VanDrisse Chelsey M, Hentchel Kristy L, Escalante-Semerena Jorge C
Department of Microbiology, University of Georgia, Athens, Georgia, USA.
Department of Microbiology, University of Georgia, Athens, Georgia, USA
Appl Environ Microbiol. 2016 Nov 21;82(24):7041-7051. doi: 10.1128/AEM.02604-16. Print 2016 Dec 15.
Acetylation of small molecules is widespread in nature, and in some cases, cells use this process to detoxify harmful chemicals. Streptomyces species utilize a Gcn5 N-acetyltransferase (GNAT), known as Bar, to acetylate and detoxify a self-produced toxin, phosphinothricin (PPT), a glutamate analogue. Bar homologues, such as MddA from Salmonella enterica, acetylate methionine analogues such as methionine sulfoximine (MSX) and methionine sulfone (MSO), but not PPT, even though Bar homologues are annotated as PPT acetyltransferases. S. enterica was used as a heterologous host to determine whether or not putative PPT acetyltransferases from various sources could acetylate PPT, MSX, and MSO. In vitro and in vivo analyses identified substrates acetylated by putative PPT acetyltransferases from Deinococcus radiodurans (DR_1057 and DR_1182) and Geobacillus kaustophilus (GK0593 and GK2920). In vivo, synthesis of DR_1182, GK0593, and GK2920 blocked the inhibitory effects of PPT, MSX, and MSO. In contrast, DR_1057 did not detoxify any of the above substrates. Results of in vitro studies were consistent with the in vivo results. In addition, phylogenetic analyses were used to predict the functionality of annotated PPT acetyltransferases in Burkholderia xenovorans, Bacillus subtilis, Staphylococcus aureus, Acinetobacter baylyi, and Escherichia coli IMPORTANCE: The work reported here provides an example of the use of a heterologous system for the identification of enzyme function. Many members of this superfamily of proteins do not have a known function, or it has been annotated solely on the basis of sequence homology to previously characterized enzymes. The critical role of Gcn5 N-acetyltransferases (GNATs) in the modulation of central metabolic processes, and in controlling metabolic stress, necessitates approaches that can reveal their physiological role. The combination of in vivo, in vitro, and bioinformatics approaches reported here identified GNATs that can acetylate and detoxify phosphinothricin.
小分子的乙酰化在自然界中广泛存在，在某些情况下，细胞利用这一过程来解毒有害化学物质。链霉菌属物种利用一种名为Bar的Gcn5 N - 乙酰转移酶（GNAT）对自身产生的毒素草丁膦（PPT，一种谷氨酸类似物）进行乙酰化并解毒。Bar的同源物，如来自肠炎沙门氏菌的MddA，可对甲硫氨酸类似物如甲硫氨酸亚砜（MSX）和甲砜霉素（MSO）进行乙酰化，但不能对PPT进行乙酰化，尽管Bar同源物被注释为PPT乙酰转移酶。肠炎沙门氏菌被用作异源宿主，以确定来自各种来源的假定PPT乙酰转移酶是否能够对PPT、MSX和MSO进行乙酰化。体外和体内分析确定了来自耐辐射球菌（DR_1057和DR_1182）和嗜热栖热菌（GK0593和GK2920）的假定PPT乙酰转移酶所乙酰化的底物。在体内，DR_1182、GK0593和GK2920的合成阻断了PPT、MSX和MSO的抑制作用。相比之下，DR_1057不能对上述任何底物进行解毒。体外研究结果与体内结果一致。此外，系统发育分析被用于预测在伯克霍尔德氏菌、枯草芽孢杆菌、金黄色葡萄球菌、拜氏不动杆菌和大肠杆菌中注释的PPT乙酰转移酶的功能。重要性：本文报道的工作提供了一个使用异源系统鉴定酶功能的例子。这个蛋白质超家族的许多成员没有已知功能，或者仅仅是根据与先前表征的酶的序列同源性进行注释。Gcn5 N - 乙酰转移酶（GNATs）在调节中心代谢过程和控制代谢应激中的关键作用，需要能够揭示其生理作用的方法。本文报道的体内、体外和生物信息学方法的结合鉴定出了能够对草丁膦进行乙酰化并解毒的GNATs。
