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

Cloning, expression, purification and preliminary X-ray diffraction studies of a putative Mycobacterium smegmatis thiolase

Thiolases are important in fatty-acid degradation and biosynthetic pathways. Analysis of the genomic sequence of Mycobacterium smegmatis suggests the presence of several putative thiolase genes. One of these genes appears to code for an SCP-x protein. Human SCP-x consists of an N-terminal domain (referred to as SCP2 thiolase) and a C-terminal domain (referred as sterol carrier protein 2). Here, the cloning, expression, purification and crystallization of this putative SCP-x protein from M. smegmatis are reported. The crystals diffracted X-rays to 2.5 Å resolution and belonged to the triclinic space group P1. Calculation of rotation functions using X-ray diffraction data suggests that the protein is likely to possess a hexameric oligomerization with 32 symmetry which has not been observed in the other six known classes of this enzyme

Crystal Structure of a Monomeric Thiolase-Like Protein Type 1 (TLP1) from <em>Mycobacterium smegmatis</em>

<div><p>An analysis of the <em>Mycobacterium smegmatis</em> genome suggests that it codes for several thiolases and thiolase-like proteins. Thiolases are an important family of enzymes that are involved in fatty acid metabolism. They occur as either dimers or tetramers. Thiolases catalyze the Claisen condensation of two acetyl-Coenzyme A molecules in the synthetic direction and the thiolytic cleavage of 3-ketoacyl-Coenzyme A molecules in the degradative direction. Some of the <em>M. smegmatis</em> genes have been annotated as thiolases of the poorly characterized SCP2-thiolase subfamily. The mammalian SCP2-thiolase consists of an N-terminal thiolase domain followed by an additional C-terminal domain called sterol carrier protein-2 or SCP2. The <em>M. smegmatis</em> protein selected in the present study, referred to here as the thiolase-like protein type 1 (<em>Ms</em>TLP1), has been biochemically and structurally characterized. Unlike classical thiolases, <em>Ms</em>TLP1 is a monomer in solution. Its structure has been determined at 2.7 Å resolution by the single wavelength anomalous dispersion method. The structure of the protomer confirms that the N-terminal domain has the thiolase fold. An extra C-terminal domain is indeed observed. Interestingly, it consists of six β-strands forming an anti-parallel β-barrel which is completely different from the expected SCP2-fold. Detailed sequence and structural comparisons with thiolases show that the residues known to be essential for catalysis are not conserved in <em>Ms</em>TLP1. Consistent with this observation, activity measurements show that <em>Ms</em>TLP1 does not catalyze the thiolase reaction. This is the first structural report of a monomeric thiolase-like protein from any organism. These studies show that <em>Ms</em>TLP1 belongs to a new group of thiolase related proteins of unknown function.</p> </div

Quality of the final electron density map.

<p>Residues 330–334 corresponding to a loop in the N-terminal thiolase domain are shown with the corresponding electron density of the (2F_o−F_c), α_c-map, contoured at 1 σ.</p

Structure based sequence alignment of <i>Z. ramigera</i> thiolase (PDB ID: 1DM3) and <i>Ms</i>TLP1 (TLP1, GenBank accession no. YP_889758.1).

<p>The secondary structure assignment above and below the sequences refer to <i>Z. ramigera</i> thiolase and <i>Ms</i>STPL1, respectively. Black coils represent α-helices and black arrows represent β-strands. Residues conserved in both proteins are highlighted in red. The sequence identity between the thiolase domains of the <i>Z. ramigera</i> enzyme and <i>Ms</i>TLP1 is 15%. The five sequence fingerprints of <i>Z. ramigera</i> thiolase and their corresponding residues in <i>Ms</i>TLP1 are colored blue, and labeled in green boxes as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041894#pone-0041894-g003" target="_blank">Figure 3</a>,. The five segments of the thiolase loop domain with specific functions are highlighted as follows: tetramerization loop (orange), covering loop (purple), cationic loop (grey), adenine loop (light pink), pantetheine loop (yellow). Disordered regions of the <i>Ms</i>TLP1 thiolase domain are highlighted in light green.</p

Sequence conservation in TLP proteins.

<p>The sequence alignment was achieved using ClustalW program. Seven unique TLP proteins were used for the analysis; TLP1 and TLP2 from <i>M. smegmatis</i> (GenBank accession nos. YP_889758.1, YP_887911.1), TLP from <i>M. tuberculosis</i> (GenBank accession no. ZP_06454776.1), <i>S. chlorophenolicum</i> (GenBank accession no. YP_004555364.1), <i>A. oryzae</i>, <i>M.acridum</i> (GenBank accession no. XP_001817576.2 ) and <i>R. erythropolis</i> (GenBank accession no. ZP_04388669.1). Conserved residues are highlighted in red. Residues structurally equivalent to the five sequence finger prints of classical thiolases are highlighted in numbered green boxes.</p

Evolutionary tree analysis of the thiolase sequences.

<p>The phylogenetic tree was constructed using the neighbor-joining method, with 10,000 bootstrap replicates in MEGA5 software. Only the region corresponding to the thiolase domain was used for these calculations. The group identifiers correspond to the nomenclature described in the text. The numbers next to each node indicate bootstrap values as percentages. The following sequences (listed with their NCBI accession codes and, for trypanosomatid sequences, their NCBI and GeneDB accession codes) were used for creating the evolutionary tree. <b>1</b>, Human: HsT1 (NP_006102.2); <b>2</b>, <i>Mus musculus</i>: MmT1 (NP_803421.1); <b>3</b>, <i>Salmo salar</i>: SsT1 (ACI33809.1); <b>4</b>, <i>Monosiga brevicollis</i>: MbT1 (XP_001748310.1); <b>5</b>, <i>Bdellovibrio bacteriovorus</i>: BbT1 (NP_967398.1); <b>6</b>, <i>Zoogloea ramigera</i>: ZrCT (1DM3); <b>7</b>, HsCT (NP_005882.2); <b>8</b>, MmCT (NP_033364.2); <b>9</b>, HsT2 (NP_000010.1); <b>10</b>, <i>Macaca fascicularis</i>: MfT2 (Q8HXY6.1); <b>11</b>, <i>Rattus norvegicus</i>: RnT2 (NP_058771.1); <b>12</b>, MmT2 (NP_659033.1); <b>13</b>, <i>Saccharomyces cerevisiae</i>: ScAB (1AFW); <b>14</b>, HsAB (NP_001598.1); <b>15</b>, <i>Arabidopsis thaliana</i>: AtAB (2WU9); <b>16</b>, <i>T. cruzi</i>: TcAB (Tc00.1047053511003.60, XP_816035.1); <b>17</b>, <i>Bodo saltans</i>: BsAB (BSA00133, ACI16032.1); <b>18</b>, HsTFE (NP_000174.1); <b>19</b>, TcTFE (Tc00.1047053511389.150, XP_814301.1); <b>20</b>, <i>L. braziliensis</i>: LbTFE (LbrM.31.1840, XP_001567180.1); <b>21</b>, <i>L. major</i>: LmTFE (LmjF.31.1640, XP_001685150.1); <b>22</b>, <i>L. mexicana</i>: LxTFE (LmxM.30.1640.1, CBZ29221.1); <b>23</b>, <i>L. infantum</i>: LiTFE (LinJ.31.1660, CAM70518.2); <b>24</b>, HsSCP2 (NP_002970.2); <b>25</b>, LmSCP2 (LmjF.23.0690, XP_001683404.1); <b>26</b>, LiSCP2 (LinJ.23.0860, XP_001465761.1); <b>27</b>, LxSCP2 (LmxM.23.0690.1, CBZ27218.1); <b>28</b>, LbSCP2 (LbrM.23.0840, XP_001565156.1); <b>29</b>, TcSCP2 (Tc00.1047053510507.20, XP_807246.1); <b>30</b>, <i>T. vivax:</i> TvSCP2 (TvY486_0802010); <b>31</b>, <i>T. congolense</i>: ToSCP2 (congo1030g10.p1k_8); <b>32</b>, <i>T. brucei</i>: TbSCP2 (Tb927.8.2540, XP_847087.1); <b>33</b>, <i>T. gambiense</i>: TgSCP2 (Tbg972.8.2020); <b>34</b>, <i>M. smegmatis</i>: <i>Ms</i>STLP2 (YP_887911.1); <b>35</b>, <i>M. tuberculosis</i>: MtSTLP (NP_216383.1); <b>36</b>, <i>R. jostii</i>: RjSTLP (YP_700393.1); <b>37</b>, <i>C. crescentus</i>: CcSTLP (YP_002517418.1); <b>38</b>, <i>H. neptunium</i>: HnSTLP (YP_759708.1); <b>39</b>, <i>Ms</i>TLP1 (YP_889758.1); <b>40</b>, <i>Erythrobacter sp.</i>: EbSTLP (ZP_01041346.1).</p

The overall fold of <i>Ms</i>TLP1.

<p>A) The TLP protomer is divided into two domains. The N-terminal thiolase domain can further be divided into an N-terminal half (green), a thiolase loop region (light blue) and a C-terminal half (dark). The C-terminal extra domain of <i>Ms</i>TLP1 is in dark green. The two domains are connected by a linker region (yellow). B) The N-terminal thiolase domain of <i>Ms</i>TLP1 has the conserved fold of the thiolase superfamily. The two β sheets are sandwiched between three layers of α helices forming the characteristic α/β/α/β/α layered structure found in classical thiolases. The numbering of the strands and helices conforms to the assignment in the classical thiolases.</p

Refinement statistics and model quality.

<p>Refinement statistics and model quality.</p

Superposition of <i>Ms</i>TLP1 (brown) and <i>Z. ramigera</i> thiolase (green).

<p>A) Also included is the bound acetyl-CoA (cyan) of the complexed <i>Z.ramigera</i> thiolase. It can be noted that the Z. ramigera thiolase CoA-binding part of the thiolase loop domain generates a binding groove in both structures. The DWTP-loop of the C-terminal domain that reaches towards the putative ligand binding site is labeled. The brown stars mark the beginning and the end of the disordered loop (135–170) of MsTLP1 at the beginning of the loop domain. B) Close up of the thiolase loop domains of <i>Z. ramigera</i> thiolase (green) and <i>Ms</i>TLP1 (brown), highlighting the 5 functional regions (blue) of the <i>Z. ramigera</i> thiolase structure.</p