Molecular Systems Engineering Lab, Department of Bioinformatics, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India.
Molecular Systems Engineering Lab, Department of Bioinformatics, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India.
Int J Biol Macromol. 2019 Nov 1;140:1214-1225. doi: 10.1016/j.ijbiomac.2019.08.194. Epub 2019 Aug 28.
Agrobacterium tumefaciens uronate dehydrogenase (AtuUdh) belongs to the short-chain dehydrogenase superfamily, specifically those acting on the CH-OH group of donor with NAD or NADP as acceptor. It is apparently required for the production of D-glucaric acid. AtuUdh-catalyzed reaction is reversible with dual substrate-specific activity (D-galacturonic acid and D-glucuronic acid) in nature. In our study, 34 mutants were pre-screened from 155 mutants generated from AtuUdh (wild-type) and selected 10 structurally stable mutants with increased substrate selectivity. The specificity, efficiency, and selectivity of these mutants for different substrates and cofactors were predicted from 121 docked models using a substrate-imprinted docking approach. Q14F, S36L, and S75T mutants have shown a high binding affinity to D-glucuronic acid and its substrate intermediates such as D-glucaro-1,4-lactone and D-glucaro-1,5-lactone. These mutants exhibited a low binding affinity to the substrate and cofactor required for D-galactaric acid. D34S, N112E and S165E mutants found to show a high selectivity of D-galacturonic acid and its substrate intermediates for D-galactaric acid production. Ser75, Ser165, and Arg174 are active residues playing an imperative role in the substrate selectivity and also contributed in the conjecture the mechanism of transition state stabilization catalyzed by AtuUdh mutants. The present approach was used to reveal the substrate binding mechanism of AtuUdh mutants for a better understanding of the structural basis for selectivity and function.
农杆菌尿苷二磷酸脱氢酶（AtuUdh）属于短链脱氢酶超家族，特别是那些以 NAD 或 NADP 为受体作用于供体 CH-OH 基团的酶。它显然是生产 D-葡萄糖酸所必需的。AtuUdh 催化的反应是可逆的，具有双重底物特异性活性（D-半乳糖醛酸和 D-葡萄糖醛酸）。在我们的研究中，从 AtuUdh（野生型）产生的 155 个突变体中预先筛选了 34 个突变体，并选择了 10 个结构稳定的突变体，这些突变体具有增加的底物选择性。使用基于印迹的对接方法，从 121 个对接模型中预测了这些突变体对不同底物和辅因子的特异性、效率和选择性。Q14F、S36L 和 S75T 突变体对 D-葡萄糖醛酸及其底物中间体（如 D-葡萄糖酸-1,4-内酯和 D-葡萄糖酸-1,5-内酯）表现出高结合亲和力。这些突变体对 D-半乳糖酸及其所需的底物和辅因子表现出低结合亲和力。D34S、N112E 和 S165E 突变体被发现对 D-半乳糖醛酸及其底物中间体具有高选择性，有利于 D-半乳糖酸的生产。Ser75、Ser165 和 Arg174 是活性残基，在底物选择性中起着至关重要的作用，也有助于推测 AtuUdh 突变体催化的过渡态稳定机制。本方法用于揭示 AtuUdh 突变体的底物结合机制，以更好地理解选择性和功能的结构基础。
