Substrate specificity of Staphylococcus hyicus lipase and Staphylococcus aureus lipase as studied by in vivo chimeragenesis.
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.
van Kampen
M D
MD
Department of Enzymology and Protein Engineering, Utrecht University, TB Utrecht, The Netherlands.
Dekker
N
N
Egmond
M R
MR
Verheij
H M
HM
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
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D008049:Lipase; D004262:DNA Restriction Enzymes