Conservative Tryptophan Residue in the Vicinity of an Active Site of the M15 Family l,d-Peptidases: A Key Element in the Catalysis

Abstract

Bioinformatics analysis of the sequences of orthologous zinc-containing peptidases of the M15_C subfamily revealed the presence of a conserved tryptophan residue near the active site, which is not involved in the formation of the protein core. Site-directed mutagenesis of this Trp114/109 residue using two representatives of the family, l-alanoyl-d-glutamate peptidases of bacteriophages T5 (calcium-activated EndoT5) and RB49 (EndoRB49, without ion regulation) as examples, and further analysis of the ¹H NMR spectra of the mutants showed that a decrease in the volume of the W → F → A residue leads to changes in the hydrophobic core and active center of the protein, and also decreases the affinity for regulatory Ca²⁺ in the EndoT5 mutants. The inactive T5W114A mutant lacks the ability to bind the substrate. In general, the conserved Trp114/109 residue, due to the spatial restrictions of its side chain, significantly affects the formation of the catalytically active form of the enzyme and is critical for catalysis.

Keywords: bacteriophage; calcium; catalysis; l,d-peptidase; substrate binding.

Figure 1

Multiple sequence alignment of amino acid sequences of orthologous peptidases of bacteriophages of the taxon Caudovirales. Conservative amino acids are colored red, similar residues are written with black bold characters and boxed in yellow; consensus is represented on a separate line. A consensus sequence is generated using criteria from MultAlin: uppercase is identity, lowercase is consensus level > 0.5, ! is any of IV, # is any of NDQE. The black stars mark the amino acids that coordinate the zinc atom, the violet star marks the catalytic aspartate. The line under consensus is the result of prediction of the secondary structure: blue rectangles mark α-helices, green arrows mark β-strands. Abbreviations denote endolysins of the following bacteriophages (in parentheses are protein numbers in GenBank): phiEcoM-GJ1—Escherichia phage phiEcoM-GJ1 (YP_001595416.1), ST32—Escherichia phage ST32 (YP_009790684.1), vB_EcoM_4HA13—Escherichia phage vB_EcoM_4HA13 (YP_009884078.1), vB_PagM_SSEM1—Pantoea phage vB_PagM_SSEM1 (YP_009859342.1), 3/49—Shewanella sp. phage 3/49 (YP_009103932.1), vB_PmiM_Pm5461—Proteus phage vB_PmiM_Pm5461 (YP_009195522.1), phiP4-3—Proteus phage phiP4-3 (YP_010093702.1), phiP27—Enterobacteria phage phiP27 (NP_543082.1), P88—Escherichia phage P88 (YP_009113074.1), RB49—Enterobacteria phage RB49 (NP_891673.1), Phil1—Escherichia phage Phi1 (YP_001469446.1), phiTE—Pectobacterium phage phiTE (YP_007392609.1), eiAU-183—Edwardsiella phage eiAU-183 (YP_009004687.1), KP15—Klebsiella phage KP15 (YP_003580002.1), KP27—Klebsiella phage KP27 (YP_007348788.1), vB_CsaM_GAP161—Cronobacter phage vB_CsaM_GAP161 (YP_006986425.1), RB43—Enterobacteria phage RB43 (YP_239135.1), T5—Escherichia phage T5 (AAS19387.1), phiR2-01—Yersinia phage phiR2-01 (YP_007237012.1), vB_Kpn_IME260—Klebsiella phage vB_Kpn_IME260 (YP_009597415.1), vB_PP_V—Pectobacterium phage DU_PP_V (YP_009795235.1), Shivani—Salmonella phage Shivani (YP_009194685.1).

Figure 2

Upfield regions of aliphatic parts of ¹H NMR spectra of EndoT5(Zn²⁺Ca²⁺) and EndoRB49(Zn²⁺) protein forms. (A) EndoT5wt and its mutants EndoT5W114F and EndoT5W114A; (B) EndoRB49wt and its mutants Endo RB49W109F and Endo RB49W109A.

Figure 3

Downfield regions of aromatic parts of ¹H NMR spectra of EndoT5(Zn²⁺Ca²⁺) and EndoRB49(Zn²⁺) protein forms. (A) EndoT5wt and its mutants EndoT5W114F and EndoT5W114A. (B) EndoRB49wt and its mutants Endo RB49W109F and Endo RB49W109A.

Figure 4

Effect of excess mutant proteins on the activity of 1 nM native EndoT5 under conditions of competition for the substrate. W114A, EndoT5 mutant for conservative tryptophan; D130A, EndoT5 mutant for catalytic aspartate; BSA, bovine serum albumin. Maximum EndoT5 activity in the absence of other proteins was taken as 100%.

Figure 5

Intrinsic disorder profiles of EndoT5wt (black solid line), EndoT5W114F (red dashed line), and EndoT5W114A (green dashed line) (A) and EndoRB49wt (black solid line), EndoRB49W109F (red dashed line), EndoRB49W109A (green dashed line) (B) generated by PONDR^® VSL2. Thresholds for protein intrinsic disorder (disorder score 0.5) and conformational flexibility (0.15) are shown by solid and dashed black lines. Positions of conserved tryptophan residues W114 and W109 in EndoT5 and EndoRB49 are shown by vertical blue lines.

Figure 6

Aligned disorder profiles of 22 members of the phage l,d-peptidases of the M15_C family. Gaps in the disorder profiles correspond to gaps in the multiple sequence alignment shown in Figure 1. Vertical pink and blue lines indicate position of the zinc-coordinating aspartate residue and conserved tryptophan residue analyzed in this study, respectively.