Mutation update on ACAT1 variants associated with mitochondrial acetoacetyl-CoA thiolase (T2) deficiency

Abstract

Mitochondrial acetoacetyl-CoA thiolase (T2, encoded by the ACAT1 gene) deficiency is an inherited disorder of ketone body and isoleucine metabolism. It typically manifests with episodic ketoacidosis. The presence of isoleucine-derived metabolites is the key marker for biochemical diagnosis. To date, 105 ACAT1 variants have been reported in 149 T2-deficient patients. The 56 disease-associated missense ACAT1 variants have been mapped onto the crystal structure of T2. Almost all these missense variants concern residues that are completely or partially buried in the T2 structure. Such variants are expected to cause T2 deficiency by having lower in vivo T2 activity because of lower folding efficiency and/or stability. Expression and activity data of 30 disease-associated missense ACAT1 variants have been measured by expressing them in human SV40-transformed fibroblasts. Only two variants (p.Cys126Ser and p.Tyr219His) appear to have equal stability as wild-type. For these variants, which are inactive, the side chains point into the active site. In patients with T2 deficiency, the genotype does not correlate with the clinical phenotype but exerts a considerable effect on the biochemical phenotype. This could be related to variable remaining residual T2 activity in vivo and has important clinical implications concerning disease management and newborn screening.

Keywords: ACAT1; T2-deficiency; genotype-phenotype correlation; mutations; structure; variants; β-ketothiolase deficiency.

Figure 1

The reactions catalyzed by the T2 thiolase. (a) The biosynthetic reaction: The substrates are two molecules of acetyl‐CoA. (b) The degradative reaction: The substrates are 2‐methylacetoacetyl‐CoA (or acetoacetyl‐CoA) and CoA. In both directions, the reaction mechanism proceeds via a covalent intermediate, in which the nucleophilic cysteine, Cys126 in human T2, becomes acetylated in the biosynthetic as well as in the degradative reactions

Figure 2

Schematic drawing showing the T2 thiolase reaction in the synthetic direction. Two molecules of acetyl‐CoA are converted into CoA and acetoacetyl‐CoA. The role of the four catalytic residues (Cys126, Asn353, His385, Cys413 of human T2) is highlighted. These residues protrude into the catalytic site from the four catalytic loops (the CxS, NEAF, GHP, and CxG loops, respectively, shown in bold). Cys126 is the nucleophilic cysteine and Cys413 is the acid/base cysteine. The side chains of Asn353 (fixing Wat98) and His385, as well as the main chain N‐atoms of the CxS and CxG loops, contribute to the two oxyanion holes (OAH1 and OAH2, shown as shaded semicircles). These oxyanion holes stabilize the negative charge that develops during the reaction on the thioester oxygen atom of the reaction intermediates, being therefore also critically important for catalysis. The short‐curved arrows visualize the breaking/forming of bonds

Figure 3

The sequence of the human mitochondrial acetoacetyl‐CoA thiolase (T2, UniProt code: P24752) with nomenclature of secondary structure, sequence fingerprints, and loops. The N‐terminal region is the mitochondrial leader sequence, which is cleaved off on entry into the mitochondria. The secondary structure is obtained from the structure of the human T2 (PDB code: 2IBW) using the ESPript 3.0 server (Robert & Gouet, 2014) and shown above the sequence. An asterisk (*) above the sequence marks every tenth residue. The mature sequence starts at Val34, indicated by a black circle (•) above the sequence. Important active site loops that are near the catalytic site are identified below the sequence with their sequence fingerprint. The nomenclature of the functional regions of the loop domain (residues 156–286) is also given below the sequence. The structural properties of the latter loop regions are visualized in Figure 7 and Figure S3

Figure 4

Schematic illustration (not to scale) of human ACAT1 gene showing the location of 105 variants associated with mitochondrial acetoacetyl‐CoA thiolase deficiency. Exons (boxes) and introns (lines) are numbered according to NCBI refseq: NG_009888.1. Shaded boxes denote the untranslated region. Numbering of complementary DNA (cDNA; above boxes) is according to NCBI refseq: NM_000019.3, with +1 as the number of A of the ATG initiation codon. Description of variants follows the HGVS nomenclature (version 15.11, http://varnomen.hgvs.org; den Dunnen et al., 2016). Missense and nonsense variants are mainly described at the protein level (NCBI refseq: NP_000010.1). Exonic variants are shown above the diagram in black (missense), red (nonsense), and green (others); those associated with aberrant splicing are underlined, and those affecting the ATG initiation codon, causing reduced translation efficiency, are shown in italics. Intronic and large deletions/insertions/duplications variants are shown below the diagram. Large deletions/insertions/duplications are shown in bold with a solid line (‒) above depicting the approximate location. A number sign (^#) marks variants attributed to Alu‐mediated unequal homologous recombination

Figure 5

The structure of the T2 tetramer (PDB entry 2IBW), complexed with CoA. The bound CoA molecules are shown as stick models. The two tight dimers (below and above; side view) are assembled into tetramers via the four tetramerization loops (in the middle). “cationic” labels one of the cationic loops, which points to the 3′‐phosphate of the CoA bound in the active site of the opposing dimer. Stereo view is provided in Figure S1

Figure 6

The structure of the T2 tight dimer (PDB entry 2IBW). (a) Top view (view approximately down the local two fold axis of the tight dimer). (b) Side view (rotated by 90° around the horizontal with respect to the top view, same view as in Figure 5). The bound CoA molecules are shown as stick models. In the left subunit, the N‐domain, loop domain, and C‐domain are colored as purple, blue, and green ribbons, respectively. In the right subunit, the N‐domain, loop domain, and C‐domain are colored as yellow, orange, and cyan ribbons, respectively. “cationic” and “tetra” identify the cationic and tetramerization loops, respectively. Stereo views are provided in Figure S2

Figure 7

The structure of the T2 loop domain (residues 156–286; PDB entry 2IBW). (a) Top view (same as Figure 6a). (b) Side view (same as Figure 6b). The loop domain protrudes out of Nβ4 and ends at Nβ5 of the N‐terminal domain (Figure 3). The covering loop “cov” is in orange, the cationic loop is in green, the adenine loop is in red and the pantetheine loop is in purple. “tetra” identifies the tetramerization loop. The Lα2 and Lα3 helices are also labeled. The bound CoA molecule is shown as a stick model. Stereo views are provided in Figure S3

Figure 8

Missense variants of residues in loops on the surface of the T2 tetramer (PDB entry 2IBW). The visualized loop residues are either in the loop domain (panels a, b, c) or interact with the loop domain (panel d). Expression of variant T2 cDNAs containing these variants produces T2 protein levels of 25% or higher compared to wild‐type T2, as discussed in the text. These panels are zoomed‐in views, of the loop domain (same view as in Figure 7b). The covering loop (“cov”) and the Lα2 and Lα3 helices are labeled in each panel. The bound CoA molecule is shown as a stick model. (a) The p.Asp186Tyr variant (T2 protein level, 33%; measured activity, 0%). Asp186 (D186) is in the covering loop (orange) and points to the Cβ4‐Cβ5 loop (light blue) of the catalytic site. (b) The p.Arg208Gln variant (T2 protein level, 50%; measured activity, 0%). Arg208 (R208) is at the beginning of helix Lα3 (cyan). The Arg208 side chain is hydrogen bonded to the loop region just after the adenine loop (“ade”, shown in red). (c) The p.Asn282His variant (T2 protein level, 50%; measured activity, 0%). Asn282 (N282) is in the pantetheine loop (purple). (d) The p.Ile323Thr variant (T2 protein level, 25%; measured activity, 20%). Ile323 (I323) is in the Cβ1‐Cα1 loop (light blue) of the C‐terminal domain, just before the DFP sequence fingerprint of the binding pocket for the 2‐methyl group of the 2‐methylacetoacetyl‐CoA substrate. cDNA, complementary DNA