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 angstrom 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

Structural characterization of a mitochondrial 3-ketoacyl-CoA (T1)-like thiolase from Mycobacterium smegmatis

Thiolases catalyze the degradation and synthesis of 3-ketoacyl-CoA molecules. Here, the crystal structures of a T1-like thiolase (MSM-13 thiolase) from Mycobacterium smegmatis in apo and liganded forms are described. Systematic comparisons of six crystallographically independent unliganded MSM-13 thiolase tetramers (dimers of tight dimers) from three different crystal forms revealed that the two tight dimers are connected to a rigid tetramerization domain via flexible hinge regions, generating an asymmetric tetramer. In the liganded structure, CoA is bound to those subunits that are rotated towards the tip of the tetramerization loop of the opposing dimer, suggesting that this loop is important for substrate binding. The hinge regions responsible for this rotation occur near Val123 and Arg149. The L alpha 1-covering loop-L alpha 2 region, together with the N beta 2-N alpha 2 loop of the adjacent subunit, defines a specificity pocket that is larger and more polar than those of other tetrameric thiolases, suggesting that MSM-13 thiolase has a distinct substrate specificity. Consistent with this finding, only residual activity was detected with acetoacetyl-CoA as the substrate in the degradative direction. No activity was observed with acetyl-CoA in the synthetic direction. Structural comparisons with other well characterized thiolases suggest that MSM-13 thiolase is probably a degradative thiolase that is specific for 3-ketoacyl-CoA molecules with polar, bulky acyl chains

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

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

The protomers are assembled with 32 symmetry in the asymmetric unit of <i>Ms</i>TLP1.

<p>The six protomers are arranged as two discs of three protomers each. A) The two discs are related by a non-crystallographic 2-fold symmetry axis. B) A non-crystallographic 3-fold symmetry axis relates the three protomers of each disk.</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

Data collection statistics for <i>Mycobacterium smegmatis</i> TLP1.

<p>Values in parenthesis refer to the highest resolution shell.</p><p><i>I</i> is the integrated intensity and σ (<i>I</i>) is the estimated standard deviation of that intensity.</p><p>R_merge = (Σ_hklΣ_i(|I_i(hkl)−|)/Σ_hklΣI_i(hkl), where I_i(hkl) is the intensity of the i^th measurement of reflection hkl and is its mean.</p

Refinement statistics and model quality.

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