Research article

Mutational analysis of the catalytic centre of the Citrobacter freundii AmpD N-acetylmuramyl-l-alanine amidase

Catherine GÉNÉREUX, Dominique DEHARENG, Bart DEVREESE, Jozef VAN BEEUMEN, Jean-Marie FRÈRE, Bernard JORIS

Abstract

Citrobacter freundii AmpD is an intracellular 1,6-anhydro-N-acetylmuramyl-l-alanine amidase involved in both peptidoglycan recycling and β-lactamase induction. AmpD exhibits a strict specificity for 1,6-anhydromuropeptides and requires zinc for enzymic activity. The AmpD three-dimensional structure exhibits a fold similar to that of another Zn2+ N-acetylmuramyl-l-alanine amidase, the T7 lysozyme, and these two enzymes define a new family of Zn-amidases which can be related to the eukaryotic PGRP (peptidoglycan-recognition protein) domains. In an attempt to assign the different zinc ligands and to probe the catalytic mechanism of AmpD amidase, molecular modelling based on the NMR structure and site-directed mutagenesis were performed. Mutation of the two residues presumed to act as zinc ligands into alanine (H34A and D164A) yielded inactive proteins which had also lost their ability to bind zinc. By contrast, the active H154N mutant retained the capacity to bind the metal ion. Three other residues which could be involved in the AmpD catalytic mechanism have been mutated (Y63F, E116A, K162H and K162Q). The E116A mutant was inactive, but on the basis of the molecular modelling this residue is not directly involved in the catalytic mechanism, but rather in the binding of the zinc by contributing to the correct orientation of His-34. The K162H and K162Q mutants retained very low activity (0.7 and 0.2% of the wild-type activity respectively), whereas the Y63F mutant showed 16% of the wild-type activity. These three latter mutants exhibited a good affinity for Zn ions and the substituted residues are probably involved in the binding of the substrate. We also describe a new method for generating the N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-tripeptide AmpD substrate from purified peptidoglycan by the combined action of two hydrolytic enzymes.

  • AmpD
  • β-lactamase induction
  • muropeptide
  • peptidoglycan recycling
  • site-directed mutagenesis
  • zinc amidase

Footnotes

  • Abbreviations used: MurNAc, N-acetylmuramic acid; GlcNAc, N-acetylglucosamine; MurNAc-l-Ala amidase, N-acetylmuramyl-l-alanine amidase; GlcNAc-anhMurNAc-tripeptide, N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-tripeptide; anhMurNAc-tripeptide, anhydro-N-acetylmuramyl-tripeptide; Tm, melting temperature; MM, molecular mechanics.