van Kampen M D, Simons J W, Dekker N, Egmond M R, Verheij H M
Department of Enzymology and Protein Engineering, Utrecht University, The Netherlands.
Chem Phys Lipids. 1998 Jun;93(1-2):39-45. doi: 10.1016/s0009-3084(98)00027-9.
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.
采用定点诱变和结构域交换技术研究了猪葡萄球菌脂肪酶（SHL）和金黄色葡萄球菌脂肪酶（SAL）的C末端结构域在底物选择性中的作用。在SHL中引入一个编码将Ser356替换为Val的单点突变，产生了一种保留了全部脂肪酶活性但磷脂酶活性降低了12倍以上的酶。从SHL的这种S356V变体开始，将C末端的40个氨基酸替换为相应的SAL序列。尽管同时引入了23个氨基酸变化，但对磷脂酶/脂肪酶活性比的影响仅为4倍。因此，我们得出结论，在C末端结构域中，主要是Ser356决定了磷脂酶活性。在SAL中引入Val357到Ser的替换并没有使SAL变成磷脂酶，这表明其他结构域的残基也对这种活性有贡献。结合脂肪酶和（溶）磷脂酶的序列同源性对结果进行了讨论。
