title: Chimeric pneumococcal cell wall lytic enzymes reveal important physiological and evolutionary traits.
abstract: Two novel chimeric pneumococcal cell wall lytic enzymes, named LC7 and CL7, have been constructed by in vitro recombination of the lytA gene encoding the major autolysin (LYTA amidase) of Streptococcus pneumoniae, a choline-dependent enzyme, and the cpl7 gene encoding the CPL7 lysozyme of phage Cp-7, a choline-independent enzyme. In remarkable contrast with previous chimeric constructions, we fused here two genes that lack nucleotide homology. The CL7 enzyme, which contains the N-terminal domain of CPL7 and C-terminal domain of LYTA, exhibited a choline-dependent lysozyme activity. This experimental rearrangement of domains might mimic the process that have generated the choline-dependent CPL1 lysozyme of phage Cp-1 during evolution, providing additional support to the modular theory of protein evolution. The LC7 enzyme, built up by fusion of the N-terminal domain of LYTA and the C-terminal domain of CPL7, exhibited an amidase activity capable of degrading ethanolamine-containing cell walls. The chimeric amidase behaved as an autolytic enzyme when it was cloned and expressed in S. pneumoniae. The chimeric enzymes provided new insights on the mechanisms involved in regulation of the host pneumococcal autolysins and on the participation of these enzymes in the process of cell separation. Furthermore, our experimental approach confirmed the basic role of the C-terminal domains in substrate recognition and revealed the influence of these domains on the optimal pH for catalytic activity.
authors.affiliation: Unidad de Genética Bacteriana, Consejo Superior de Investigaciones Cientificas, Madrid, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Diaz
authors.forename: R
authors.initials: R
authors.lastname: López
authors.forename: J L
authors.initials: JL
authors.lastname: Garcia
mesh_terms: D001483:Base Sequence; D005075:Biological Evolution; D015153:Blotting, Western; D002678:Chimera; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics* / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics* / Q000502:physiology; D010957:Plasmids; D011995:Recombination, Genetic; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000254:growth & development
chemical_list: C067928:CPL7 lysozyme; D009113:Muramidase; D009238:N-Acetylmuramoyl-L-alanine Amidase