Comparative biochemical and molecular analysis of the Staphylococcus hyicus, Staphylococcus aureus and a hybrid lipase. Indication for a C-terminal phospholipase domain

Eur J Biochem. 1995 Mar 15;228(3):732-8.

Abstract

The lipase gene, geh, from Staphylococcus aureus NCTC8530 was cloned in Staphylococcus carnosus. DNA sequencing revealed an open reading frame (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 overexpression of a 83-kDa lipase found in the culture supernatant. N-terminal protein sequencing and sequence comparison with three other staphylococcal lipases suggest that this lipase is organised as a pre-pro-enzyme. The substrate specificity of this lipase is different from the Staphylococcus hyicus lipase. The S. hyicus lipase expressed both a high Ca(2+)-dependent phospholipase and lipase activity while the S. aureus lipase lacked this phospholipase activity and its activity with tributyrylglycerol 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-terminal of the S. aureus lipase, which contains the proposed active-site amino acids Asp602 and His641. The hybrid enzyme was still active and revealed an intermediary enzymic activity. The most striking effect was that it had lost the S. hyicus-specific phospholipase activity and that, in contrast to the two parental enzymes, its activity with p-nitrophenyl octanoate became highly sensitive to the presence of Ca2+. These observations suggest that the C-terminal domain of the S. hyicus lipase strongly contributes to the binding pocket of the polar headgroup of phospholipids. The Ca(2+)-binding site seems to be located in the N-terminal fragment of the S. hyicus lipase. The fact that two closely related enzymes differ in the need for Ca2+ underscores the notion that it plays a structural rather than a catalytic role.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • DNA, Bacterial
  • Molecular Sequence Data
  • Monoacylglycerol Lipases / genetics*
  • Monoacylglycerol Lipases / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Staphylococcus / enzymology
  • Staphylococcus / genetics
  • Staphylococcus / metabolism*
  • Staphylococcus aureus / enzymology
  • Staphylococcus aureus / genetics
  • Staphylococcus aureus / metabolism*
  • Substrate Specificity

Substances

  • DNA, Bacterial
  • Recombinant Fusion Proteins
  • Monoacylglycerol Lipases

Associated data

  • GENBANK/M90693