COMPARATIVE BIOCHEMICAL AND MOLECULAR ANALYSIS OF THE STAPHYLOCOCCUS-HYICUS, STAPHYLOCOCCUS-AUREUS AND A HYBRID LIPASE - INDICATION FOR A C-TERMINAL PHOSPHOLIPASE DOMAIN

The lipase gene, geh, from Staphylococcus aureus NCTC8530 was cloned i n Staphylococcus carnosus. DNA sequencing revealed an open reading fra me (ORF) of 2046 nucleotides encoding a 682-amino-acid protein with a molecular mass of 76900 Da. Determination of the transcriptional start site revealed a 203-nucleotide mRNA leader. Expression of geh in the protease-negative S. carnosus (pT181copSA22) resulted in overexpressio n of a 83-kDa lipase found in the culture supernatant. N-terminal prot ein sequencing and sequence comparison with three other staphylococcal lipases suggest that this lipase is organised as a pre-pro-enzyme. Th e substrate specificity of this lipase is different from the Staphyloc occus hyicus Lipase. The S. hyicus lipase expressed both a high Ca2+-d ependent phospholipase and lipase activity while the S. aureus lipase lacked this phospholipase activity and its activity with tributyrylgly cerol or p-nitrophenyl octanoate is hardly stimulated by Ca2+ ions. A hybrid protein was constructed in which the C-terminal 146 residues of the S. hyicus lipase were substituted by 145 residues of the C-termin al of the S. aureus lipase, which contains the proposed active-site am ino acids Asp602 and His641. The hybrid enzyme was still active and re vealed an intermediary enzymic activity. The most striking effect was that it had lost the S. hyicus-specific phospholipase activity and tha t, in contrast to the two parental enzymes, its activity with p-nitrop henyl octanoate became highly sensitive to the presence of Ca2+. These observations suggest that the C-terminal domain of the S. hycius lipa se strongly contributes to the binding pocket of the polar headgroup o f phospholipids. The Ca2+-binding site seems to be located in the N-te rminal fragment of the S. hycius lipase. The fact that two closely rel ated enzymes differ in the need for Ca2+ underscores the notion that i t plays a structural rather than a catalytic role.