The NOX Family of Proteins Is Also Present in Bacteria.
The NOX Family of Proteins Is Also Present in Bacteria.
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DOI：
10.1128/mbio.01487-17
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发表时间：
2017-11-07
期刊：
影响因子：
6.4
通讯作者：
Dupuy J
中科院分区：
文献类型：
--
作者：
Hajjar C;Cherrier MV;Dias Mirandela G;Petit-Hartlein I;Stasia MJ;Fontecilla-Camps JC;Fieschi F;Dupuy J
到目前为止，跨膜NADPH氧化酶(NOX)酶只在真核生物中得到了鉴定。在大多数这样的生物体中，它们将分子氧还原为超氧化物，根据其他结构域的存在，被称为NOX或双重氧化酶(DUOX)。活性氧(ROS)，包括超氧化物，传统上被认为是有氧代谢的意外有毒副产物。然而，在过去的十年中，O2·−和H2O2都是复杂的信号网络和防御中的关键角色，这一点已经变得明显。一个被充分研究的例子是吞噬细胞在杀菌呼吸爆发过程中产生O2·−；这种产生是由NOX2催化的。在这里，我们设计并应用了一种新的算法来在基因组数据库中搜索额外的NOX基因。这一过程使我们能够从细菌中发现大约23%的新序列(相对于作者识别的与NOX相关的序列的数量)，我们已经将这些序列添加到现有的真核细胞NOX家族中，并用于构建扩展的系统发育树。我们克隆并高效表达了肺炎链球菌的NOx基因，证实其编码NADPH氧化酶。肺炎链球菌NOX蛋白膜(SpNOX)与真核生物有许多共同的特性，如对NADPH和黄素腺嘌呤二核苷酸的亲和力、超氧化物歧化酶和二苯基碘抑制、抗氰化物、耗氧和产生超氧化物。传统上，真核生物中的NOX酶与与多细胞有关的功能有关。因此，在细菌世界中发现了一个与NOX相关的酶大家族，这就带来了关于它们在这种新的生物学背景下所扮演的角色的有趣的问题。NADPH氧化酶(NOX)在细菌中的研究尚未见报道。在这里，我们进行了计算和实验研究，首次对原核生物NOX进行了表征。在显示NOX特征的996个原核蛋白中，我们最初选择、克隆并过度表达了其中的四个蛋白。随后，在初步测试的基础上，我们将重点放在了链球菌SpNOX上，它与NOX酶的参照模型NOX2有许多共同的生化特征。我们的工作首次使研究这一重要酶家族的纯形式成为可能，允许以前所未有的方式进行生物物理和分子表征。在其他膜蛋白家族方面的类似进展导致了新的结构、进一步的机制研究和抑制剂的改进。此外，这些新描述的细菌酶的生物学功能在不久的将来肯定会被发现。
Transmembrane NADPH oxidase (NOX) enzymes have been so far only characterized in eukaryotes. In most of these organisms, they reduce molecular oxygen to superoxide and, depending on the presence of additional domains, are called NOX or dual oxidases (DUOX). Reactive oxygen species (ROS), including superoxide, have been traditionally considered accidental toxic by-products of aerobic metabolism. However, during the last decade it has become evident that both O2•− and H2O2 are key players in complex signaling networks and defense. A well-studied example is the production of O2•− during the bactericidal respiratory burst of phagocytes; this production is catalyzed by NOX2. Here, we devised and applied a novel algorithm to search for additional NOX genes in genomic databases. This procedure allowed us to discover approximately 23% new sequences from bacteria (in relation to the number of NOX-related sequences identified by the authors) that we have added to the existing eukaryotic NOX family and have used to build an expanded phylogenetic tree. We cloned and overexpressed the identified nox gene from Streptococcus pneumoniae and confirmed that it codes for an NADPH oxidase. The membrane of the S. pneumoniae NOX protein (SpNOX) shares many properties with its eukaryotic counterparts, such as affinity for NADPH and flavin adenine dinucleotide, superoxide dismutase and diphenylene iodonium inhibition, cyanide resistance, oxygen consumption, and superoxide production. Traditionally, NOX enzymes in eukaryotes are related to functions linked to multicellularity. Thus, the discovery of a large family of NOX-related enzymes in the bacterial world brings up fascinating questions regarding their role in this new biological context. NADPH oxidase (NOX) enzymes have not yet been reported in bacteria. Here, we carried out computational and experimental studies to provide the first characterization of a prokaryotic NOX. Out of 996 prokaryotic proteins showing NOX signatures, we initially selected, cloned, and overexpressed four of them. Subsequently, and based on preliminary testing, we concentrated our efforts on Streptococcus SpNOX, which shares many biochemical characteristics with NOX2, the referent model of NOX enzymes. Our work makes possible, for the first time, the study of pure forms of this important family of enzymes, allowing for biophysical and molecular characterization in an unprecedented way. Similar advances regarding other membrane protein families have led to new structures, further mechanistic studies, and the improvement of inhibitors. In addition, biological functions of these newly described bacterial enzymes will be certainly discovered in the near future.