Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy.
Arch Biochem Biophys. 2013 Jan 15;529(2):55-65. doi: 10.1016/j.abb.2012.11.011. Epub 2012 Dec 3.
Cyclosporin A, a cyclic peptide produced by the fungus Tolypocladium inflatum, is a widely employed immunosuppressant drug. Its biosynthesis is strictly dependent on the action of the pyridoxal 5'-phosphate-dependent enzyme alanine racemase, which produces the d-alanine incorporated in the cyclic peptide. This enzyme has a different fold with respect to bacterial alanine racemases. The interest elicited by T. inflatum alanine racemase not only relies on its biotechnological relevance, but also on its evolutionary and structural similarity to the promiscuous enzymes serine hydroxymethyltransferase and threonine aldolase. The three enzymes represent a model of divergent evolution from an ancestral enzyme that was able to catalyse all the reactions of the modern enzymes. A protocol to express and purify with high yield recombinant T. inflatum alanine racemase was developed. The catalytic properties of the enzyme were characterized. Similarly to serine hydroxymethyltransferase and threonine aldolase, T. inflatum alanine racemase was able to catalyse retroaldol cleavage and transamination reactions. This observation corroborates the hypothesis of the common evolutionary origin of these enzymes. A three-dimensional model of T. inflatum alanine racemase was constructed on the basis of threonine aldolase crystal structure. The model helped rationalise the experimental data and explain the catalytic properties of the enzymes.
环孢菌素 A 是一种由土曲霉产生的环状肽，是一种广泛应用的免疫抑制剂药物。它的生物合成严格依赖于吡哆醛 5'-磷酸依赖的丙氨酸消旋酶的作用，该酶产生环状肽中掺入的 d-丙氨酸。与细菌丙氨酸消旋酶相比，该酶具有不同的折叠。土曲霉丙氨酸消旋酶之所以引起人们的兴趣，不仅是因为它具有生物技术相关性，还因为它在进化和结构上与混杂酶丝氨酸羟甲基转移酶和苏氨酸醛缩酶相似。这三种酶代表了从能够催化现代酶所有反应的祖先酶发散进化的模型。开发了一种表达和纯化高产量重组土曲霉丙氨酸消旋酶的方案。对酶的催化特性进行了表征。与丝氨酸羟甲基转移酶和苏氨酸醛缩酶类似，土曲霉丙氨酸消旋酶能够催化反醛裂解和转氨基反应。这一观察结果证实了这些酶共同进化起源的假设。基于苏氨酸醛缩酶晶体结构构建了土曲霉丙氨酸消旋酶的三维模型。该模型有助于合理化实验数据并解释酶的催化特性。
