PDBsum entry 1qt3

Hydrolase

PDB id: 1qt3

Contents

Protein chain

164 a.a. *

Ligands

HED

Metals

_CL ×2

Waters ×159

* Residue conservation analysis

PDB id:

1qt3

Name:

Hydrolase

Title:

T26d mutant of t4 lysozyme

Structure:

Protein (t4 lysozyme). Chain: a. Engineered: yes. Mutation: yes

Source:

Enterobacteria phage t4. Organism_taxid: 10665. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Monomer (from PDB file)

Resolution:

1.85Å R-factor: 0.155

Authors:

R.Kuroki,L.H.Weaver,B.W.Matthews

Key ref:

R.Kuroki et al. (1999). Structural basis of the conversion of T4 lysozyme into a transglycosidase by reengineering the active site. Proc Natl Acad Sci U S A, 96, 8949-8954. PubMed id: 10430876 DOI: 10.1073/pnas.96.16.8949

Date:

30-Jun-99 Release date: 08-Jul-99

Protein chain

P00720  (ENLYS_BPT4) -  Endolysin from Enterobacteria phage T4

Seq: 164 a.a.

Struc: 164 a.a.*

* PDB and UniProt seqs differ at 5 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.2.1.17 - lysozyme.
• Reaction: Hydrolysis of the 1,4-beta-linkages between N-acetyl-D-glucosamine and N-acetylmuramic acid in peptidoglycan heteropolymers of the prokaryotes cell walls.

DOI no: 10.1073/pnas.96.16.8949

Proc Natl Acad Sci U S A 96:8949-8954 (1999)

PubMed id: 10430876

Structural basis of the conversion of T4 lysozyme into a transglycosidase by reengineering the active site.

R.Kuroki, L.H.Weaver, B.W.Matthews.

ABSTRACT

In contrast to hen egg-white lysozyme, which retains the beta-configuration of the substrate in the product, T4 lysozyme (T4L) is an inverting glycosidase. The substitution Thr-26 --> His, however, converts T4L from an inverting to a retaining enzyme. It is shown here that the Thr-26 --> His mutant is also a transglycosidase. Indeed, the transglycosylation reaction can be more effective than hydrolysis. In contrast, wild-type T4L has no detectable transglycosidase activity. The results support the prior hypothesis that catalysis by the Thr-26 --> His mutant proceeds via a covalent intermediate. Further mutations (Glu-11 --> His, Asp-20 --> Cys) of the T26H mutant lysozyme indicate that the catalytic mechanism of this mutant requires Glu-11 as a general acid but Asp-20 is not essential. The results help provide an overall rationalization for the activity of glycosidases, in which a highly conserved acid group (Glu-11 in T4L, Glu-35 in hen egg-white lysozyme) on the beta-side of the substrate acts as a proton donor, whereas alterations in the placement and chemical identity of residues on the alpha-side of the substrate can lead to catalysis with or without retention of the configuration, to transglycosidase activity, or to the formation of a stable enzyme-substrate adduct.

Selected figure(s)

Figure 2.
Fig. 2. Comparison of the products formed by the digestion of the substrate shown in Fig. 1 with WT lysozyme and mutant T26H. (a) Substrate alone. (b) Digestion of 2 × 10^ 6 M substrate by 8 × 10^ 8 M WT* lysozyme for 5 min at 25°C (14). (c) Digestion of 2 × 10^ 6 M substrate by 5 × 10^ 7 M T26H for 5 min at 25°C (14). (d) Digestion of 8 × 10^ 5 M substrate by 5 × 10^ 7 M T26H for 60 min at 0°C. See text for additional details and explanation of peaks.

Figure 3.
Fig. 3. Superposition of 12 structures of mutant lysozymes (see text). The figure includes WT*, E11H, E11N, D20A, D20S, D20N, D20C, D20E, T26D, T26H, T26Q, and T26E with the covalent adduct formed by this mutant (13). Water molecules clustered together at the two sites discussed in the text are shown as red spheres. The water molecule present in the covalent adduct also is shown as a red sphere. Other solvent molecules in the vicinity of the active site are shown as red crosses.

Figures were selected by an automated process.