Comparative Biochemical and Molecular Analysis of the Staphylococcus hyicus, Staphylococcus aureus and a Hybrid Lipase. Indication for a C-Terminal Phospholipase Domain

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.

Heterologous expression and N-terminal His-tagging processes affect the catalytic properties of staphylococcal lipases: A monolayer study

“…It has emerged from several kinetic studies on lipases that the free N-terminal NH2 group may play either an important or an essential role in the expression of the maximum catalytic activity measured in vitro [50][51][52].…”

The N-terminal His-tag affects the enantioselectivity of staphylococcal lipases: A monolayer study

“…The 40-46 kDa mature parts of these lipases are closely related suggesting a comparable tertiary structure. All these enzymes are able to hydrolyse triacylglycerols but only the enzyme of S. Hyicus has a high phospholipase activity as well, distinguishing the enzyme from other bacterial lipases [14,[18][19][20]. Due to their high degree of similarity, the staphylococcal lipases provide an interesting system to study the significance of particular regions and positions for their substrate selectivity.…”

Secretion of human growth hormone by the food-grade bacterium Staphylococcus carnosus requires a propeptide irrespective of the signal peptide used

“…The unusually slow electrophoretic migration of hGH fused to the Lip, Lss, or Geh propeptides is reminescent of the native Lip, Lss, and Geh proteins (Demleitner and Götz 1994;Nikoleit et al 1995;Thumm and Götz 1997). It is likely to be caused by an unusual behavior of the propeptides in SDS-PAGE gels.…”

“…Secretion of human growth hormone by the food-grade bacterium Staphylococcus carnosus requires a propeptide irrespective of the signal peptide used S. carnosus, such as bacterial enzymes (Liebl and Götz 1986;Nikoleit et al 1995), antigens from various pathogens (Samuelson et al 1995(Samuelson et al , 1999, and antibody fragments (Pschorr et al 1994;Schnappinger et al 1995). S. carnosus cells have proven to be particularly useful for cell-surface anchoring of secretory proteins (Robert et al 1996;Strauss and Götz 1996) and as live vaccines (Cano et al 2000).…”

“…The propeptides of the lipase (Lip) of Staphylococcus hyicus, the staphylolytic endopeptidase lysostaphin (Lss) of Staphylococcus simulans, and the glycerol ester hydrolase (Geh) of S. aureus are essential for efficient secretion of the proteins. They have been proposed to constitute intramolecular chaperones that maintain a secretion-competent conformation of the mature proteins (Demleitner and Götz 1994;Nikoleit et al 1995;Thumm and Götz 1997). The signal peptides of staphylococcal proteins without a propeptide are usually shorter (25-30 amino acid residues compared to 35-40 amino acid residues in propeptide-bearing proteins) and they contain fewer positive charges at the N-terminus (Fig.…”