ABSTRACT
PylB is a radical S-adenosyl-l-methionine (SAM) enzyme predicted to convert l-lysine into (3R)-3-methyl-d-ornithine, a precursor in the biosynthesis of the 22nd proteogenic amino acid pyrrolysine. This protein highly resembles that of the radical SAM tyrosine and tryptophan lyases, which activate their substrate by abstracting a H atom from the amino-nitrogen position. Here, combining in vitro assays, analytical methods, electron paramagnetic resonance spectroscopy, and theoretical methods, we demonstrated that instead, PylB activates its substrate by abstracting a H atom from the Cγ position of l-lysine to afford the radical-based ß-scission. Strikingly, we also showed that PylB catalyzes the reverse reaction, converting (3R)-3-methyl-d-ornithine into l-lysine and using catalytic amounts of the 5'-deoxyadenosyl radical. Finally, we identified significant in vitro production of 5'-thioadenosine, an unexpected shunt product that we propose to result from the quenching of the 5'-deoxyadenosyl radical species by the nearby [Fe4S4] cluster.
Subject(s)
Methionine , Ornithine/analogs & derivatives , S-Adenosylmethionine , S-Adenosylmethionine/metabolism , Lysine , Racemethionine , Electron Spin Resonance SpectroscopyABSTRACT
Naturally occurring protein nanocages like ferritin are self-assembled from multiple subunits. Because of their unique cage-like structure and biocompatibility, there is a growing interest in their biomedical use. A multipurpose and straightforward engineering approach does not exist for using nanocages to make drug-delivery systems by encapsulating hydrophilic or hydrophobic drugs and developing vaccines by surface functionalization with a protein like an antigen. Here, a versatile engineering approach is described by mimicking the HIV-1 Gap polyprotein precursor. Various PREcursors of nanoCages (PREC) are designed and created by linking two ferritin subunits via a flexible linker peptide containing a protease cleavage site. These precursors can have additional proteins at their N-terminus, and their protease cleavage generates ferritin-like nanocages named protease-induced nanocages (PINCs). It is demonstrated that PINC formation allows concurrent surface decoration with a protein and hydrophilic or hydrophobic drug encapsulation up to fourfold more than the amount achieved using other methods. The PINCs/Drug complex is stable and efficiently kills cancer cells. This work provides insight into the precursors' design rules and the mechanism of PINCs formation. The engineering approach and mechanistic insight described here will facilitate nanocages' applications in drug delivery or as a platform for making multifunctional therapeutics like mosaic vaccines.
Subject(s)
Ferritins , Humans , Ferritins/chemistry , Surface Properties , HIV-1 , Hydrophobic and Hydrophilic Interactions , Drug Delivery Systems/methods , Nanostructures/chemistry , Biomimetic Materials/chemistry , Biomimetics/methodsABSTRACT
[FeFe]-hydrogenases efficiently catalyze the reversible oxidation of molecular hydrogen. Their prowess stems from the intricate H-cluster, combining a [Fe4 S4 ] center with a binuclear iron center ([2Fe]H ). In the latter, each iron atom is coordinated by a CO and CN ligand, connected by a CO and an azadithiolate ligand. The synthesis of this active site involves a unique multiprotein assembly, featuring radical SAM proteins HydG and HydE. HydG initiates the transformation of L-tyrosine into cyanide and carbon monoxide to generate complex B, which is subsequently transferred to HydE to continue the biosynthesis of the [2Fe]H -subcluster. Due to its instability, complex B isolation for structural or spectroscopic characterization has been elusive thus far. Nevertheless, the use of a biomimetic analogue of complex B allowed circumvention of the need for the HydG protein during in vitro functional investigations, implying a similar structure for complex B. Herein, we used the HydE protein as a nanocage to encapsulate and stabilize the complex B product generated by HydG. Using X-ray crystallography, we successfully determined its structure at 1.3â Å resolution. Furthermore, we demonstrated that complex B is directly transferred from HydG to HydE, thus not being released into the solution post-synthesis, highlighting a transient interaction between the two proteins.
Subject(s)
Hydrogenase , Iron-Sulfur Proteins , Hydrogenase/metabolism , Ligands , Electron Spin Resonance Spectroscopy , Proteins/metabolism , Iron/chemistry , Ferrous Compounds/metabolism , Iron-Sulfur Proteins/chemistryABSTRACT
YabA negatively regulates initiation of DNA replication in low-GC Gram-positive bacteria. The protein exerts its control through interactions with the initiator protein DnaA and the sliding clamp DnaN. Here, we combined X-ray crystallography, X-ray scattering (SAXS), modeling and biophysical approaches, with in vivo experimental data to gain insight into YabA function. The crystal structure of the N-terminal domain (NTD) of YabA solved at 2.7 Å resolution reveals an extended α-helix that contributes to an intermolecular four-helix bundle. Homology modeling and biochemical analysis indicates that the C-terminal domain (CTD) of YabA is a small Zn-binding domain. Multi-angle light scattering and SAXS demonstrate that YabA is a tetramer in which the CTDs are independent and connected to the N-terminal four-helix bundle via flexible linkers. While YabA can simultaneously interact with both DnaA and DnaN, we found that an isolated CTD can bind to either DnaA or DnaN, individually. Site-directed mutagenesis and yeast-two hybrid assays identified DnaA and DnaN binding sites on the YabA CTD that partially overlap and point to a mutually exclusive mode of interaction. Our study defines YabA as a novel structural hub and explains how the protein tetramer uses independent CTDs to bind multiple partners to orchestrate replication initiation in the bacterial cell.
Subject(s)
Bacterial Proteins/metabolism , DNA Replication , DNA-Binding Proteins/metabolism , Multiprotein Complexes/metabolism , Amino Acid Motifs , Amino Acid Sequence , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Intracellular Space , Models, Molecular , Molecular Sequence Data , Mutation , Position-Specific Scoring Matrices , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Protein Interaction Mapping/methods , Protein Multimerization , Protein Transport , Sequence Alignment , Structure-Activity Relationship , Zinc/metabolismABSTRACT
Replicative helicases are essential ATPases that unwind DNA to initiate chromosomal replication. While bacterial replicative DnaB helicases are hexameric, Helicobacter pylori DnaB (HpDnaB) was found to form double hexamers, similar to some archaeal and eukaryotic replicative helicases. Here we present a structural and functional analysis of HpDnaB protein during primosome formation. The crystal structure of the HpDnaB at 6.7 Å resolution reveals a dodecameric organization consisting of two hexamers assembled via their N-terminal rings in a stack-twisted mode. Using fluorescence anisotropy we show that HpDnaB dodecamer interacts with single-stranded DNA in the presence of ATP but has a low DNA unwinding activity. Multi-angle light scattering and small angle X-ray scattering demonstrate that interaction with the DnaG primase helicase-binding domain dissociates the helicase dodecamer into single ringed primosomes. Functional assays on the proteins and associated complexes indicate that these single ringed primosomes are the most active form of the helicase for ATP hydrolysis, DNA binding and unwinding. These findings shed light onto an activation mechanism of HpDnaB by the primase that might be relevant in other bacteria and possibly other organisms exploiting dodecameric helicases for DNA replication.
Subject(s)
Bacterial Proteins/chemistry , DNA Primase/metabolism , DnaB Helicases/chemistry , Helicobacter pylori/enzymology , Adenosine Triphosphatases/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA/metabolism , DNA Primase/chemistry , DNA, Single-Stranded/metabolism , DnaB Helicases/genetics , DnaB Helicases/metabolism , Enzyme Activation , Models, Molecular , Protein Multimerization , Protein Structure, TertiaryABSTRACT
Herein, we describe an uncommon example of a manganese-thiolate complex, which is capable of activating dioxygen and catalyzing its two-electron reduction to generate H2O2. The structurally characterized dimercapto-bridged Mn(II) dimer [Mn(II)2(LS)(LSH)]ClO4 (Mn(II)2SH) is formed by reaction of the LS ligand (2,2'-(2,2'-bipyridine-6,6'-diyl)bis(1,1-diphenylethanethiolate)) with Mn(II). The unusual presence of a pendant thiol group bound to one Mn(II) ion in Mn(II)2SH is evidenced both in the solid state and in solution. The Mn(II)2SH complex reacts with dioxygen in CH3CN, leading to the formation of a rare mono-µ-hydroxo dinuclear Mn(III) complex, [(Mn(III)2(LS)2(OH)]ClO4 (Mn(III)2OH), which has also been structurally characterized. When Mn(II)2SH reacts with O2 in the presence of a proton source, 2,6-lutidinium tetrafluoroborate (up to 50 equiv), the formation of a new Mn species is observed, assigned to a bis-µ-thiolato dinuclear Mn(III) complex with two terminal thiolate groups (Mn(III)2), with the concomitant production of H2O2 up to â¼40% vs Mn(II)2SH. The addition of a catalytic amount of Mn(II)2SH to an air-saturated solution of MenFc (n = 8 or 10) and 2,6-lutidinium tetrafluoroborate results in the quantitative and efficient oxidation of MenFc by O2 to afford the respective ferrocenium derivatives (MenFc(+), with n = 8 or 10). Hydrogen peroxide is mainly produced during the catalytic reduction of dioxygen with 80-84% selectivity, making the Mn(II)2SH complex a rare Mn-based active catalyst for two-electron O2 reduction.
ABSTRACT
Quinolinate synthase (NadA) is a Fe4S4 cluster-containing dehydrating enzyme involved in the synthesis of quinolinic acid (QA), the universal precursor of the essential nicotinamide adenine dinucleotide (NAD) coenzyme. A previously determined apo NadA crystal structure revealed the binding of one substrate analog, providing partial mechanistic information. Here, we report on the holo X-ray structure of NadA. The presence of the Fe4S4 cluster generates an internal tunnel and a cavity in which we have docked the last precursor to be dehydrated to form QA. We find that the only suitably placed residue to initiate this process is the conserved Tyr21. Furthermore, Tyr21 is close to a conserved Thr-His-Glu triad reminiscent of those found in proteases and other hydrolases. Our mutagenesis data show that all of these residues are essential for activity and strongly suggest that Tyr21 deprotonation, to form the reactive nucleophilic phenoxide anion, is mediated by the triad. NadA displays a dehydration mechanism significantly different from the one found in archetypical dehydratases such as aconitase, which use a serine residue deprotonated by an oxyanion hole. The X-ray structure of NadA will help us unveil its catalytic mechanism, the last step in the understanding of NAD biosynthesis.
Subject(s)
Hydrolases/chemistry , Multienzyme Complexes/chemistry , Tyrosine/chemistry , Crystallography, X-Ray , Dehydration , Hydrolases/metabolism , Models, Molecular , Molecular Structure , Multienzyme Complexes/metabolism , Tyrosine/metabolismABSTRACT
Flaviviruses are a group of human pathogens causing severe encephalitic or hemorrhagic diseases that include West Nile, dengue and yellow fever viruses. Here, using X-ray crystallography we have defined the structure of the flavivirus cross-reactive antibody E53 that engages the highly conserved fusion loop of the West Nile virus envelope glycoprotein. Using cryo-electron microscopy, we also determined that E53 Fab binds preferentially to spikes in noninfectious, immature flavivirions but is unable to bind significantly to mature virions, consistent with the limited solvent exposure of the epitope. We conclude that the neutralizing impact of E53 and likely similar fusion-loop-specific antibodies depends on its binding to the frequently observed immature component of flavivirus particles. Our results elucidate how fusion-loop antibodies, which comprise a significant fraction of the humoral response against flaviviruses, can function to control infection without appreciably recognizing mature virions. As these highly cross-reactive antibodies are often weakly neutralizing they also may contribute to antibody-dependent enhancement and flavi virus pathogenesis thereby complicating development of safe and effective vaccines.
Subject(s)
Antibodies, Viral/immunology , Flavivirus/immunology , Flavivirus/ultrastructure , Antibodies, Viral/chemistry , Cryoelectron Microscopy , Crystallography, X-Ray , Flavivirus/chemistry , Glycoproteins/chemistry , Glycoproteins/immunology , Models, Molecular , Protein Structure, Secondary , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/immunologyABSTRACT
Understanding the interaction of a protein with a relevant ligand is crucial for the design of an artificial metalloenzyme. Our own interest is focused on the synthesis of artificial monooxygenases. In an initial effort, we have used the periplasmic nickel-binding protein NikA from Escherichia coli and iron complexes in which N(2)Py(2) ligands (where Py is pyridine) have been varied in terms of charge, aromaticity, and size. Six "NikA/iron complex" hybrids have been characterized by X-ray crystallography, and their interactions and solution properties have been studied. The hybrids are stable as indicated by their K (d) values, which are all in the micromolar range. The X-ray structures show that the ligands interact with NikA through salt bridges with arginine residues and π-stacking with a tryptophan residue. We have further characterized these interactions using quantum mechanical calculations and determined that weak CH/π hydrogen bonds finely modulate the stability differences between hybrids. We emphasize the important role of the tryptophan residues. Thus, our study aims at the complete characterization of the factors that condition the interaction of an artificial ligand and a protein and their implications for catalysis. Besides its potential usefulness in the synthesis of artificial monooxygenases, our approach should be generally applicable in the field of artificial metalloenzymes.
Subject(s)
ATP-Binding Cassette Transporters/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/chemistry , Iron Compounds/chemistry , Metalloproteins/chemistry , Nickel/metabolism , ATP-Binding Cassette Transporters/metabolism , Biomimetic Materials/chemistry , Biomimetic Materials/metabolism , Crystallography, X-Ray , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Iron Compounds/metabolism , Ligands , Metalloproteins/metabolism , Models, Molecular , Pyridines/chemistry , Pyridines/metabolism , Water/chemistryABSTRACT
Paramecium bursaria Chlorella virus-1 is an icosahedrally shaped, 1,900-A-diameter virus that infects unicellular eukaryotic green algae. A 5-fold symmetric, 3D reconstruction using cryoelectron microscopy images has now shown that the quasiicosahedral virus has a unique vertex, with a pocket on the inside and a spike structure on the outside of the capsid. The pocket might contain enzymes for use in the initial stages of infection. The unique vertex consists of virally coded proteins, some of which have been identified. Comparison of shape, size, and location of the spike with similar features in bacteriophages T4 and P22 suggests that the spike might be a cell-puncturing device. Similar asymmetric features may have been missed in previous analyses of many other viruses that had been assumed to be perfectly icosahedral.
Subject(s)
Phycodnaviridae/ultrastructure , Capsid/ultrastructure , Cryoelectron MicroscopyABSTRACT
This Review focuses on the structure-function relationship of radical S-adenosyl-l-methionine (SAM) enzymes involved in the assembly of metallocofactors corresponding to the active sites of [FeFe]-hydrogenase and nitrogenase [MoFe]-protein. It does not claim to correspond to an extensive review on the assembly machineries of these enzyme active sites, for which many good reviews are already available, but instead deals with the contribution of structural data to the understanding of their chemical mechanism (Buren et al. Chem. Rev.2020, 142 ( (25), ) 11006-11012; Britt et al. Chem. Sci.2020, 11 ( (38), ), 10313-10323). Hence, we will present the history and current knowledge about the radical SAM maturases HydE, HydG, and NifB as well as what, in our opinion, should be done in the near future to overcome the existing barriers in our understanding of this fascinating chemistry that intertwine organic radicals and organometallic complexes.
ABSTRACT
Metalloproteins are involved in key cell processes such as photosynthesis, respiration, and oxygen transport. However, the presence of transition metals (notably iron as a component of [Fe-S] clusters) often makes these proteins sensitive to oxygen-induced degradation. Consequently, their study usually requires strict anaerobic conditions. Although X-ray crystallography has been the method of choice for solving macromolecular structures for many years, recently electron microscopy has also become an increasingly powerful structure-solving technique. We have used our previous experience with cryo-crystallography to develop a method to prepare cryo-EM grids in an anaerobic chamber and have applied it to solve the structures of apoferritin and the 3 [Fe4S4]-containing pyruvate ferredoxin oxidoreductase (PFOR) at 2.40 Å and 2.90 Å resolution, respectively. The maps are of similar quality to the ones obtained under air, thereby validating our method as an improvement in the structural investigation of oxygen-sensitive metalloproteins by cryo-EM.
Subject(s)
Metalloproteins , Apoferritins , Cryoelectron Microscopy/methods , Crystallography, X-Ray , OxygenABSTRACT
The nitrogenase MoFe protein contains two different FeS centers, the P-cluster and the iron-molybdenum cofactor (FeMo-co). The former is a [Fe8S7] center responsible for conveying electrons to the latter, a [MoFe7S9C-(R)-homocitrate] species, where N2 reduction takes place. NifB is arguably the key enzyme in FeMo-co assembly as it catalyzes the fusion of two [Fe4S4] clusters and the insertion of carbide and sulfide ions to build NifB-co, a [Fe8S9C] precursor to FeMo-co. Recently, two crystal structures of NifB proteins were reported, one containing two out of three [Fe4S4] clusters coordinated by the protein which is likely to correspond to an early stage of the reaction mechanism. The other one was fully complemented with the three [Fe4S4] clusters (RS, K1 and K2), but was obtained at lower resolution and a satisfactory model was not obtained. Here we report improved processing of this crystallographic data. At odds with what was previously reported, this structure contains a unique [Fe8S8] cluster, likely to be a NifB-co precursor resulting from the fusion of K1- and K2-clusters. Strikingly, this new [Fe8S8] cluster has both a structure and coordination sphere geometry reminiscent of the fully reduced P-cluster (PN-state) with an additional µ2-bridging sulfide ion pointing toward the RS cluster. Comparison of available NifB structures further unveils the plasticity of this protein and suggests how ligand reorganization would accommodate cluster loading and fusion in the time-course of NifB-co synthesis.
ABSTRACT
Phospholipase D (PLD) is a ubiquitous enzyme that cleaves the distal phosphoester bond of phospholipids generating phosphatidic acid (PA). In plants, PA is involved in numerous cell responses triggered by stress. Similarly, in mammals, PA is also a second messenger involved in tumorigenesis. PLD is nowadays considered as a therapeutic target and blocking its activity with specific inhibitors constitutes a promising strategy to treat cancers. Starting from already described PLD inhibitors, this study aims to investigate the effect of their structural modifications on the enzyme's activity, as well as identifying new potent inhibitors of eukaryotic PLDs. Being able to purify the plant PLD from Vigna unguiculata (VuPLD), we obtained a SAXS model of its structure. We then used a fluorescence-based test suitable for high-throughput screening to review the effect of eukaryotic PLD inhibitors described in the literature. In this regard, we found that only few molecules were in fact able to inhibit VuPLD and we confirmed that vanadate is the most potent of all with an IC50 around 58 µM. Moreover, the small-scale screening of a chemical library of 3120 compounds allowed us to optimize the different screening's steps and paved the way towards the discovery of new potent inhibitors.
Subject(s)
Drug Evaluation, Preclinical , Enzyme Inhibitors/analysis , Enzyme Inhibitors/pharmacology , Phospholipase D/antagonists & inhibitors , Alcohols/pharmacology , High-Throughput Screening Assays , Humans , Hydrolysis , Phospholipase D/metabolism , Salts/pharmacology , Scattering, Small Angle , Vanadates/pharmacology , Vigna/enzymology , X-Ray DiffractionABSTRACT
Escherichia coli and related bacteria require nickel for the synthesis of hydrogenases, enzymes involved in hydrogen oxidation and proton reduction. Nickel transport to the cytoplasm depends on five proteins, NikA-E. We have previously reported the three-dimensional structure of the soluble periplasmic nickel transporter NikA in a complex with FeEDTA(H 2O) (-). We have now determined the structure of EDTA-free NikA and have found that it binds a small organic molecule that contributes three ligands to the coordination of a transition metal ion. Unexpectedly, His416, which was far from the metal-binding site in the FeEDTA(H 2O) (-)-NikA complex, becomes the fourth observed ligand to the metal. The best match to the omit map electron density is obtained for butane-1,2,4-tricarboxylate (BTC). Our attempts to obtain a BTC-Ni-NikA complex using apo protein and commercial reagents resulted in nickel-free BTC-NikA. Overall, our results suggest that nickel transport in vivo requires a specific metallophore that may be BTC.
Subject(s)
ATP-Binding Cassette Transporters/chemistry , Chelating Agents/chemistry , Escherichia coli Proteins/chemistry , Nickel , Periplasm , Chelating Agents/metabolism , Crystallography, X-Ray , Escherichia coli Proteins/metabolism , Nickel/metabolism , Periplasm/metabolism , Protein Structure, Secondary/physiologySubject(s)
Biocompatible Materials/metabolism , Mixed Function Oxygenases/metabolism , ATP-Binding Cassette Transporters/chemistry , Binding Sites , Biocompatible Materials/chemistry , Catalysis , Catalytic Domain , Crystallography, X-Ray , Escherichia coli Proteins/chemistry , Mixed Function Oxygenases/chemistry , Molecular Docking Simulation , Oxidation-Reduction , Substrate Specificity , Sulfides/chemistryABSTRACT
The X-ray structure of an aerated achiral Ru-papain conjugate has revealed the hydroxylation of two tyrosine residues found near the ruthenium ion. The most likely mechanism involves a ruthenium-bound superoxide as the reactive species responsible for the first hydroxylation and the resulting high valent Ru(iv)[double bond, length as m-dash]O species for the second one.
Subject(s)
Papain/chemistry , Papain/metabolism , Ruthenium/chemistry , Ruthenium/metabolism , Superoxides/chemistry , Tyrosine/chemistry , Crystallography, X-Ray , Hydroxylation , Models, Molecular , Molecular Structure , Oxidation-Reduction , Protein ConformationABSTRACT
The production of pure enzymes in high quantities is a proven strategy to study the catalytic mechanism as well as the solving of structure at the atomic scale for therapeutic or industrial purposes. Phospholipase D (PLD, EC 3.1.4.4) is found in a wide majority of living organisms and has been shown to be involved in signal transduction, vesicle trafficking, and membrane metabolism processes. Located at the membrane-cytoplasm interface, plant PLDs are soluble but also bear an evident hydrophobic aspect making challenging its expression and its purification in large quantity. So far there is no high-resolution three-dimensional structure for a eukaryotic PLD. The protocols herein describe the cloning of the eukaryotic recombinant PLDα of Vigna unguiculata (cowpea) into the yeast expression system Pichia pastoris and its two-step purification process. This allowed us to purify to homogeneity hundreds of micrograms of highly pure protein to conduct in fine structural studies.
Subject(s)
Phospholipase D/genetics , Phospholipase D/metabolism , Pichia/genetics , Recombinant Proteins , Vigna/enzymology , Vigna/genetics , Chromatography/methods , Cloning, Molecular , Enzyme Activation , Gene Expression , Phospholipase D/chemistry , Phospholipase D/isolation & purification , Plasmids , Transformation, GeneticABSTRACT
The more severe strains of the bacterial human pathogen Helicobacter pylori produce a type IV secretion system (cagT4SS) to inject the oncoprotein cytotoxin-associated gene A (CagA) into gastric cells. This syringe-like molecular apparatus is prolonged by an external pilus that exploits integrins as receptors to mediate the injection of CagA. The molecular determinants of the interaction of the cagT4SS pilus with the integrin ectodomain are still poorly understood. In this study, we have used surface plasmon resonance (SPR) to generate a comprehensive analysis of the protein-protein interactions between purified CagA, CagL, CagI, CagY repeat domain II (CagYRRII ), CagY C-terminal domain (CagYB10 ) and integrin α5ß1 ectodomain (α5ß1E ) or headpiece domain (α5ß1HP ). We found that CagI, CagA, CagL and CagYB10 but not CagYRRII were able to interact with α5ß1E with affinities similar to the one observed for α5ß1E interaction with its physiological ligand fibronectin. We further showed that integrin activation and its associated conformational change increased CagA, CagL and CagYB10 affinities for the receptor. Furthermore, CagI did not interact with integrin unless the receptor was in open conformation. CagI, CagA but not CagL and CagYB10 interacted with the α5ß1HP . Our SPR study also revealed novel interactions between CagA and CagL, CagA and CagYB10 , and CagA and CagI. Altogether, our data map the network of interactions between host-cell α5ß1 integrin and the cagT4SS proteins and suggest that activation of the receptor promotes interactions with the secretion apparatus and possibly CagA injection.
Subject(s)
Bacterial Proteins/metabolism , Helicobacter pylori/metabolism , Integrin alpha5beta1/metabolism , Protein Interaction Mapping/methods , Type IV Secretion Systems/metabolism , Animals , Antigens, Bacterial/chemistry , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , CHO Cells , Cricetinae , Cricetulus , Helicobacter pylori/genetics , Humans , Integrin alpha5beta1/chemistry , Integrin alpha5beta1/genetics , Protein Binding , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Scattering, Small Angle , Surface Plasmon Resonance , Type IV Secretion Systems/chemistry , Type IV Secretion Systems/genetics , X-Ray DiffractionABSTRACT
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 O2â¢- and H2O2 are key players in complex signaling networks and defense. A well-studied example is the production of O2â¢- 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.IMPORTANCE 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.