Tholander Fredrik, Kull Filippa, Ohlson Eva, Shafqat Jawed, Thunnissen Marjolein M G M, Haeggström Jesper Z
Department of Medical Biochemistry and Biophysics, Divisions of Chemistry 1 and 2, Karolinska Institutet, Stockholm S-171 77, Sweden.
J Biol Chem. 2005 Sep 30;280(39):33477-86. doi: 10.1074/jbc.M506821200. Epub 2005 Jul 15.
Mammalian leukotriene A4 (LTA4) hydrolase is a bifunctional zinc metalloenzyme possessing an Arg/Ala aminopeptidase and an epoxide hydrolase activity, which converts LTA4 into the chemoattractant LTB4. We have previously cloned an LTA4 hydrolase from Saccharomyces cerevisiae with a primitive epoxide hydrolase activity and a Leu aminopeptidase activity, which is stimulated by LTA4. Here we used a modeled structure of S. cerevisiae LTA4 hydrolase, mutational analysis, and binding studies to show that Glu-316 and Arg-627 are critical for catalysis, allowing us to a propose a mechanism for the epoxide hydrolase activity. Guided by the structure, we engineered S. cerevisiae LTA4 hydrolase to attain catalytic properties resembling those of human LTA4 hydrolase. Thus, six consecutive point mutations gradually introduced a novel Arg aminopeptidase activity and caused the specific Ala and Pro aminopeptidase activities to increase 24 and 63 times, respectively. In contrast to the wild type enzyme, the hexuple mutant was inhibited by LTA4 for all tested substrates and to the same extent as for the human enzyme. In addition, these mutations improved binding of LTA4 and increased the relative formation of LTB4, whereas the turnover of this substrate was only weakly affected. Our results suggest that during evolution, the active site of an ancestral eukaryotic zinc aminopeptidase has been reshaped to accommodate lipid substrates while using already existing catalytic residues for a novel, gradually evolving, epoxide hydrolase activity. Moreover, the unique ability to catalyze LTB4 synthesis appears to be the result of multiple and subtle structural rearrangements at the catalytic center rather than a limited set of specific amino acid substitutions.
哺乳动物白三烯A4（LTA4）水解酶是一种双功能锌金属酶，具有精氨酸/丙氨酸氨基肽酶和环氧化物水解酶活性，可将LTA4转化为趋化因子白三烯B4（LTB4）。我们之前从酿酒酵母中克隆了一种LTA4水解酶，它具有原始的环氧化物水解酶活性和亮氨酸氨基肽酶活性，且受LTA4刺激。在此，我们利用酿酒酵母LTA4水解酶的模拟结构、突变分析和结合研究表明，Glu-316和Arg-627对催化作用至关重要，这使我们能够提出一种环氧化物水解酶活性的机制。在该结构的指导下，我们对酿酒酵母LTA4水解酶进行改造，以获得类似于人LTA4水解酶的催化特性。因此，六个连续的点突变逐渐引入了一种新的精氨酸氨基肽酶活性，并使特定的丙氨酸和脯氨酸氨基肽酶活性分别增加了24倍和63倍。与野生型酶不同，六重突变体对所有测试底物均受LTA4抑制，且抑制程度与人酶相同。此外，这些突变改善了LTA4的结合并增加了LTB4的相对生成量，而该底物的周转仅受到微弱影响。我们的结果表明，在进化过程中，祖先真核锌氨基肽酶的活性位点已被重塑，以适应脂质底物，同时利用现有的催化残基实现一种新的、逐渐进化的环氧化物水解酶活性。此外，催化LTB4合成的独特能力似乎是催化中心多个细微结构重排的结果，而非有限的一组特定氨基酸取代的结果。
