Abstract：
:The lipase from Staphylococcus hyicus (SHL) displays a high phospholipase activity whereas the homologous S. aureus lipase (SAL) is not active or hardly active on phospholipid substrates. Previously, it has been shown that elements within the region comprising residues 254-358 are essential for the recognition of phospholipids by SHL. To specifically identify the important residues, nine small clusters of SHL were individually replaced by the corresponding SAL sequence within region 254-358. For cloning convenience, a synthetic gene fragment of SHL was assembled, thereby introducing restriction sites into the SHL gene and optimizing the codon usage. All nine chimeras were well-expressed as active enzymes. Eight chimeras showed lipase and phospholipase activities within a factor of 2 comparable to WT-SHL in standard activity assays. Exchange of the polar SHL region 293-300 by the more hydrophobic SAL region resulted in a 32-fold increased k(cat)/K(m) value for lipase activity and a concomitant 68-fold decrease in k(cat)/K(m) for phospholipase activity. Both changes are due to effects on catalytic turnover as well as on substrate affinity. Subsequently, six point mutants were generated; G293N, E295F, T297P, K298F, I299V, and L300I. Residue E295 appeared to play a minor role whereas K298 was the major determinant for phospholipase activity. The mutation K298F caused a 60-fold decrease in k(cat)/K(m) on the phospholipid substrate due to changes in both k(cat) and K(m). Substitution of F298 by a lysine in SAL resulted in a 4-fold increase in phospholipase activity. Two additional hydrophobic to polar substitutions further increased the phospholipase activity 23-fold compared to WT-SAL.
journal_name
Biochemistryjournal_title
Biochemistryauthors
van Kampen MD,Verheij HM,Egmond MRdoi
10.1021/bi990096dsubject
Has Abstractpub_date
1999-07-20 00:00:00pages
9524-32issue
29eissn
0006-2960issn
1520-4995pii
bi990096djournal_volume
38pub_type
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