MUTATION IN ASPARTIC-ACID RESIDUES MODIFIES CATALYTIC AND HEMOLYTIC ACTIVITIES OF BACILLUS-CEREUS SPHINGOMYELINASE

Four aspartic acid residues (Asp(126), Asp(156), Asp(233) and Asp(295) ) of Bacillus cereus sphingomyelinase (SMase) in the conservative regi ons were changed to glycine by in vitro mutagenesis, and the mutant SM ases [D126G (Asp(126) --> Gly etc.), D156G, D233G and D295G] were prod uced in Bacillus brevis 47, a protein-producing strain. The sphingomye lin (SM)-hydrolysing activity of D295G was completely abolished and th ose of D126G and D156G were reduced by more than 80 %, whereas that of D233G was not so profoundly affected. Two mutant enzymes (D126G and D 156G) were purified and characterized further. The hydrolytic activiti es of D126G and D156G toward four phosphocholine-containing substrates with different hydrophobicities, SM, exadecanoylamino-4-nitrophenylph osphocholine(HNP), lysophosphatidylcholine (lysoPC) and p-nitrophenylp hosphocholine (p-NPPC), were compared with those of the wild-type. The activity of D126G toward water-soluble p-NPPC was comparable with tha t of the wild-type. On the other hand, D156G catalysed the hydrolysis of hydrophilic substrates such as HNP and p-NPPC more efficiently (> 4 -fold) than the wild-type. These results suggested that Asp(126) and A sp(156), located in the highly conserved region, may well be involved in a substrate recognition process rather than catalytic action. Haemo lytic activities of the mutant enzymes were found to be parallel with their SM-hydrolysing activities. Two regions, including the C-terminal region containing Asp(295), were found to show considerable sequence identity with the corresponding regions of bovine pancreatic DNase I. Structural predictions indicated structural similarity between SMase a nd DNase I. An evolutionary relationship based on the catalytic functi on was suggested between the structures of these two phosphodiesterase s.