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.
Department of Enzymology and Protein Engineering, Utrecht University, The Netherlands.
M D
MD
van Kampen
J W
JW
Simons
N
N
Dekker
M R
MR
Egmond
H M
HM
Verheij
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