title: Substrate specificity of Staphylococcus hyicus lipase and Staphylococcus aureus lipase as studied by in vivo chimeragenesis.
abstract: 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.
authors.affiliation: Department of Enzymology and Protein Engineering, Utrecht University, TB Utrecht, The Netherlands.
authors.forename: M D
authors.initials: MD
authors.lastname: van Kampen
authors.forename: N
authors.initials: N
authors.lastname: Dekker
authors.forename: M R
authors.initials: MR
authors.lastname: Egmond
authors.forename: H M
authors.initials: HM
authors.lastname: Verheij
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013210:Staphylococcus / Q000201:enzymology* / Q000235:genetics; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D008049:Lipase; D004262:DNA Restriction Enzymes