Structural Basis for Binding of Fluorinated Glucose and Galactose to Trametes multicolor Pyranose 2-Oxidase Variants with Improved Galactose Conversion

Each year, about six million tons of lactose are generated from liquid whey as industrial byproduct, and optimally this large carbohydrate waste should be used for the production of value-added products. Trametes multicolor pyranose 2-oxidase (TmP2O) catalyzes the oxidation of various monosaccharides to the corresponding 2-keto sugars. Thus, a potential use of TmP2O is to convert the products from lactose hydrolysis, D-glucose and D-galactose, to more valuable products such as tagatose. Oxidation of glucose is however strongly favored over galactose, and oxidation of both substrates at more equal rates is desirable. Characterization of TmP2O variants (H450G, V546C, H450G/V546C) with improved D-galactose conversion has been given earlier, of which H450G displayed the best relative conversion between the substrates. To rationalize the changes in conversion rates, we have analyzed high-resolution crystal structures of the aforementioned mutants with bound 2- and 3-fluorinated glucose and galactose. Binding of glucose and galactose in the productive 2-oxidation binding mode is nearly identical in all mutants, suggesting that this binding mode is essentially unaffected by the mutations. For the competing glucose binding mode, enzyme variants carrying the H450G replacement stabilize glucose as the α-anomer in position for 3-oxidation. The backbone relaxation at position 450 allows the substrate-binding loop to fold tightly around the ligand. V546C however stabilize glucose as the β-anomer using an open loop conformation. Improved binding of galactose is enabled by subtle relaxation effects at key active-site backbone positions. The competing binding mode for galactose 2-oxidation by V546C stabilizes the β-anomer for oxidation at C1, whereas H450G variants stabilize the 3-oxidation binding mode of the galactose α-anomer. The present study provides a detailed description of binding modes that rationalize changes in the relative conversion rates of D-glucose and D-galactose and can be used to refine future enzyme designs for more efficient use of lactose-hydrolysis byproducts.每年，工业副产物液体乳清可产生约600万吨乳糖，这种大量产出的碳水化合物副产物本应被用于制备高附加值产品。彩绒革盖菌（Trametes multicolor）的吡喃糖2-氧化酶（pyranose 2-oxidase，简称TmP2O）可催化多种单糖氧化生成对应的2-酮糖。因此，利用TmP2O将乳糖水解产物D-葡萄糖与D-半乳糖转化为塔格糖等更高价值产物具备潜在应用价值。不过，该酶对葡萄糖的氧化偏好显著强于半乳糖，因此亟需实现两种底物以更均衡的速率被氧化。此前已有研究报道了可提升D-半乳糖转化效率的TmP2O突变体（H450G、V546C、H450G/V546C），其中H450G在两种底物间展现出最优的相对转化效率。为阐明转化速率变化的分子机制，我们解析了上述突变体结合2-氟代与3-氟代葡萄糖、半乳糖的高分辨率晶体结构。分析结果表明，在所有突变体中，葡萄糖与半乳糖以催化产生活性的2-氧化结合模式的构象几乎完全一致，提示该结合模式基本不受突变影响。对于竞争性结合模式，携带H450G替换的酶变体可将葡萄糖的α-异头体稳定在3-氧化的催化位点上；450位的主链松弛使得底物结合环能够紧密包裹配体。而V546C突变则通过开放环构象将葡萄糖稳定为β-异头体。半乳糖结合能力的提升源于关键活性位点主链位置的细微松弛效应。V546C变体介导的半乳糖2-氧化竞争性结合模式可稳定β-异头体，使其在C1位发生氧化；而H450G突变体则稳定半乳糖α-异头体的3-氧化结合模式。本研究详细阐明了可解释D-葡萄糖与D-半乳糖相对转化速率变化的结合模式，可为未来优化酶设计以更高效利用乳糖水解副产物提供理论指导。