6-磷酸葡萄糖脱氢酶在基于烟酰胺的仿生体上的辅酶工程及其作为葡萄糖生物传感器的应用,ACS Catalysis

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6-磷酸葡萄糖脱氢酶在基于烟酰胺的仿生体上的辅酶工程及其作为葡萄糖生物传感器的应用
ACS Catalysis ( IF 11.3 ) Pub Date : 2023-01-20 , DOI: 10.1021/acscatal.2c04707
Dongdong Meng ₁ , Meixia Liu _{1,

2} , Hao Su _{1,

3} , Haiyan Song ₁ , Lijie Chen _{1,

4} , Qiangzi Li _{1,

2} , Ya-nan Liu ₁ , Zhiguang Zhu ₁ , Weidong Liu ₁ , Xiang Sheng _{1,

2,

3} , Chun You _{1,

2,

3} , Yi-Heng P. Job Zhang _{1,

5}

Affiliation

烟酰胺腺嘌呤二核苷酸 (NAD(P) ⁺ ) 依赖性氧化还原酶已被广泛用作许多应用的生物催化剂，例如体外生物制造和生物传感器。仿生烟酰胺辅酶 (BNC) 在酶促氧化还原级联中的应用构成了一种有前途的替代方案，可以消除对昂贵的天然 NAD(P) ⁺辅酶的需求。在此，我们证明了来自运动发酵单胞菌₍ ZmG6PDH ) 的葡萄糖 6-磷酸脱氢酶的辅酶工程提高了其^对氧化_烟酰胺单核苷酸 ( NMN +). 与野生型 (WT) 酶相比，最佳突变体 R4 对 NMN ⁺的催化效率提高了 112 倍，两个辅酶特异性值分别增加了 4.7 × 10 ³和 2.6 × 10 ³倍 ([ k _cat / K _m ] _NMN⁺ /[ k _cat / K _m ] _NAD⁺和 [ k _cat / K _m ] _NMN⁺ /[ k _cat / K _m ] _NADP⁺），分别。为了深入了解酶及其突变体的结构-功能关系，我们确定了 WT G6PDH (apo-WT) 和突变体 R4 和 NMN ⁺ (R4:NMN ⁺ ) 的复合物的晶体结构，并进行了分子动力学模拟G6PDH/底物（葡萄糖-6-磷酸，G6P）/辅酶的三元复合物。结果表明，突变体 R4 中辅酶结合 Rossmann 样结构域的螺旋区域比野生型酶更接近活性位点，从而产生更紧凑的底物/辅酶结合位点，有利于底物的结合G6P 和 NMN ⁺。此外，基于葡萄糖激酶 (GK)/G6PDH-NMN ^{+的葡萄糖生物传感器}以NMN ⁺为辅酶构建了无需冷冻保存的生物系统。这些结果表明BNCs和氧化还原酶的组合可以广泛应用于生物传感器、生物催化和体外生物制造。

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Coenzyme Engineering of Glucose-6-phosphate Dehydrogenase on a Nicotinamide-Based Biomimic and Its Application as a Glucose Biosensor

Nicotinamide adenine dinucleotide (NAD(P)⁺)-dependent oxidoreductases have been widely employed as biocatalysts for numerous applications, such as in vitro biomanufacturing and biosensors. The application of biomimetic nicotinamide coenzymes (BNCs) in an enzymatic redox cascade constitutes a promising alternative that can eliminate the need for expensive natural NAD(P)⁺ coenzymes. Herein, we demonstrated that the coenzyme engineering of glucose-6-phosphate dehydrogenase from Zymomonas mobilis (ZmG6PDH) enhanced its catalytic efficiency (k_cat/K_m) on oxidized nicotinamide mononucleotide (NMN⁺). Compared with the wild-type (WT) enzyme, the optimal mutant R4 exhibited a 112-fold enhancement in catalytic efficiency on NMN⁺, with 4.7 × 10³ and 2.6 × 10³-fold change in the two coenzyme specificity values ([k_cat/K_m]_NMN⁺/[k_cat/K_m]_NAD⁺ and [k_cat/K_m]_NMN⁺/[k_cat/K_m]_NADP⁺), respectively. To obtain insights into the structure–function relationship of the enzyme and its mutant, we determined the crystal structures of WT G6PDH (apo-WT) and the complex of mutant R4 and NMN⁺ (R4:NMN⁺) and performed molecular dynamics simulations of the ternary complexes of the G6PDH/substrate (glucose-6-phosphate, G6P)/coenzyme. The results revealed that the helix region of the coenzyme-binding Rossmann-like domain in mutant R4 was nearer to the active site than the wild-type enzyme, thus creating a more compact substrate/coenzyme-binding site to favor the binding of the substrate G6P and NMN⁺. Furthermore, a glucose biosensor based on the glucokinase (GK)/G6PDH-NMN⁺ biosystem without cryopreservation was constructed using NMN⁺ as the coenzyme. These results indicate that the combination of BNCs and oxidoreductases can be widely employed in biosensors, biocatalysis, and in vitro biomanufacturing.

更新日期：2023-01-20

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