INTERCHANGE OF FUNCTIONAL DOMAINS SWITCHES ENZYME SPECIFICITY - CONSTRUCTION OF A CHIMERIC PNEUMOCOCCAL-CLOSTRIDIAL CELL-WALL LYTIC ENZYME

Bacterial autolysins are endogenous enzymes that specifically cleave c ovalent bonds in the cell wall. These enzymes show both substrate and bond specificities. The former is related to their interaction with th e insoluble substrate whereas the latter determine their site of actio n. The bond specificity allows their classification as muramidases (ly sozymes), glucosaminidases, amidases, and endopeptidases. To demonstra te that the autolysin (LYC muramidase) of Clostridium acetobutylicum A TCC824 presents a domainal organization, a chimeric gene (clc) contain ing the regions coding for the catalytic domain of the LYC muramidase and the choline-binding domain of the pneumococcal phage CPL1 muramida se has been constructed by in vitro recombination of the corresponding gene fragments. This chimeric construction codes for a choline-bindin g protein (CLC) that has been purified using affinity chromatography o n DEAE-cellulose. Several biochemical tests demonstrate that this rear rangement of domains has generated an enzyme with a choline-dependent muramidase activity on pneumococcal cell walls. Since the parental LYC muramidase was choline-independent and unable to degrade pneumococcal cell walls, the formation of this active chimeric enzyme by exchangin g protein domains between two enzymes that specifically hydrolyse cell walls of bacteria belonging to different genera shows that a switch o n substrate specificity has been achieved. The chimeric CLC muramidase behaved as an autolytic enzyme when it was adsorbed onto a live autol ysin-defective mutant of Streptococcus pneumoniae. The construction de scribed here provides experimental support for the theory of modular e volution which assumes that novel proteins have evolved by the assembl y of preexisting polypeptide units.