N-acetylmuramoyl-L-alanine amidase

 

T7 lysosome is a bifunctional protein found only in T7 phage. It has amidase activity in cleaving the amide bond between N-acetyl-muramic acid and L-alanine in the bacterial cell wall. It also acts as an inhibitor of T7 RNA polymerase, which provides a feedback mechanism that shuts off late transcription during infection and stimulates DNA replication. T7 lysosyme differs from the previously well-studied egg-white and phage T4 lysosymes not only in having an interaction with T7 RNA polymerase but also in the chemistry of lysis: it cuts the amide bond between N-acetylmuramic acid and L-alanine rather than the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan layer of bacterial cell walls.

 

Reference Protein and Structure

Sequence
P00806 UniProt (3.5.1.28) IPR034689 (Sequence Homologues) (PDB Homologues)
Biological species
Enterobacteria phage T7 (Virus) Uniprot
PDB
1lba - THE STRUCTURE OF BACTERIOPHAGE T7 LYSOZYME, A ZINC AMIDASE AND AN INHIBITOR OF T7 RNA POLYMERASE (2.2 Å) PDBe PDBsum 1lba
Catalytic CATH Domains
3.40.80.10 CATHdb (see all for 1lba)
Cofactors
Zinc(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:3.5.1.28)

water
CHEBI:15377ChEBI
+
N-acetyl-alpha-D-muramoyl-L-alanine
CHEBI:28920ChEBI
→
2-acetamido-3-O-[(1R)-1-carboxylatoethyl]-2-deoxy-D-glucopyranose
CHEBI:28881ChEBI
+
hydron
CHEBI:15378ChEBI
+
L-alanine zwitterion
CHEBI:57972ChEBI
Alternative enzyme names: N-acetylmuramic acid L-alanine amidase, N-acetylmuramoyl-L-alanine amidase type I, N-acetylmuramoyl-L-alanine amidase type II, N-acetylmuramyl-L-alanine amidase, N-acetylmuramylalanine amidase, N-acylmuramyl-L-alanine amidase, Acetylmuramoyl-alanine amidase, Acetylmuramyl-L-alanine amidase, Acetylmuramyl-alanine amidase, Murein hydrolase,

Enzyme Mechanism

Introduction

A tentative mechanism has been put forward by analogy with the other zinc proteases, specifically caboxypeptidase A. A positively charged lysine forms a bond to the carbonyl group of the amide link polarising it and allowing nucleophilic attack by a zinc bound hydroxide activated by a negatively charged tyrosine. This is followed by bond cleavage and donation of a proton to the leaving amino group.

Catalytic Residues Roles

UniProt PDB* (1lba)
Tyr47 Tyr46(42)A Negatively charged tyrosine abstracts a proton from a zinc-bound water moelcue to form a nucleophile for attack on the amide bond. proton shuttle (general acid/base)
His18, Cys131, His123 His17(13)A, Cys130(126)A, His122(118)A Forms the zinc binding site
Lys129 Lys128(124)A Positively charged lysine polarises carbonyl group of the amide bond, to activate it for nucleophilic attack via hydrogen bonding. Stabilises the transition state. electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

References

  1. Cheng X et al. (1994), Proc Natl Acad Sci U S A, 91, 4034-4038. The structure of bacteriophage T7 lysozyme, a zinc amidase and an inhibitor of T7 RNA polymerase. DOI:10.2210/pdb1lba/pdb. PMID:8171031.

Step 1.

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Catalytic Residues Roles

Residue Roles
Lys128(124)A electrostatic stabiliser
Tyr46(42)A proton shuttle (general acid/base)

Chemical Components

Step 1.

Contributors

Anna Waters, Craig Porter, Gemma L. Holliday, Charity Hornby