Giuseppe Priscila Oliveira de, Santos Marcelo Leite Dos, Sousa Sylvia Morais de, Koch Karen E, Yunes José Andrés, Aparicio Ricardo, Murakami Mario Tyago
Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials (CNPEM), Campinas, SP, Brazil.
Laboratory of Structural Biology and Crystallography, Institute of Chemistry, University of Campinas, Campinas, SP, Brazil.
Biochem Biophys Res Commun. 2016 Jun 10;474(4):696-701. doi: 10.1016/j.bbrc.2016.05.011. Epub 2016 May 3.
Plant aldo-keto reductases of the AKR4C subfamily play key roles during stress and are attractive targets for developing stress-tolerant crops. However, these AKR4Cs show little to no activity with previously-envisioned sugar substrates. We hypothesized a structural basis for the distinctive cofactor binding and substrate specificity of these plant enzymes. To test this, we solved the crystal structure of a novel AKR4C subfamily member, the AKR4C7 from maize, in the apo form and in complex with NADP(+). The binary complex revealed an intermediate state of cofactor binding that preceded closure of Loop B, and also indicated that conformational changes upon substrate binding are required to induce a catalytically-favorable conformation of the active-site pocket. Comparative structural analyses of homologues (AKR1B1, AKR4C8 and AKR4C9) showed that evolutionary redesign of plant AKR4Cs weakened interactions that stabilize the closed conformation of Loop B. This in turn decreased cofactor affinity and altered configuration of the substrate-binding site. We propose that these structural modifications contribute to impairment of sugar reductase activity in favor of other substrates in the plant AKR4C subgroup, and that catalysis involves a three-step process relevant to other AKRs.
AKR4C亚家族的植物醛糖 - 酮糖还原酶在胁迫过程中发挥关键作用，是培育耐胁迫作物的有吸引力的靶点。然而，这些AKR4C对先前设想的糖类底物几乎没有活性。我们推测了这些植物酶独特的辅因子结合和底物特异性的结构基础。为了验证这一点，我们解析了一种新型AKR4C亚家族成员——玉米中的AKR4C7——的无配体形式以及与NADP(+)形成复合物的晶体结构。二元复合物揭示了辅因子结合的中间状态，该状态先于环B的闭合，并且还表明底物结合时的构象变化是诱导活性位点口袋形成催化有利构象所必需的。同源物（AKR1B1、AKR4C8和AKR4C9）的比较结构分析表明，植物AKR4C的进化重新设计削弱了稳定环B闭合构象的相互作用。这反过来降低了辅因子亲和力并改变了底物结合位点的构型。我们提出，这些结构修饰导致植物AKR4C亚组中糖还原酶活性受损，转而有利于其他底物，并且催化涉及与其他AKR相关的三步过程。
