Impact of Leucine 278 Residue on Fatty Acid Length Specificity of Candida antarctica Lipase B
Impact of Leucine 278 Residue on Fatty Acid Length Specificity of Candida antarctica Lipase B
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DOI：
10.4236/aim.2015.57050
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发表时间：
2015-07
期刊：
影响因子：
0.9
通讯作者：
Fanghua Wang;Shulin Hou;Qian Wang;Pu Wang;Jinsong Liu;Bo Yang;Yonghua Wang
中科院分区：
文献类型：
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作者：
Fanghua Wang;Shulin Hou;Qian Wang;Pu Wang;Jinsong Liu;Bo Yang;Yonghua Wang
对南极假丝酵母脂肪酶B（CALB）的结构分析表明，278位亮氨酸侧链位于催化口袋入口的上方，这表明它可能对酶的底物特异性起作用。为了验证这一假设，合理设计了甘氨酸或脯氨酸的短侧链，并通过定点突变的方法构建了突变体。采用不同链长的脂肪酸对硝基苯酯比色法研究了脂肪酶的底物选择性。结果表明，L278 G或L278 P突变均导致CALB的底物特异性从辛酸对硝基苯基酯（pNP-C8）漂移到更长碳链的癸酸对硝基苯基酯（pNP-C10）。同时，两个突变体对pNP-C10的Vmax值均高于野生型。对接结果还表明，在该位点进行甘氨酸侧链缩短或脯氨酸残基取代，可以消除催化口袋上方存在的空间阻滞，使长链底物（pNP-C10）更容易进入催化口袋。观察到的特异性的调制允许建立底物结合模型，并为设计配体特异性脂肪酶开辟了新的可能性。
Structural analysis of Candida antarctica lipase B (CALB) indicates that side chain of leucine at 278 site lies above the entrance of the catalytic pocket, which prognosticates its potential role on substrate specificity of the enzyme. To verify this presumption, shortened side chain of glycine or proline was rational designed and mutants were constructed by site-directed mutagenesis method. The colorimetric assay using p-nitrophenyl esters of fatty acids with various chain-lengths was used to study the substrate preference of lipases. Results indicated that L278G or L278P mutations both induced the drift of substrate specificity of CALB from p-nitrophenyl caprylate (pNP-C8) to longer carbon chain length of p-nitrophenyl caprate (pNP-C10). Meanwhile, Vmax value of two mutants to pNP-C10 was both higher than that of wild-type. Docking results also indicated that shortened side chain of glycine or proline residues substitution at this site could get rid of the space block present above the catalytic pocket, and made longer chain substrate (pNP-C10) enter into the catalytic pocket easier. The modulation of specificity observed allowed for building substrate binding model and opened new possibilities for designing ligand specific lipases.