Substrate Specificity of Staphylococcus hyicus Lipase and Staphylococcus aureus Lipase As Studied by in Vivo Chimeragenesis

• Published in: Biochemistry (Easton) 1998-03, Vol.37 (10), p.3459-3466
• Main Authors: van Kampen, Muriel D, Dekker, Niek, Egmond, Maarten R, Verheij, Hubertus M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Binding Sites; DNA Restriction Enzymes; Escherichia coli - genetics; Kinetics; Lipase - chemistry; Lipase - genetics; Lipase - metabolism; Molecular Sequence Data; Oligodeoxyribonucleotides - genetics; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Sequence Homology, Amino Acid; Species Specificity; Staphylococcus - enzymology; Staphylococcus - genetics; Staphylococcus aureus - enzymology; Staphylococcus aureus - genetics; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9725430

Staphylococcus hyicus lipase (SHL) and Staphylococcus aureus lipase (SAL) are highly homologous enzymes, yet they show remarkable differences in their biochemical characteristics. SHL displays a high phospholipase activity, hydrolyses neutral lipids, and has no chain length preference, whereas SAL only degrades short-chain fatty acid esters. To identify the regions in the primary sequence of SHL responsible for phospholipase activity and chain length selectivity, a set of histidine-tagged SAL/SHL chimeras was generated by in vivo recombination in Escherichia coli. Several classes of chimeric enzymes were identified on the basis of restriction site analysis. All chimeras were well-expressed as active enzymes. They were characterized for their specific activities on both phospholipids and p-nitrophenyl esters of various chain lengths. Phospholipase activity appeared to be determined by three regions, all located in the C-terminal domain of SHL. Testing of the enzymatic activity of the chimeras toward p-nitrophenyl esters showed that chain length selectivity is defined by elements within the region of residues 180−253. Moreover, also residues along the stretch 275−358 contribute to the binding of acyl chains. Interestingly, several chimeras were even more active than the parent enzymes on long-chain p-nitrophenyl esters.