Lessons from the structure of a bacterial homologue
During bacterial infections, neutrophils play a crucial role in defending the body by engulfing and destroying microorganisms at the site of infection. They achieve this by producing large quantities of superoxide ions and other derived products like hydrogen peroxide (H₂O₂) and hypochlorous acid (HOCl), collectively known as Reactive Oxygen Species (ROS). This production is facilitated by the membrane-bound enzyme NADPH oxidase (NOX) located within the phagosomal membrane.
Beyond their established role in the immune system—discovered a century ago—various isoforms of the NOX enzyme have been identified in many other organs. In these tissues, ROS serve as either signaling molecules or reactants in biosynthetic processes, depending on their physiological and tissue-specific contexts. NOX enzymes catalyze the electron transfer from cytosolic NAD(P)H to external oxygen (O₂) through internal electron relays involving a flavin cofactor (FAD) and two heme-b groups within the protein (Figure 1).
Given the potential damage ROS can inflict, their production needs to be tightly regulated. In eukaryotic systems, the activation of NOX enzymes typically depends on their assembly with cytosolic factors. However, the precise molecular mechanisms of this activation have been elusive, particularly since the necessary redox cofactors and substrate binding sites are present within the NOX enzymes even before they form the active complex. NOX enzymes have become important drug targets for the pharmaceutical industry, but progress has been slow due to limited structural understanding of their activation. Recent structures of human NOX isoforms in their resting states have not fully elucidated the initiation of electron transfer, leaving the process incompletely understood.
The Fieschi group at the Institute of Structural Biology (IBS) has been studying this family of membrane enzymes and their cytosolic partners for over two decades. They identified several years ago that some prokaryotic NOX homologs are constitutively active, meaning they do not rely on additional factors for their activation. One such homolog, SpNOX from Streptococcus pneumoniae, serves as a robust model to illuminate the molecular features required for an active state.
Both the full-length SpNOX and its soluble dehydrogenase (DH) domain, which contains the NAD(P)H binding site and the FAD cofactor, have been crystallized and their structure solved in collaboration with the HTX lab (EMBL) (Figure 2) [3]. Enzymatic studies revealed that the isolated DH domain is capable of catalyzing the transfer of hydride ions from NAD(P)H to FAD independently and that this step is rate-limiting for the entire enzyme [3]. A critical phenylalanine residue in the active site facilitates the access of NADPH to the flavin ring. The FAD binding at the interface between the DH and transmembrane (TM) domains allows electron transfer to the heme groups within the membrane [3].
Comparing the structure of the constitutively active SpNOX with the resting states of related human enzymes reveals a key difference: the relative positioning of the NAD(P)H binding site. In SpNOX, NAD(P)H is positioned close enough to facilitate hydride transfer to FAD. In contrast, human NOX enzymes exhibit a 10 Å distance between the redox centers, which impedes such transfer (Figure 2B) [3]. This research suggests that the activation of human NOX enzymes through cytosolic factor assembly likely induces a conformational change within the DH domain, bringing NAD(P)H closer to FAD and transitioning the enzyme from a resting to an active state.
This new mechanistic insight will aid in developing strategies for drug design aimed at better controlling ROS-producing enzymes in various physiological contexts, from immune response and hormone synthesis to cardiovascular regulation.
I. Petit-Hartlein, (IBS), A. Vermot (IBS, now EMBL), J.A. Marquez(EMBL), F. Fieschi (IBS)
[1] Vermot A, Petit-Härtlein I, Smith SME, Fieschi F (2021) Antioxidants, 10, 890
[2] Hajjar C, Cherrier MV, Dias Mirandela G, Petit-Hartlein I, et al. (2017) mBio, 8, e01487-17
[3] Petit-Hartlein I, Vermot A, Thepaut M, Humm AS, et al. (2024) Elife, 13, RP93759
Figure 1: Scheme of the core domain conserved amongst all NOX family members. As a function of the specific cellular/tissular environment, the ROS generated can have several fate illustrating the wide pleiotropic impact that these enzymes can have depending on their location.
Figure 2: A) Structure of SpNOX from Streptococcus pneumoniae. B) Structural comparison of the DH domain of SpNOX with that of the human neutrophil NOX enzyme with NADPH anchored in its respective binding sites.
