Purification, characterization, and primary structure of a novel cell wall hydrolytic amidase, CwhA, from Achromobacter lyticus.
A novel bacteriolytic enzyme CwhA (cell wall hydrolytic amidase) was purified by ion exchange and gel-filtration chromatographies from a commercial bacteriolytic preparation from Achromobacter lyticus. CwhA exhibited optimal pH at 8.5 and lysed CHCl(3)-treated Escherichia coli more efficiently than Micrococcus luteus, Staphylococcus aureus, Enterococcus faecalis, and Pediococcus acidilactici. The enzyme was inhibited by 1,10-phenanthroline strongly and by EDTA to a lesser extent, suggesting that it is probably a metalloenzyme. Amino acid composition and mass spectrometric analyses for the CwhA-derived M. luteus muropeptides revealed that CwhA is N-acetylmuramoyl-L-alanine amidase [EC 3.5.1. 28]. The complete amino acid sequence of CwhA was established by a combination of Edman degradation and mass spectrometry for peptides obtained by Achromobacter protease I (API) digestion and cyanogen bromide (CNBr) cleavage. The enzyme consists of a single polypeptide chain of 177 amino acid residues with one disulfide bond, Cys114-Cys121. CwhA was found to be homologous to N-acetylmuramoyl-L-alanine amidase from bacteriophage T7 (BPT7). Its sequence identity with BPT7 is 35%, but the amino acid residues functioning as zinc ligands in BPT7 are absent in CwhA. These results suggest that CwhA is a new type of N-acetylmuramoyl-L-alanine amidase.
Li
S
S
Division of Protein Chemistry, Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan. lishao@protein.osaka-u.ac
Norioka
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S
Sakiyama
F
F
D000421:Alcaligenes / Q000201:enzymology*; D000595:Amino Acid Sequence; D001433:Bacteriolysis; D017123:Bacteriophage T7 / Q000201:enzymology; D002473:Cell Wall / Q000201:enzymology; D002614:Chelating Agents / Q000494:pharmacology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000737:chemistry; D006094:Gram-Positive Bacteria / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry*; D010457:Peptidoglycan / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry*
D002614:Chelating Agents; D010457:Peptidoglycan; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D009238:N-Acetylmuramoyl-L-alanine Amidase