Dual Active Site in the Endolytic Transglycosylase gp144 of Bacteriophage phiKZ

Abstract

Lytic transglycosylases are abundant peptidoglycan lysing enzymes that degrade the heteropolymers of bacterial cell walls in metabolic processes or in the course of a bacteriophage infection. The conventional catalytic mechanism of transglycosylases involves only the Glu or Asp residue. Endolysin gp144 of Pseudomonas aeruginosa bacteriophage phiKZ belongs to the family of Gram-negative transglycosylases with a modular composition and C-terminal location of the catalytic domain. Glu115 of gp144 performs the predicted role of a catalytic residue. However, replacement of this residue does not completely eliminate the activity of the mutant protein. Site-directed mutagenesis has revealed the participation of Tyr197 in the catalytic mechanism, as well as the presence of a second active site involving Glu178 and Tyr147. The existence of the dual active site was supported by computer modeling and monitoring of the molecular dynamics of the changes in the conformation and surface charge distribution as a consequence of point mutations.

Keywords: bacteriophage phiKZ; endolysin; enzyme active site; molecular dynamics; site-directed mutagenesis; transglycosylase.

Fig. 1

The side chains of amino acid residues forming the active sites of phiKZ gp144 and the possible location of the NAM–NAG molecule. The top image shows the general view of the enzyme without (top left) and with the substrate (top right). Bottom images provide a close-up view of the binding sites of the enzyme without (bottom left) and with the substrate (bottom right).

Fig. 2

Activities of single- and double-point phiKZ gp144 mutants compared to that of the wild-type enzyme taken as 100%.

Fig. 3

A – The root-mean-square deviation of the protein backbone of transglycosylases frozen at 77 K. The deviation was calculated only for the area in contact with the substrate. B – Changes in spacing between the Cα atoms of the amino acids 126 and 229 during freezing up to 77K.

Fig. 4

Surface modeling of the phiKZ gp144 mutants compared to the native form: A – E115A, B – E115A/ E178A, C – E115A/Y197F, and D – E178A/Y197F. The translucent surface belongs to the structure of the wild-type enzyme. The domain not involved in binding to the substrate is deleted in the images. The side view of the groove is provided.

Fig. 5

Charge distribution on the surface of the mutant phiKZ gp144 isoforms. The substrate-binding site is shown with a circle. A) native structure, B) E115A, C) E178A/ Y197F, D) E115A/E178A, and E) E115A Y197F.

Fig. 6

The possible directions of substrate orientation in the groove of phiKZ gp144. The substrate (N-acetylmuramyl-N-acetylglucosamine) is shown above the arrow.