Unraveling the stereochemical and dynamic aspects of the catalytic site of bacterial peptidyl-tRNA hydrolase

Abstract

Bacterial peptidyl-tRNA hydrolase (Pth; EC 3.1.1.29) hydrolyzes the peptidyl-tRNAs accumulated in the cytoplasm and thereby prevents cell death by alleviating tRNA starvation. X-ray and NMR studies of Vibrio cholerae Pth (VcPth) and mutants of its key residues involved in catalysis show that the activity and selectivity of the protein depends on the stereochemistry and dynamics of residues H24, D97, N118, and N14. D97-H24 interaction is critical for activity because it increases the nucleophilicity of H24. The N118 and N14 have orthogonally competing interactions with H24, both of which reduce the nucleophilicity of H24 and are likely to be offset by positioning of a peptidyl-tRNA substrate. The region proximal to H24 and the lid region exhibit slow motions that may assist in accommodating the substrate. Helix α3 exhibits a slow wobble with intermediate time scale motions of its N-cap residue N118, which may work as a flypaper to position the scissile ester bond of the substrate. Overall, the dynamics of interactions between the side chains of N14, H24, D97, and N118, control the catalysis of substrate by this enzyme.

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

“…The B ‐factor diagram indicated that except for the N‐ and C‐terminal ends, the base loop has the highest B ‐factor values (Figure A,B). Previous nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulation studies of Mycobacterium smegmatis Pth ( Ms Pth) and Vibrio cholerae Pth ( Vc Pth) also indicated the high flexibility of this region . The B ‐factor diagram revealed that helix α6 also has high B ‐factor values (Figure A,B), consistent with the NMR and MD simulation studies of Mycobacterium tuberculosis Pth ( Mt Pth) .…”

“…The B-factor diagram indicated that except for the N-and C-terminal ends, the base loop has the highest B-factor values ( Figure 4A,B). Previous nuclear magnetic resonance (NMR) and 29,37 The B-factor diagram revealed that helix α6 also has high B-factor values ( Figure 4A,B), consistent with the NMR and MD simulation studies of Mycobacterium tuberculosis Pth (MtPth). 27 In addition, relatively high B-factors were observed in the loop-helix α4 region (termed the "lid region"), which covers the active site cleft, and in the region following the lid region ( Figure 4A,B).…”

“…In the present crystal structure, a citrate ion was bound to the back side of the lid region and reinforced it (Figure B and Supplementary Information Figure S3A and B). We expect that if this citrate ion had not been bound, the lid region of Tt Pth would have exhibited higher B ‐factor values, as observed in Mt Pth, Ms Pth, and Vc Pth . The loop connecting strands β3 and β4 also exhibited relatively high B ‐factor values (Figure B).…”

“…We expect that if this citrate ion had not been bound, the lid region of TtPth would have exhibited higher B-factor values, as observed in MtPth, MsPth, and VcPth. 27,29,37 The loop connecting strands β3 and β4 also exhibited relatively high B-factor values ( Figure 4B). This is probably due to the two consecutive glycine residues (Figure 2).…”

“…25 Therefore, to elucidate the structural basis for the thermostability of a protein, comparisons of the structure of a thermophilic protein with those of its mesophilic homologues are needed. In the case of Pth, the structures from many mesophilic bacteria have been determined; 12,[26][27][28][29][30][31][32][33][34][35][36][37] however, no structure from a thermophilic bacterium has been determined so far.…”