Structural and Kinetic Analysis of Schwanniomyces occidentalis Invertase Reveals a New Oligomerization Pattern and the Role of Its Supplementary Domain in Substrate Binding
Structural and Kinetic Analysis of Schwanniomyces occidentalis Invertase Reveals a New Oligomerization Pattern and the Role of Its Supplementary Domain in Substrate Binding
复制标题
DOI：
10.1074/jbc.m109.095430
复制
发表时间：
2010-04-30
影响因子：
4.8
通讯作者：
Sanz-Aparicio, Julia
中科院分区：
文献类型：
--
作者：
Alvaro-Benito, Miguel;Polo, Aitana;Sanz-Aparicio, Julia
西雪旺酵母转化酶是一种胞外酶，可水解蔗糖并从各种寡糖和菊粉等必需的储存果聚糖聚合物中释放 β-果糖。我们在这里报告 Sw 的三维结构。 occidentalis 转化酶的分辨率为 2.9 埃，其与果糖的复合物的分辨率为 1.9 埃。该单体呈现出与其他 GH32 酶常见的双模块排列，具有 N 端 5 重 β 螺旋桨催化结构域和 C 端 β 夹心结构域，其功能迄今为止尚不清楚。然而，Sw 的二聚体性质。 occidentalis 转化酶揭示了由两个亚基形成的独特活性位点裂缝，这可能代表了据报道为多聚体的其他酵母酶。通过对接分析探索了四糖制霉菌糖和聚合物菊粉的结合，这表明中等大小和长的底物可以被两个亚基的残基识别。对鉴定出的残基进行突变，并通过动力学分析研究突变体针对蔗糖、制霉菌糖和菊粉的酶活性。对催化效率影响最大的替换是 Q228V（一种推测参与制霉菌糖和菊粉结合的残基）和 S281I（参与二聚体界面的极性连接）。此外，在突变体 Q435A 和 Y462A 中观察到针对菊粉的催化效率显着降低，这两个突变体都位于第二单体的 β-夹心结构域中。这凸显了寡聚化在底物特异性中发挥的重要作用，并首次将直接催化作用分配给 GH32 酶的补充结构域。
Schwanniomyces occidentalis invertase is an extracellular enzyme that hydrolizes sucrose and releases beta-fructose from various oligosaccharides and essential storage fructan polymers such as inulin. We report here the three-dimensional structure of Sw. occidentalis invertase at 2.9 angstrom resolution and its complex with fructose at 1.9 angstrom resolution. The monomer presents a bimodular arrangement common to other GH32 enzymes, with an N-terminal 5-fold beta-propeller catalytic domain and a C-terminal beta-sandwich domain for which the function has been unknown until now. However, the dimeric nature of Sw. occidentalis invertase reveals a unique active site cleft shaped by both subunits that may be representative of other yeast enzymes reported to be multimeric. Binding of the tetrasaccharide nystose and the polymer inulin was explored by docking analysis, which suggested that medium size and long substrates are recognized by residues from both subunits. The identified residues were mutated, and the enzymatic activity of the mutants against sucrose, nystose, and inulin were investigated by kinetic analysis. The replacements that showed the largest effect on catalytic efficiency were Q228V, a residue putatively involved in nystose and inulin binding, and S281I, involved in a polar link at the dimer interface. Moreover, a significant decrease in catalytic efficiency against inulin was observed in the mutants Q435A and Y462A, both located in the beta-sandwich domain of the second monomer. This highlights the essential function that oligomerization plays in substrate specificity and assigns, for the first time, a direct catalytic role to the supplementary domain of a GH32 enzyme.