Mutational analysis of peptidoglycan amidase MepA.
Murein endopeptidase A (MepA) from Escherichia coli is a periplasmic peptidoglycan amidase that cleaves d,d amide bonds between d-alanine and meso-2,6-diaminopimelic acid in E. coli peptidoglycan. MepA and its homologues in other proteobacteria share overall structural similarity with d-Ala-d-Ala metallopeptidases and local similarity around the active site with lysostaphin-type enzymes, which has prompted the classification of these enzymes as LAS enzymes. LAS enzymes contain a single divalent cation in the active site, which is tetracoordinated in the crystal structures. Three of the metal ligands are identical in all structures, but the identity of the fourth ligand varies. Two residues in proximity to the metal might act as a general acid/base, but their role is not clear. Here, we report a new MepA expression system, which allows the separation of MepA variants from the endogenous wild-type enzyme, and an HPLC assay with a defined peptidoglycan fragment, which allows assessment of MepA activity without a refolding step. We find that the conserved metal ligands are required for folding (D120) or catalysis (H113, H211). Separate mutations of the candidate catalytic residues H206 or H209 and of the "fourth" metal ligand H110 are tolerated for folding but drastically reduce activity. Mutation of residue W203 to aspartate impairs substrate binding.
Firczuk
Małgorzata
M
International Institute of Molecular and Cell Biology, Ks Trojdena 4, 02-109 Warsaw, Poland.
Bochtler
Matthias
M
D001665:Binding Sites; D002384:Catalysis; D002473:Cell Wall / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019281:Dimerization; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D009154:Mutation; D010457:Peptidoglycan / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism
D029968:Escherichia coli Proteins; D008024:Ligands; D010457:Peptidoglycan; D010450:Endopeptidases; C021813:mepA protein, E coli; D015032:Zinc