Hermann Lucas, Dempwolff Felix, Steinchen Wieland, Freibert Sven-Andreas, Smits Sander H J, Seubert Andreas, Bremer Erhard
Faculty of Biology, Philipps-University Marburg, Marburg, Germany.
Department of Biochemistry and Synthetic Metabolism, Max-Planck-Institute for Terrestrial Microbiology, Marburg, Germany.
Front Microbiol. 2021 Dec 24;12:764731. doi: 10.3389/fmicb.2021.764731. eCollection 2021.
The compatible solutes ectoine and 5-hydroxyectoine are widely synthesized by bacteria as osmostress protectants. These nitrogen-rich tetrahydropyrimidines can also be exploited as nutrients by microorganisms. Many ectoine/5-hydroxyectoine catabolic gene clusters are associated with a regulatory gene (: ectoine nutrient utilization regulator) encoding a repressor protein belonging to the MocR/GabR sub-family of GntR-type transcription factors. Focusing on EnuR from the marine bacterium , we show that the dimerization of EnuR is mediated by its aminotransferase domain. This domain can fold independently from its amino-terminal DNA reading head and can incorporate pyridoxal-5'-phosphate (PLP) as cofactor. The covalent attachment of PLP to residue Lys302 of EnuR was proven by mass-spectrometry. PLP interacts with system-specific, ectoine and 5-hydroxyectoine-derived inducers: alpha-acetyldiaminobutyric acid (alpha-ADABA), and hydroxy-alpha-acetyldiaminobutyric acid (hydroxy-alpha-ADABA), respectively. These inducers are generated in cells actively growing with ectoines as sole carbon and nitrogen sources, by the EutD hydrolase and targeted metabolic analysis allowed their detection. EnuR binds these effector molecules with affinities in the low micro-molar range. Studies addressing the evolutionary conservation of EnuR, modelling of the EnuR structure, and docking experiments with the inducers provide an initial view into the cofactor and effector binding cavity. In this cavity, the two high-affinity inducers for EnuR, alpha-ADABA and hydroxy-alpha-ADABA, are positioned such that their respective primary nitrogen group can chemically interact with PLP. Purified EnuR bound with micro-molar affinity to a 48 base pair DNA fragment containing the sigma-70 type substrate-inducible promoter for the ectoine/5-hydroxyectoine importer and catabolic gene cluster. Consistent with the function of EnuR as a repressor, the core elements of the promoter overlap with two predicted EnuR operators. Our data lend themselves to a straightforward regulatory model for the initial encounter of EnuR-possessing ectoine/5-hydroxyectoine consumers with environmental ectoines and for the situation when the external supply of these compounds has been exhausted by catabolism.
相容性溶质四氢嘧啶和5-羟基四氢嘧啶被细菌广泛合成为渗透胁迫保护剂。这些富含氮的四氢嘧啶也可被微生物用作营养物质。许多四氢嘧啶/5-羟基四氢嘧啶分解代谢基因簇与一个调控基因（：四氢嘧啶营养利用调控因子）相关联，该调控基因编码一种属于GntR型转录因子的MocR/GabR亚家族的阻遏蛋白。以海洋细菌中的EnuR为研究对象，我们发现EnuR的二聚化是由其转氨酶结构域介导的。该结构域可以独立于其氨基末端的DNA读取头折叠，并且可以结合磷酸吡哆醛（PLP）作为辅因子。通过质谱法证实了PLP与EnuR的赖氨酸残基302共价结合。PLP分别与系统特异性的、源自四氢嘧啶和5-羟基四氢嘧啶的诱导剂相互作用：α-乙酰二氨基丁酸（α-ADABA）和羟基-α-乙酰二氨基丁酸（羟基-α-ADABA）。这些诱导剂是在以四氢嘧啶作为唯一碳源和氮源进行活跃生长的细胞中，由EutD水解酶产生的，靶向代谢分析使其得以检测。EnuR以低微摩尔范围内的亲和力结合这些效应分子。针对EnuR的进化保守性、EnuR结构建模以及与诱导剂的对接实验的研究，为辅助因子和效应分子结合腔提供了初步见解。在这个腔内，EnuR的两种高亲和力诱导剂α-ADABA和羟基-α-ADABA的定位方式使得它们各自的伯氨基能够与PLP发生化学相互作用。纯化后的EnuR以微摩尔亲和力与一个48个碱基对的DNA片段结合，该片段包含用于四氢嘧啶/5-羟基四氢嘧啶转运体和分解代谢基因簇的σ-70型底物诱导型启动子。与EnuR作为阻遏蛋白的功能一致，启动子的核心元件与两个预测的EnuR操纵子重叠。我们的数据有助于建立一个直接的调控模型，用于解释具有EnuR的四氢嘧啶/5-羟基四氢嘧啶消费者初次接触环境中的四氢嘧啶的情况，以及这些化合物的外部供应因分解代谢而耗尽时的情况。
