Implication of Molecular Constraints Facilitating the Functional Evolution of Pseudomonas aeruginosa KPR2 into a Versatile α-Keto-Acid Reductase,Biochemistry

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Implication of Molecular Constraints Facilitating the Functional Evolution of Pseudomonas aeruginosa KPR2 into a Versatile α-Keto-Acid Reductase
Biochemistry ( IF 2.9 ) Pub Date : 2024-07-04 , DOI: 10.1021/acs.biochem.4c00087
Gourab Basu Choudhury _{1,

2} , Saumen Datta _{1,

2}

Affiliation

Theoretical concepts linking the structure, function, and evolution of a protein, while often intuitive, necessitate validation through investigations in real-world systems. Our study empirically explores the evolutionary implications of multiple gene copies in an organism by shedding light on the structure–function modulations observed in Pseudomonas aeruginosa’s second copy of ketopantoate reductase (PaKPR2). We demonstrated with two apo structures that the typical active site cleft of the protein transforms into a two-sided pocket where a molecular gate made up of two residues controls the substrate entry site, resulting in its inactivity toward the natural substrate ketopantoate. Strikingly, this structural modification made the protein active against several important α-keto-acid substrates with varied efficiency. Structural constraints at the binding site for this altered functional trait were analyzed with two binary complexes that show the conserved residue microenvironment faces restricted movements due to domain closure. Finally, its mechanistic highlights gathered from a ternary complex structure help in delineating the molecular perspectives behind its kinetic cooperativity toward these broad range of substrates. Detailed structural characteristics of the protein presented here also identified four key amino acid residues responsible for its versatile α-keto-acid reductase activity, which can be further modified to improve its functional properties through protein engineering.

中文翻译：

分子约束的含义促进铜绿假单胞菌 KPR2 功能进化为多功能的 α-酮酸还原酶

将蛋白质的结构、功能和进化联系起来的理论概念虽然通常很直观，但需要通过在现实世界系统中的研究来验证。我们的研究通过阐明在铜绿假单胞菌的第二个酮泛酸还原酶（PaKPR2）拷贝中观察到的结构-功能调节，实证地探讨了生物体中多个基因拷贝的进化意义。我们用两个载脂蛋白结构证明，蛋白质的典型活性位点裂隙转化为一个双面口袋，其中由两个残基组成的分子门控制底物进入位点，导致其对天然底物酮泛酸酯无活性。引人注目的是，这种结构修饰使该蛋白质对几种重要的α酮酸底物具有活性，效率各不相同。用两个二元复合物分析了这种改变的功能性状的结合位点的结构约束，这些复合物显示由于结构域关闭，保守的残基微环境面临受限的运动。最后，它从三元复合物结构中收集的机理亮点有助于描绘其对这些广泛底物的动力学协同性背后的分子视角。这里介绍的蛋白质的详细结构特征还确定了负责其多功能 α-酮酸还原酶活性的四个关键氨基酸残基，这些残基可以通过蛋白质工程进一步修饰以改善其功能特性。

更新日期：2024-07-04

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