Studies on the structure and function of the N-terminal domain of the pneumococcal murein hydrolases.

The structures of the choline-dependent pneumococcal murein hydrolases, LYTA amidase and CPL1 lysozyme, and the choline-independent CPL7 lysozyme were analysed by controlled proteolytic digestions. The trypsin cleavage of the CPL1 and CPL7 lysozymes produced two resistant polypeptides, F1 and F7 respectively, corresponding to the N-terminal domain of the enzymes, whereas the amidase LYTA was completely hydrolysed by the protease. Interestingly, the F1 and F7 fragments showed a low, but significant, choline-independent lysozyme activity. Choline reduced the rate of proteolytic hydrolysis of choline-dependent enzymes, suggesting that the C-terminal choline-binding domain adopts a more resistant conformation in the presence of the ligand. On the other hand, the regions encoding the N-terminal domains of the three enzymes have been cloned and expressed in Escherichia coli, showing that these domains adopt an active conformation even in the absence of their C-terminal domains. The lower activity shown by the catalytic domains when compared with that of the complete enzymes suggests that the acquisition of a substrate-binding domain represents a noticeable evolutionary advantage for enzymes that interact with polymeric substrates, allowing them to achieve a higher catalytic efficiency. These results strongly reinforce the hypothesis that the pneumococcal murein hydrolases have been originated by fusion of two structural and functional independent domains, and provide new experimental support to the theory of modular evolution of proteins.