Molecular properties of the putative autolysin Atl(WM) encoded by Staphylococcus warneri M: mutational and biochemical analyses of the amidase and glucosaminidase domains.
The putative autolysin Atl(WM) of Staphylococcus warneri M is a modular protein exhibiting two enzyme activities, an N-terminal side amidase (ami(atlwm)-R1-R2) and a C-terminal side glucosaminidase (R3-glu(atlwm)). 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(atlwm) (or glu(atlwm)) had the pH and temperature optima of about 7.0 (5.5) and 41 (50) degrees C, respectively. ami(atlwm) was inactivated by EDTA, and was stimulated by such salts as CoCl(2), MnCl(2), CaCl(2), or ZnCl(2). Six mutations within ami(atlwm), (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(atlwm) 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(WM) might be primarily processed at two specific sites (one between pro and ami(atlwm), and the other between R2 and R3) to yield the mature amidase and glucosaminidase.
Yokoi
Ken-Ji
KJ
Molecular Biology Group, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan.
Sugahara
Kazuki
K
Iguchi
Akinori
A
Nishitani
Go
G
Ikeda
Masahide
M
Shimada
Takako
T
Inagaki
Nobuya
N
Yamakawa
Ayanori
A
Taketo
Akira
A
Kodaira
Ken-Ichi
K
D000581:Amidohydrolases / Q000032:analysis* / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics; D006094:Gram-Positive Bacteria / Q000187:drug effects; D006596:Hexosaminidases / Q000032:analysis* / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000032:analysis* / Q000235:genetics*; D013210:Staphylococcus / Q000235:genetics*; D013696:Temperature; D014162:Transfection
D001426:Bacterial Proteins; D006596:Hexosaminidases; D000581:Amidohydrolases; D009238:N-Acetylmuramoyl-L-alanine Amidase; C019665:amidase