Molecular properties of the putative autolysin Atl sub(W) sub(M) encoded by Staphylococcus warneri M: Mutational and biochemical analyses of the amidase and glucosaminidase domains

• Published in: Gene Vol. 416; no. 1-2; pp. 66 - 76
• Main Authors: Yokoi, Kj, Sugahara, K, Iguchi, A, Nishitani, G, Ikeda, M, Shimada, T, Inagaki, N, Yamakawa, A, Taketo, A, Kodaira, KI
• Format: Journal Article
• Language: English
• Published: 15-06-2008
• Subjects: Escherichia coli; Lactobacillus; Staphylococcus warneri
• ISSN: 0378-1119
• DOI: 10.1016/j.gene.2008.03.004

The putative autolysin Atl sub(W) sub(M) of Staphylococcus warneri M is a modular protein exhibiting two enzyme activities, an N-terminal side amidase (ami sub(a) sub(t) sub(l) sub(w) sub(m)-R1-R2) and a C-terminal side glucosaminidase (R3-glu sub(a) sub(t) sub(l) sub(w) sub(m)). Zymographic analysis of the protein overproduced in Escherichia coli showed that both enzymes were active toward 17 Gram-positive bacteria, including staphylococci, lactobacilli, lactococci, enterococci, and micrococci. The purified enzyme core ami sub(a) sub(t) sub(l) sub(w) sub(m) (or glu sub(a) sub(t) sub(l) sub(w) sub(m)) had the pH and temperature optima of about 7.0 (5.5) and 41 (50) super(o)C, respectively. ami sub(a) sub(t) sub(l) sub(w) sub(m) was inactivated by EDTA, and was stimulated by such salts as CoCl sub(2), MnCl sub(2), CaCl sub(2), or ZnCl sub(2). Six mutations within ami sub(a) sub(t) sub(l) sub(w) sub(m), (H362A, E421A, H467A, H479, D481A, and Y491D) drastically reduced cell-lytic activity. Comparative analysis with other related amidases suggested that the three residues H362, H467, and D481 likely act as ligands (and/or active sites). The lytic activity of glu sub(a) sub(t) sub(l) sub(w) sub(m) markedly declined in four mutants (E1238A, E1238Q, T1239A, and Y1332A). For determination of the putative cell-recognition regions, four domains (R1-R2, R1, R2, and R3) were purified; all the proteins substantially bound to S. warneri M cells from exponential to stationary growth phases, and R1-R2 aggregated the cells. Protein sequencing and immunoblot analysis suggested that the extacellular Atl sub(W) sub(M) might be primarily processed at two specific sites (one between pro and ami sub(a) sub(t) sub(l) sub(w) sub(m), and the other between R2 and R3) to yield the mature amidase and glucosaminidase.