High‐resolution crystal structure of peptidyl‐tRNA hydrolase from Thermus thermophilus

Abstract

Peptidyl‐tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl‐tRNA molecules, which are the products of defective translation, to recycle the tRNA for further rounds of protein synthesis. Pth is ubiquitous in nature, and its activity is essential for bacterial viability. Here, we have determined the crystal structure of Pth from Thermus thermophilus (TtPth) at 1.00 Å resolution. This is the first structure of a Pth from a thermophilic bacterium and the highest resolution Pth structure reported so far. The present atomic resolution data enabled the calculation of anisotropic displacement parameters for all atoms, which revealed the directionality of the fluctuations of key regions for the substrate recognition. Comparisons between TtPth and mesophilic bacterial Pths revealed that their structures are similar overall. However, the structures of the N‐ and C‐terminal, loop‐helix α4, and helix α6 regions are different. In addition, the helix α1 to strand β4 region of TtPth is remarkably different from those of the mesophilic bacterial Pths, because this region is 9 or 10 amino acid residues shorter than those of the mesophilic bacterial Pths. This shortening seems to contribute to the thermostability of TtPth. To further understand the determinants for the thermostability of TtPth, we compared various structural factors of TtPth with those of mesophilic bacterial Pths. The data suggest that the decreases in accessible surface area and thermolabile amino acid residues, and the increases in ion pairs, hydrogen bonds, and proline residues cooperatively contribute to the thermostability of TtPth.

Carboxylic Ester Hydrolases in Bacteria: Active Site, Structure, Function and Application

“…As described in Figure 5, catalytic His-Asp (denoted by an asterisk), two oxyanion hole-forming Asn residues (O-marked) and Asn involved in substrate binding (S-marked) are also conserved in other peptidyl-tRNA hydrolases, such as those from Mycobacterium tuberculosis (PDB code: 2Z2I) [124], M. smegmatis (PDB code: 3P2J), E. coli (PDB code: 3VJR, 2PTH, and 3OFV) [123], S. typhimurium (PDB code: 4P7B) [132], Acinetobacter baumannii (PDB code: 4LWQ), Pseudomonas aeruginosa (PDB code: 4FYJ) [131], Francisella tularensis (PDB code: 3NEA) [125], Streptococcus pyogenes (PDB code: 4QT4) [133], Staphylococcus aureus (PDB code: 4YLY) [135] and Burkholderia thailandensis (PDB code: 3V2I) [127]. In the Thermus thermophilus peptidyl-tRNA hydrolase (PDB code: 5ZX8), Arg103 plays a role in substrate binding and oxyanion formation similar to Asn118 in V. cholerae ( Figure 5) [138]. Important residues in this group are shown in Figure 3F.…”

“…[135] and Burkholderia thailandensis (PDB code: 3V2I) [127]. In the Thermus thermophilus peptidyl-tRNA hydrolase (PDB code: 5ZX8), Arg103 plays a role in substrate binding and oxyanion formation similar to Asn118 in V. cholerae ( Figure 5) [138]. Important residues in this group are shown in Figure 3F.…”

Crystal Structure of Peptidyl-tRNA Hydrolase from <i>Acinetobacter baumannii</i> at 1.00 Å Resolution

“…There are seven structures from Gramnegative bacteria which include the current structure of AbPth and that of PaPth [29], EcPth [11], StPth [30], BtPth [31], VcPth [32] and FtPth [33]. Four structures are from Gram-positive bacteria which include structures of MsPth [34], SaPth [35], TtPth [36] and SpPth [37]. The structure of MtPth [17] is also available.…”

“…The crystal structures of Pth enzyme from nearly a dozen of bacteria including Acinetobacter baumannii (AbPth) [28], Pseudomonas aeruginosa (PaPth) [29], Escherichia coli (EcPth) [11], Salmonella typhimurium (StPth) [30], Burkholderia structome (BtPth) [31], Vibrio cholerae (VcPth) [32], Francisella tularensis (FtPth) [33], Mycobacterium tuberculosis (MtPth) [17], Mycobacterium smegmatis (MsPth) [34], Staphylococcus aureus (SaPth) [35], Thermus thermophilus (TtPth) [36], Streptococcus pyogenes (SpPth) [37] are known. First seven structures of Pth from the above list are from Gram-negative bacteria while the last four structures belong to Gram-positive bacteria.…”

“…First seven structures of Pth from the above list are from Gram-negative bacteria while the last four structures belong to Gram-positive bacteria. Most of these structures have been determined at moderate resolutions except the structures of EcPth [11], the complex of AbPth with cyatarabine [38] and TtPth [36], which were determined at 1.20 Å, 1.10 Å and 1.00 Å respectively. Here, we report a structure of AbPth at 1.00 Å resolution giving several new insights.…”