RidA proteins prevent metabolic damage inflicted by PLP-dependent dehydratases in all domains of life.
RidA proteins prevent metabolic damage inflicted by PLP-dependent dehydratases in all domains of life.
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DOI：
10.1128/mbio.00033-13
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发表时间：
2013-02-05
期刊：
影响因子：
6.4
通讯作者：
Downs DM
中科院分区：
文献类型：
--
作者：
Lambrecht JA;Schmitz GE;Downs DM
5‘-磷酸吡哆醛(PLP)是一种由各种生物合成的辅酶。与这里报道的工作相关的是PLP依赖的苏氨酸/丝氨酸脱水酶的机制，它产生反应性的烯胺/亚胺中间体，这些中间体被RIDA家族的成员转化为酮酸。鼠伤寒沙门氏菌LT2的Rida蛋白是这个广泛保守的蛋白质家族(以前称为YjgF/YER057c/UK114)的创始成员。最近发现RIDA蛋白是一种烯胺脱氨酶。在这里，我们展示了在没有RIDA蛋白的情况下，烯胺的破坏潜力。值得注意的是，缺乏RIDA的肠杆菌菌株降低了PLP依赖的转氨酶B酶ILVE的活性，ILVE是一种参与支链氨基酸生物合成的酶。我们在体外重组了依赖苏氨酸/丝氨酸脱水酶(ILVA)对ILVE的抑制，表明体外系统反映了体内RIDA功能的机制，并表明RIDA蛋白从生命的各个领域阻止了对ILVE的抑制。我们的结论是，2-氨基丙烯酸酯(2AA)抑制代表了一种新的代谢损伤类型，这一发现为无处不在的RIDA家族在预防2AA损伤中的作用提供了重要的生理背景。破坏新陈代谢成分的外部压力会扰乱细胞功能，影响生长。如果内源性代谢物是反应性的并持续存在于细胞环境中，预计也会出现类似的后果。在这里，我们表明，代谢中间体2-氨基丙烯酸酯(2AA)如果允许异常积累，会导致显着的细胞损伤。此外，我们还表明，广泛保守的蛋白质RIDA通过促进2AA转化为稳定的代谢物来防止这种积累。这项工作证明，反应性代谢物2AA以前被认为在细胞中是无害的，因为它在水溶液中的半衰期很短，可以在细胞环境中存活足够长的时间来造成损害。这项工作为活性代谢物在体内的作用和持久性提供了见解，并表明RIDA家族蛋白质能够防止由于PLP依赖的化学作用而产生的活性中间体造成的损害。
Pyridoxal 5′-phosphate (PLP) is a coenzyme synthesized by all forms of life. Relevant to the work reported here is the mechanism of the PLP-dependent threonine/serine dehydratases, which generate reactive enamine/imine intermediates that are converted to keto acids by members of the RidA family of enzymes. The RidA protein of Salmonella enterica serovar Typhimurium LT2 is the founding member of this broadly conserved family of proteins (formerly known as YjgF/YER057c/UK114). RidA proteins were recently shown to be enamine deaminases. Here we demonstrate the damaging potential of enamines in the absence of RidA proteins. Notably, S. enterica strains lacking RidA have decreased activity of the PLP-dependent transaminase B enzyme IlvE, an enzyme involved in branched-chain amino acid biosynthesis. We reconstituted the threonine/serine dehydratase (IlvA)-dependent inhibition of IlvE in vitro, show that the in vitro system reflects the mechanism of RidA function in vivo, and show that IlvE inhibition is prevented by RidA proteins from all domains of life. We conclude that 2-aminoacrylate (2AA) inhibition represents a new type of metabolic damage, and this finding provides an important physiological context for the role of the ubiquitous RidA family of enamine deaminases in preventing damage by 2AA. External stresses that disrupt metabolic components can perturb cellular functions and affect growth. A similar consequence is expected if endogenously generated metabolites are reactive and persist in the cellular environment. Here we show that the metabolic intermediate 2-aminoacrylate (2AA) causes significant cellular damage if allowed to accumulate aberrantly. Furthermore, we show that the widely conserved protein RidA prevents this accumulation by facilitating conversion of 2AA to a stable metabolite. This work demonstrates that the reactive metabolite 2AA, previously considered innocuous in the cell due to a short half-life in aqueous solution, can survive in the cellular environment long enough to cause damage. This work provides insights into the roles and persistence of reactive metabolites in vivo and shows that the RidA family of proteins is able to prevent damage caused by a reactive intermediate that is created as a consequence of PLP-dependent chemistry.