between the crystallized structures and sequences retrieved by blastp (see also Table 1). The “*” symbol indicates CPT1 that has not a crystallized counterpart. The alignment was obtained by using ClustalW 2.1 (a free software currently maintained at the Conway Institute UCD Dublin by Des Higgins, Fabian Sievers, David Dineen, and Andreas Wilm) in the Jalview 2.11.4.1 package (a free software released under GPLv3, developed by The Barton Group, University of Dundee, Scotland UK). Amino acid colors reflect their physico-chemical properties according the Jalview “zappo” color scheme: aliphatic/hydrophobic residues Alanine (A), Isoleucine (I), Leucine (L), Methionine(M), Valine (V) are colored in light-pink; aromatic residues Phenylalanine (F), Tryptophan (W), Tyrosine (Y) are colored in orange; conformationally special Gycine (G) and Proline (P) are colored in magenta; Cysteine (C) is colored in yellow; hydrophilic residues Asparagine (N), Glutamine (Q), Serine (S), Threonine (T) are colored in green; acidic negatively charged residues, Aspartate (D), Glutamate (E) are colored in red; basic positively charged residues Arginine (R), Histidine (H), Lysine (K) are colored in blue [55,56].

Figure 3

Three-dimensional comparative analysis of the human carnitine acyltransferases and choline acyltransferase. The crystallized structures of CRAT, CROT, CPT2, and ChAT are reported in cyan, dark-pink, dark-green, and blue cartoon representations (by PyMOL [63]) in panels (a–d), respectively. CoA is reported in yellow sticks in all the a-d panels. Acetylcarnitine is reported in magenta sticks in panel (a), octanoylcarnitine is reported in green sticks in panel (b), carnitine is reported in salmon sticks in panel (c), whereas choline is reported in orange sticks in panel (d). The binding sites of acetylcarnitine, octanoylcarnitine, carnitine, and choline in the four crystallized structures are reported in panels (e–h), whereas CoA binding sites are reported in panels (i–l). The ligands and protein side chains are shown in ball-and-stick representation, with the ligand bonds colored in purple, according to the default style of LigPlot [64]. Hydrogen bonds are shown as green dotted lines, while the spoked arcs represent protein residues making nonbonded contacts with the ligand. Residues numbering in panels (e) and (i) reflects the numbering of the crystallized structure of CRAT lacking 21 residues at the N-terminal., i.e., the real residues numbering of CRAT residues is obtained by adding 21 residues (to be considered for comparative purposes with reference to residues numbering reported in the multiple sequence alignment).

Figure 1

Scheme of the cross-talk between a mitochondrion and a peroxisome with a set of representative proteins, pathways and cycles. CRAT, CROT, protein of the carnitine shuttle, respiratory chain complexes, mitochondrial transporters and other proteins are reported in surf representation and labeled. CRAT (based on the human crystallized structure 1nm8.pdb) is reported in orange surface representation; the peroxisomal CROT (based on the human crystallized structure 1xl8.pdb) is reported in brown surface representation; the peroxisomal very long chain fatty acid (VLCFA) transporter ABCD1 (based on the crystallized structure 7rr9.pdb) is reported in magenta-blue surface representation; the mitochondrial branched chain amino acid aminotransferase BCAT2 (based on the crystallized structure 5mpr.pdb) is reported in blue-green surface representation; the mitochondrial branched chain alpha-ketoacid dehydrogenase BCKDH (based on the crystallized structure 1u5b.pdb) is reported in dark magenta-blue surface representation; CPT1 and CPT2 (both based on the R. norvegicus crystallized structure 4ep9.pdb) in dark violet; ATP synthase (CV) is reported in blue (based on the Bos taurus crystallized structure 6zqn.pdb). Mitochondrial carriers are reported in cyan (based on the 3D structure of the bovine ADP/ATP carrier, 1okc.pdb). VDAC is reported as a reference protein of the outer mitochondrial membrane in light-pink surface representation (based on the 3D structure of the human 2jk4.pdb). MPC (an in-house developed 3D comparative model, data not published) is reported in black; PDH in light green (based on the human crystallized structure 6cfo.pdb); AIF in white (based on the human crystallized structure 4bur.pdb). Complex I (CI, based on the Ovis aries crystallized structure 5lnk.pdb), complex II (CII, based on the Sus scrofa 3aef.pdb), complex III (CIII, based on the O. aries 6q9e.pdb), complex IV (CIV) (together with CytC in red, based on the bovine crystallized structure 5iy5.pdb) are reported in green, yellow, magenta and gray, respectively, according to PyMOL colors. Black circular arrows indicate cyclic pathways. Red arrows indicate impaired pathways or reactions. Black solid/dashed lines indicate the possible direction of the reported reactions. Abbreviations: C2-CoA, acetyl-CoA; C2-carnitine, acetyl-carnitine; SC-CoA, short chain acyl-CoA; LCFA, long chain fatty acids; VLCFA, very long chain fatty acids; BCFA, branched chain fatty acids; BCKA, branched chain ketoacids; MIM: mitochondrial inner membrane; MOM, mitochondrial outer membrane; IMS, intermembrane space; UQ, ubiquinone; CFNC, the peroxisomal CoA, FAD, NAD⁺/PAP, FMN, AMP carrier, coded in H. sapiens by SLC25A17; AAC, ADP/ATP carrier, coded in H. sapiens by SLC25A4, SLC25A5, SLC25A6, SLC25A31; TPC, thiamine pyrophosphate carrier, coded by SLC25A19; CAC, carnitine/acyl-carnitine carrier, coded by SLC25A20; ORC, ornithine carrier, coded by SLC25A15 (or SLC25A2); AGC, aspartate/glutamate carrier, coded by SLC25A12 and SLC25A13; DIC, dicarboxylate carrier, coded by SLC25A10; NDT, assumed to be the NAD+ carrier, coded by SLC25A51; MFT, assumed to be the FAD (folate/riboflavin) carrier, coded by SLC25A32; OGC, malate/2-oxoglutarate carrier, coded by SLC25A11; CIC, citrate carrier, coded by SLC25A1; PiC, phosphate carrier, coded by SLC25A3; CoAC, CoA carrier, coded by SLC25A42; MAS, malate/aspartate shuttle; TCA, tricarboxylic acid cycle; Bax, Bcl-2 associated X protein; Bak, Bcl-2 antagonist/killer-1; Bcl-2, B-cell lymphoma-2; MDH1, cytosolic malate dehydrogenase 1; ME1, malic enzyme 1; MPC, mitochondrial pyruvate carrier; PDH, pyruvate dehydrogenase; CypD, cyclophilin D; CytC, cytochrome C; VDAC, voltage-dependent anion channel; AIF, apoptosis-inducing factor; PNC, pyrimidine nucleotide carrier, coded in H. sapiens by SLC25A33 and SLC25A36. The green arrow for Artemisinin and the magenta arrow for suramin indicate the ability of artemisinin to stimulate the activity of p.Tyr110Cys variant and the ability of suramin to inhibit both the WT-CRAT and the p.Tyr110Cys variant. The other two magenta arrows for mildronate, ranolazine and thioridazine indicate their ability in partially inhibiting in a selective way CRAT, mitochondrial beta oxidation or peroxisomal beta oxidation, respectively. The reported 2D structures of the mentioned drugs have been collected from the KEGG-DRUG database (https://www.kegg.jp/kegg/drug/, accessed last time on the 23 January 2025). For all the mentioned drugs, the KEGG-DRUG identification number has been reported.