Schmitt Jutta, Brocca Stefania, Schmid Rolf D, Pleiss Jürgen
Institute of Technical Biology, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
Protein Eng. 2002 Jul;15(7):595-601. doi: 10.1093/protein/15.7.595.
The molecular basis of chain length specificity of Candida rugosa lipase 1 was investigated by molecular modeling and site-directed mutagenesis. The synthetic lip1 gene and the lipase mutants were expressed in Pichia pastoris and assayed for their chain length specificity in single substrate assays using triglycerides as well as in a competitive substrate assay using a randomized oil. Mutation of amino acids at different locations inside the tunnel (P246F, L413F, L410W, L410F/S300E, L410F/S365L) resulted in mutants with a different chain length specificity. Mutants P246F and L413F have a strong preference for short chain lengths whereas substrates longer than C10 are hardly hydrolyzed. Increasing the bulkiness of the amino acid at position 410 led to mutants that show a strong discrimination of chain lengths longer than C14. The results obtained can be explained by a simple mechanical model: the activity for a fatty acid sharply decreases as it becomes long enough to reach the mutated site. In contrast, a mutation at the entrance of the tunnel (L304F) has a strong impact on C4 and C6 substrates. This mutant is nevertheless capable of hydrolyzing chain lengths longer than C8.
通过分子建模和定点诱变研究了皱褶假丝酵母脂肪酶1链长特异性的分子基础。合成的lip1基因和脂肪酶突变体在毕赤酵母中表达，并在使用甘油三酯的单底物测定以及使用随机化油的竞争性底物测定中检测其链长特异性。通道内不同位置的氨基酸突变（P246F、L413F、L410W、L410F/S300E、L410F/S365L）产生了具有不同链长特异性的突变体。突变体P246F和L413F对短链长度有强烈偏好，而长于C10的底物几乎不被水解。增加410位氨基酸的体积导致突变体对长于C14的链长表现出强烈的区分。所得结果可用一个简单的力学模型解释：当脂肪酸长到足以到达突变位点时，其活性急剧下降。相比之下，通道入口处的突变（L304F）对C4和C6底物有强烈影响。不过，该突变体仍能够水解长于C8的链长。
