Lan Dongming, Wang Qian, Popowicz Grzegorz Maria, Yang Bo, Tang Qingyun, Wang Yonghua
College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510641, P.R. China.
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, P.R. China.
J Microbiol Biotechnol. 2015 Nov;25(11):1827-34. doi: 10.4014/jmb.1506.06079.
The SMG1 lipase from Malassezia globosa is a newly found mono- and diacylglycerol (DAG) lipase that has a unique lid in the loop conformation that differs from the common alpha-helix lid. In the present study, we characterized the contribution of three residues, L103 and F104 in the lid and F278 in the rim of the binding site groove, on the function of SMG1 lipase. Sitedirected mutagenesis was conducted at these sites, and each of the mutants was expressed in the yeast Pichia pastoris, purified, and characterized for their activity toward DAG and pnitrophenol (pNP) ester. Compared with wild-type SMG1, F278A retained approximately 78% of its activity toward DAG, but only 11% activity toward pNP octanoate (pNP-C8). L103G increased its activity on pNP-C8 by approximately 2-fold, whereas F104G showed an approximate 40% decrease in pNP-C8 activity, and they both showed decreased activity on the DAG emulsion. The deletion of 103-104 retained approximately 30% of its activity toward the DAG emulsion, with an almost complete loss of pNP-C8 activity. The deletion of 103-104 showed a weaker penetration ability to a soybean phosphocholine monolayer than wild-type SMG1. Based on the modulation of the specificity and activity observed, a pNP-C8 binding model for the ester (pNP-C8, N102, and F278 form a flexible bridge) and a specific lipidanchoring mechanism for DAG (L103 and F104 serve as "anchors" to the lipid interface) were proposed.
来自球形马拉色菌的SMG1脂肪酶是一种新发现的单酰基甘油和二酰基甘油（DAG）脂肪酶，其具有独特的环状构象盖子，与常见的α-螺旋盖子不同。在本研究中，我们表征了结合位点凹槽边缘的盖子中的三个残基L103和F104以及F278对SMG1脂肪酶功能的贡献。在这些位点进行定点诱变，每个突变体在毕赤酵母中表达、纯化，并对其对DAG和对硝基苯酚（pNP）酯的活性进行表征。与野生型SMG1相比，F278A对DAG保留了约78%的活性，但对对硝基苯酚辛酸酯（pNP-C8）仅保留11%的活性。L103G对对硝基苯酚辛酸酯的活性增加了约2倍，而F104G对对硝基苯酚辛酸酯的活性降低了约40%，并且它们对DAG乳液的活性均降低。缺失103-104对DAG乳液保留了约30%的活性，对硝基苯酚辛酸酯的活性几乎完全丧失。缺失103-104对大豆磷酸胆碱单层的穿透能力比野生型SMG1弱。基于观察到的特异性和活性调节，提出了酯（pNP-C8、N102和F278形成柔性桥）的对硝基苯酚辛酸酯结合模型以及DAG的特定脂质锚定机制（L103和F104作为脂质界面的“锚”）。
