Forlani Giuseppe, Nocek Boguslaw, Chakravarthy Srinivas, Joachimiak Andrzej
Department of Life Science and Biotechnology, University of FerraraFerrara, Italy.
Center for Structural Genomics of Infectious Diseases, University of ChicagoChicago, IL, United States.
Front Microbiol. 2017 Aug 2;8:1442. doi: 10.3389/fmicb.2017.01442. eCollection 2017.
In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of δ-pyrroline-5-carboxylate (P5C) catalyzed by P5C reductase (EC 1.5.1.2). In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidative stress conditions. Genome analysis of identifies the presence of four genes (, and ) that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that expression may be beneficial to satisfy an increased need for proline.
在大多数生物中，氨基酸脯氨酸是从谷氨酸和鸟氨酸开始合成的。在原核生物中，由于迄今仅在少数土壤和植物细菌中发现鸟氨酸环脱氨酶，这些途径共享最后一步，即由P5C还原酶（EC 1.5.1.2）催化的δ-吡咯啉-5-羧酸（P5C）的还原。在几个物种中，已报道了多种形式的P5C还原酶，这可能反映了脯氨酸的双重功能。除了作为蛋白质组成部分的常见作用外，脯氨酸确实还参与细胞对渗透和氧化应激条件的反应。对[物种名称]的基因组分析确定存在四个基因（[基因名称1]、[基因名称2]、[基因名称3]和[基因名称4]），基于生物信息学和系统发育研究，它们分别被定义为编码一种假定的P5C还原酶。在这里，我们描述了这四种亲和纯化蛋白的克隆、异源表达、功能分析和小角X射线散射研究。结果表明，其中两种，即ProI和ComER，失去了催化效率或发生了亚功能化。就ComER而言，这可能是由于失去了形成二聚体的能力，此前已表明二聚体是具有催化活性的P5C还原酶的基本结构特征。这两种活性酶的特性与ProG的组成性作用一致，并表明[基因名称]的表达可能有利于满足对脯氨酸增加的需求。
