Construction of a multifunctional pneumococcal murein hydrolase by module assembly.
A chimeric trifunctional pneumococcal peptidoglycan hydrolase (CHL) has been constructed by fusing a choline-binding domain with two catalytic modules that provide lysozyme and amidase activity. The chimeric enzymes behaves as a choline-dependent enzyme and its activity is comparable to that of the parent enzymes. Site-directed mutagenesis of CHL produced a mutated enzyme [D9A,36A]CHL) that only exhibited an amidase activity. Comparative biochemical analyses of CHL and [D9A, E36A]CHL strongly suggest that the lysozyme catalytic module confers 88% of the total activity of CHL, whereas 12% of the activity can be ascribed to the amidase module. Both enzymatic activities are affected by the process of activation or conversion induced by choline suggesting that the conversion process is produced by a conformational change in the choline-binding domain. Our findings demonstrate that the three modules can acquire the proper folding conformation in the-three domain chimeric CHL enzyme. This experimental evidence supports the modular theory of protein evolution, and demonstrates that modular assembly of functional domains can be a rational approach to construct fully active chimeric enzymes with novel biological or biotechnological properties.
Sanz
J M
JM
Department of Molecular Microbiology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
García
P
P
García
J L
JL
D000581:Amidohydrolases / Q000378:metabolism; D005822:Genetic Vectors; D009113:Muramidase / Q000037:antagonists & inhibitors / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000201:enzymology / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D009113:Muramidase; D000581:Amidohydrolases; D009238:N-Acetylmuramoyl-L-alanine Amidase; C019665:amidase