van Kampen M D, Dekker N, Egmond M R, Verheij H M
Department of Enzymology and Protein Engineering, Utrecht University, TB Utrecht, The Netherlands.
Biochemistry. 1998 Mar 10;37(10):3459-66. doi: 10.1021/bi9725430.
Staphylococcus hyicus lipase (SHL) and Staphylococcus aureus lipase (SAL) are highly homologous enzymes, yet they show remarkable differences in their biochemical characteristics. SHL displays a high phospholipase activity, hydrolyses neutral lipids, and has no chain length preference, whereas SAL only degrades short-chain fatty acid esters. To identify the regions in the primary sequence of SHL responsible for phospholipase activity and chain length selectivity, a set of histidine-tagged SAL/SHL chimeras was generated by in vivo recombination in Escherichia coli. Several classes of chimeric enzymes were identified on the basis of restriction site analysis. All chimeras were well-expressed as active enzymes. They were characterized for their specific activities on both phospholipids and p-nitrophenyl esters of various chain lengths. Phospholipase activity appeared to be determined by three regions, all located in the C-terminal domain of SHL. Testing of the enzymatic activity of the chimeras toward p-nitrophenyl esters showed that chain length selectivity is defined by elements within the region of residues 180-253. Moreover, also residues along the stretch 275-358 contribute to the binding of acyl chains. Interestingly, several chimeras were even more active than the parent enzymes on long-chain p-nitrophenyl esters.
猪葡萄球菌脂肪酶（SHL）和金黄色葡萄球菌脂肪酶（SAL）是高度同源的酶，但它们在生化特性上表现出显著差异。SHL具有较高的磷脂酶活性，能水解中性脂质，且对链长没有偏好，而SAL仅降解短链脂肪酸酯。为了确定SHL一级序列中负责磷脂酶活性和链长选择性的区域，通过在大肠杆菌中进行体内重组产生了一组带有组氨酸标签的SAL/SHL嵌合体。基于限制性酶切位点分析鉴定出了几类嵌合酶。所有嵌合体均作为活性酶得到良好表达。对它们针对不同链长的磷脂和对硝基苯酯的比活性进行了表征。磷脂酶活性似乎由三个区域决定，所有这些区域都位于SHL的C端结构域。对嵌合体针对对硝基苯酯的酶活性测试表明，链长选择性由残基180 - 253区域内的元件定义。此外，275 - 358区段的残基也有助于酰基链的结合。有趣的是，几种嵌合体在长链对硝基苯酯上甚至比亲本酶更具活性。
