title: Mutational and biochemical analyses of the endolysin Lys(gaY) encoded by the Lactobacillus gasseri JCM 1131T phage phi gaY.
abstract: The Lys(gaY) of Lactobacillus gasseri JCM 1131(T) phage phigaY endolysin was purified to homogeneity using the Escherichia coli/His.Tag system. Zymographic and spectrophotometric assays showed that Lys(gaY) lysed over 20 heated Gram-positive bacterial species as the substrates, including lactobacilli, lactococci, enterococci, micrococci, and staphylococci. The enzymatic activity had the pH and temperature optima of about 6.5 and 37 degrees C, respectively. Amino-acid substitution analysis revealed that 13 residues of Lys(gaY) were involved in cell-lytic activity: in the beta/alpha(gaY) domain, G10, D12, E33, D36, H60, Y61, D96, E98, V124, L132, and D198; in the SH3b(gaY) domain, Y272 and W284. In addition, deletion analysis demonstrated that the beta/alpha(gaY) domain of N-terminal 216 residues is the core enzyme portion, although the cell-lytic ability is lower than that of Lys(gaY). These mutational experiments suggested that beta/alpha(gaY) (in which two acidic residues of D12 and E98 likely act as catalytic residues) is responsible for cell-lytic activity, and SH3b(gaY) promotes beta/alpha(gaY) possibly through cell-wall binding function. The purified His-tagged SH3b(gaY) domain containing 94 residues from S217 to K310 (i) bound to Gram-positive bacteria susceptible to Lys(gaY), (ii) induced aggregation of exponentially growing cells of L. gasseri JCM 1131(T), L. casei IAM 1045, Lactococcus lactis C2, L. lactis MG 1363, and Enterococcus hirae IAM 1262 by forming thread-like chained cells, (iii) inhibited lytic activity of Lys(gaY), and (iv) impeded autolysis of L. gasseri JCM 1131(T) in buffer systems. A truncated protein HDeltaSH3b(gaY) lacking in N-terminal 21 residues (from S217 to E237) of SH3b(gaY) and an amino-acid substituted protein HSH3b(gaY)G (W284G) lost the activities of HSH3b(gaY), showing that the N-terminal region and W284 probably play important roles in the SH3b(gaY) function(s).
authors.affiliation: Molecular Biology Group, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Sugahara
authors.forename: Ken-ji
authors.initials: KJ
authors.lastname: Yokoi
authors.forename: Yuuko
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Taito
authors.initials: T
authors.lastname: Nishino
authors.forename: Ayanori
authors.initials: A
authors.lastname: Yamakawa
authors.forename: Akira
authors.initials: A
authors.lastname: Taketo
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Kodaira
mesh_terms: D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000201:enzymology* / Q000235:genetics; D004252:DNA Mutational Analysis; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007778:Lactobacillus / Q000821:virology*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary
chemical_list: D010450:Endopeptidases; C001143:endolysin