THE PHOSPHOLIPASE-ACTIVITY OF STAPHYLOCOCCUS-HYICUS LIPASE STRONGLY DEPENDS ON A SINGLE SER TO VAL MUTATION

Site-directed mutagenesis and domain exchange were used to investigate the role of the C-terminal domains of Staphylococcus hyicus lipase (S HL) and S. aureus lipase (SAL) in substrate selectivity. The introduct ion of a single point mutation coding for the substitution of Val for Ser356 in SHL yields an enzyme which has retained full lipase activity , but with more than 12-fold lower phospholipase activity. Starting wi th this S356V variant of SHL the C-terminal 40 amino acids were replac ed by the corresponding SAL sequence. Although 23 amino acid changes w ere introduced simultaneously the impact on the phospholipase/lipase a ctivity ratio was only 4-fold. We therefore conclude that in the C-ter minal domain it is Ser356 which mainly determines phospholipase activi ty. The introduction of a Val357 to Ser substitution in SAL did not tu rn SAL into a phospholipase, showing that residues from other domains contribute to this activity as well. The results are discussed in view of the sequence homology of lipases and (lyso)phospholipases. (C) 199 8 Elsevier Science Ireland Ltd. All rights reserved.