The phospholipase activity of Staphylococcus hyicus lipase strongly depends on a single Ser to Val mutation.
Site-directed mutagenesis and domain exchange were used to investigate the role of the C-terminal domains of Staphylococcus hyicus lipase (SHL) and S. aureus lipase (SAL) in substrate selectivity. The introduction of a single point mutation coding for the substitution of Val for Ser356 in SHL yields an enzyme which has retained full lipase activity, but with more than 12-fold lower phospholipase activity. Starting with this S356V variant of SHL the C-terminal 40 amino acids were replaced by the corresponding SAL sequence. Although 23 amino acid changes were introduced simultaneously the impact on the phospholipase/lipase activity ratio was only 4-fold. We therefore conclude that in the C-terminal domain it is Ser356 which mainly determines phospholipase activity. The introduction of a Val357 to Ser substitution in SAL did not turn SAL into a phospholipase, showing that residues from other domains contribute to this activity as well. The results are discussed in view of the sequence homology of lipases and (lyso)phospholipases.
van Kampen
M D
MD
Department of Enzymology and Protein Engineering, Utrecht University, The Netherlands.
Simons
J W
JW
Dekker
N
N
Egmond
M R
MR
Verheij
H M
HM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D008049:Lipase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D010740:Phospholipases / Q000235:genetics* / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D017354:Point Mutation; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D013210:Staphylococcus / Q000201:enzymology*; D013379:Substrate Specificity; D014633:Valine / Q000378:metabolism
D001426:Bacterial Proteins; D010710:Phosphates; D010743:Phospholipids; D012694:Serine; D010740:Phospholipases; D008049:Lipase; D014633:Valine