Dong Yi-Ning, Chen Hai-Qin, Sun Yan-Hui, Zhang Hao, Chen Wei
College of Biology and Food Engineering, Chuzhou University, Chuzhou 239000, P. R. China; State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, P. R. China.
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, P. R. China; The Synergistic Innovation Center for Food Safety and Nutrition, Wuxi 214122, P. R. China.
J Dairy Sci. 2015 Apr;98(4):2268-76. doi: 10.3168/jds.2014-9117. Epub 2015 Feb 11.
The glycoside hydrolase family 42 (GH42) of thermophilic microorganisms consists of thermostable β-galactosidases that display significant variations in their temperature optima and stabilities. In this study, we compared the substrate binding modes of 2 GH42 β-galactosidases, BgaB from Geobacillus stearothermophilus and A4-β-Gal from Thermus thermophilus A4. The A4-β-Gal has a catalytic triad (Glu312-Arg32-Glu35) with an extended hydrogen bond network that has not been observed in BgaB. In this study, we performed site-saturation mutagenesis of Ile42 in BgaB (equivalent to Glu312 in A4-β-Gal) to study the effects of different residues on thermostability, catalytic function, and the extended hydrogen bond network. Our experimental results suggest that substitution of Ile42 with polar AA enhanced the thermostability but decreased the catalytic efficiency of BgaB. Polar AA substitution for Ile42 simultaneously affected thermostability, catalytic efficiency, and the hydrogen bond network, suggesting that Ile42 is responsible for functional discrimination between members of the GH42 family. These observations could lead to a novel strategy for investigating the functional evolution of the GH42 β-galactosidases.
嗜热微生物的糖苷水解酶家族42（GH42）由热稳定的β-半乳糖苷酶组成，这些酶在最适温度和稳定性方面表现出显著差异。在本研究中，我们比较了两种GH42β-半乳糖苷酶的底物结合模式，即嗜热栖热放线菌的BgaB和嗜热栖热菌A4的A4-β-Gal。A4-β-Gal具有一个催化三联体（Glu312-Arg32-Glu35），其氢键网络延伸，而在BgaB中未观察到。在本研究中，我们对BgaB中的Ile42（相当于A4-β-Gal中的Glu312）进行了位点饱和诱变，以研究不同残基对热稳定性、催化功能和延伸氢键网络的影响。我们的实验结果表明，用极性氨基酸取代Ile42可提高BgaB的热稳定性，但会降低其催化效率。用极性氨基酸取代Ile42同时影响热稳定性、催化效率和氢键网络，这表明Ile42负责GH42家族成员之间的功能区分。这些观察结果可能会导致一种研究GH42β-半乳糖苷酶功能进化的新策略。
