van Kampen M D, Verheij H M, Egmond M R
Department of Enzymology and Protein Engineering, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, The Netherlands.
Biochemistry. 1999 Jul 20;38(29):9524-32. doi: 10.1021/bi990096d.
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.
来自猪葡萄球菌的脂肪酶（SHL）表现出较高的磷脂酶活性，而同源的金黄色葡萄球菌脂肪酶（SAL）对磷脂底物无活性或活性极低。此前已表明，包含254 - 358位残基的区域内的元件对于SHL识别磷脂至关重要。为了具体鉴定重要残基，在254 - 358区域内，将SHL的九个小簇分别替换为相应的SAL序列。为便于克隆，组装了SHL的合成基因片段，从而在SHL基因中引入限制酶切位点并优化密码子使用。所有九个嵌合体均作为活性酶得到良好表达。在标准活性测定中，八个嵌合体的脂肪酶和磷脂酶活性与野生型SHL相比，活性因子在2倍范围内。将极性的SHL区域293 - 300替换为疏水性更强的SAL区域，导致脂肪酶活性的k(cat)/K(m)值增加32倍，同时磷脂酶活性的k(cat)/K(m)值相应降低68倍。这两种变化均归因于对催化周转以及底物亲和力的影响。随后，产生了六个点突变体；G293N、E295F、T297P、K298F、I299V和L300I。残基E295似乎起次要作用，而K298是磷脂酶活性的主要决定因素。突变K298F由于k(cat)和K(m)的变化，导致在磷脂底物上的k(cat)/K(m)降低60倍。在SAL中用赖氨酸取代F298导致磷脂酶活性增加4倍。与野生型SAL相比，另外两个从疏水性到极性的取代使磷脂酶活性进一步增加2到3倍。
