Madhuprakash Jogi, Tanneeru Karunakar, Karlapudi Bhavana, Guruprasad Lalitha, Podile Appa Rao
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad 500046, A.P., India.
School of Chemistry, University of Hyderabad, Gachibowli, Hyderabad 500046, A.P., India.
Biochim Biophys Acta. 2014 Sep;1840(9):2685-94. doi: 10.1016/j.bbagen.2014.06.014. Epub 2014 Jun 25.
Transglycosylation (TG) activity is a property of glycosyl hydrolases (GHs) with which new glycosidic bonds are introduced between donor and acceptor sugar molecules. This special property of the GHs has potential to generate longer chain chitooligosaccharides (CHOS) that show elicitor activity in plants. We hypothesize that TG activity could be improved by retaining the substrate for a longer duration in the catalytic site.
Four variants of chitinase D from Serratia proteamaculans (SpChiD) i.e. G119S, G119W, W120A and G201W were analyzed in detail for improved TG activity using high performance liquid chromatography (HPLC) and high resolution mass spectrometry (HRMS). The results were strongly supported by 50ns molecular dynamics (MD) simulations and estimated solvated interaction energies (SIE).
The mutant G119W lost much of both hydrolytic and TG activities, while the mutant G201W displayed increased TG. The trajectory of MD simulations of the mutant G119W showed that the indole rings of two adjacent Trp residues create a major hindrance for the DP4 movement towards the catalytic center. Increased van der Waals (vdW) and coulombic interactions between DP4 substrate and the Trp-201 resulted in enhanced TG activity with the mutant G201W. The average number of hydrogen bonds observed for the DP4 substrate was increased for the mutants G119W and G201W compared to SpChiD.
The increase in TG activity could be due to partial blocking of product exit of SpChiD.
This new approach can be used for generating mutants of GHs with improved TG activity to produce longer chain oligosaccharides.
转糖基化（TG）活性是糖基水解酶（GHs）的一种特性，利用该特性可在供体糖分子和受体糖分子之间引入新的糖苷键。GHs的这种特殊性质有潜力生成在植物中表现出诱导活性的长链壳寡糖（CHOS）。我们推测，通过将底物在催化位点保留更长时间可以提高TG活性。
利用高效液相色谱（HPLC）和高分辨率质谱（HRMS）详细分析了粘质沙雷氏菌几丁质酶D（SpChiD）的四个变体，即G119S、G119W、W120A和G201W，以提高其TG活性。50纳秒的分子动力学（MD）模拟和估计的溶剂化相互作用能（SIE）有力地支持了这些结果。
突变体G119W的水解活性和TG活性均大幅丧失，而突变体G201W的TG活性有所增加。突变体G119W的MD模拟轨迹表明，两个相邻色氨酸残基的吲哚环对DP4向催化中心的移动造成了主要阻碍。DP4底物与色氨酸-201之间增加的范德华（vdW）相互作用和库仑相互作用导致突变体G201W的TG活性增强。与SpChiD相比，突变体G119W和G201W的DP4底物观察到的氢键平均数量增加。
TG活性的增加可能是由于SpChiD产物出口的部分受阻。
这种新方法可用于生成具有改善的TG活性的GHs突变体，以生产更长链的寡糖。
