Helicobacter pylori adhesin HopQ disrupts trans dimerization in human CEACAMs.

The human gastric pathogen Helicobacter pylori is a major causative agent of gastritis, peptic ulcer disease, and gastric cancer. As part of its adhesive lifestyle, the bacterium targets members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family by the conserved outer membrane adhesin HopQ. The HopQ-CEACAM1 interaction is associated with inflammatory responses and enables the intracellular delivery and phosphorylation of the CagA oncoprotein via a yet unknown mechanism. Here, we generated crystal structures of HopQ isotypes I and II bound to the N-terminal domain of human CEACAM1 (C1ND) and elucidated the structural basis of H. pylori specificity toward human CEACAM receptors. Both HopQ alleles target the ß-strands G, F, and C of C1ND, which form the trans dimerization interface in homo- and heterophilic CEACAM interactions. Using SAXS, we show that the HopQ ectodomain is sufficient to induce C1ND monomerization and thus providing H. pylori a route to influence CEACAM-mediated cell adherence and signaling events.

Moco Carrier and Binding Proteins.

The molybdenum cofactor (Moco) is the active site prosthetic group found in numerous vitally important enzymes (Mo-enzymes), which predominantly catalyze 2 electron transfer reactions. Moco is synthesized by an evolutionary old and highly conserved multi-step pathway, whereby the metal insertion reaction is the ultimate reaction step here. Moco and its intermediates are highly sensitive towards oxidative damage and considering this, they are believed to be permanently protein bound during synthesis and also after Moco maturation. In plants, a cellular Moco transfer and storage system was identified, which comprises proteins that are capable of Moco binding and release but do not possess a Moco-dependent enzymatic activity. The first protein described that exhibited these properties was the Moco carrier protein (MCP) from the green alga Chlamydomonas reinhardtii. However, MCPs and similar proteins have meanwhile been described in various plant species. This review will summarize the current knowledge of the cellular Moco distribution system.

Function of Molybdenum Insertases.

For most organisms molybdenum is essential for life as it is found in the active site of various vitally important molybdenum dependent enzymes (Mo-enzymes). Here, molybdenum is bound to a pterin derivative called molybdopterin (MPT), thus forming the molybdenum cofactor (Moco). Synthesis of Moco involves the consecutive action of numerous enzymatic reaction steps, whereby molybdenum insertases (Mo-insertases) catalyze the final maturation step, i.e., the metal insertion reaction yielding Moco. This final maturation step is subdivided into two partial reactions, each catalyzed by a distinctive Mo-insertase domain. Initially, MPT is adenylylated by the Mo-insertase G-domain, yielding MPT-AMP which is used as substrate by the E-domain. This domain catalyzes the insertion of molybdate into the MPT dithiolene moiety, leading to the formation of Moco-AMP. Finally, the Moco-AMP phosphoanhydride bond is cleaved by the E-domain to liberate Moco from its synthesizing enzyme. Thus formed, Moco is physiologically active and may be incorporated into the different Mo-enzymes or bind to carrier proteins instead.

The functional principle of eukaryotic molybdenum insertases.

The molybdenum cofactor (Moco) is a redox-active prosthetic group found in the active site of Moco-dependent enzymes, which are vitally important for life. Moco biosynthesis involves several enzymes that catalyze the subsequent conversion of GTP into cyclic pyranopterin monophosphate (cPMP), molybdopterin (MPT), adenylated MPT (MPT-AMP), and finally Moco. While the underlying principles of cPMP, MPT, and MPT-AMP formation are well understood, the molybdenum insertase (Mo-insertase)-catalyzed final Moco maturation step is not. In the present study, we analyzed high-resolution X-ray datasets of the plant Mo-insertase Cnx1E that revealed two molybdate-binding sites within the active site, hence improving the current view on Cnx1E functionality. The presence of molybdate anions in either of these sites is tied to a distinctive backbone conformation, which we suggest to be essential for Mo-insertase molybdate selectivity and insertion efficiency.

Dimerization of the plant molybdenum insertase Cnx1E is required for synthesis of the molybdenum cofactor.

The molybdenum cofactor (Moco) is a redox active prosthetic group, essentially required for numerous enzyme-catalyzed two electron transfer reactions. Moco is synthesized by an evolutionarily old and highly conserved multistep pathway. In the last step of Moco biosynthesis, the molybdenum center is inserted into the final Moco precursor adenylated molybdopterin (MPT-AMP). This unique and yet poorly characterized maturation reaction finally yields physiologically active Moco. In the model plant Arabidopsis, the two domain enzyme, Cnx1, is required for Moco formation. Recently, a genetic screen identified novel Arabidopsis cnx1 mutant plant lines each harboring a single amino acid exchange in the N-terminal Cnx1E domain. Biochemical characterization of the respective recombinant Cnx1E variants revealed two different amino acid exchanges (S197F and G175D) that impair Cnx1E dimerization, thus linking Cnx1E oligomerization to Cnx1 functionality. Analysis of the Cnx1E structure identified Cnx1E active site-bound molybdate and magnesium ions, which allowed to fine-map the Cnx1E MPT-AMP-binding site.

Direct electrochemistry of nitrate reductase from the fungus Neurospora crassa.

We report the first direct (unmediated) catalytic electrochemistry of a eukaryotic nitrate reductase (NR). NR from the filamentous fungus Neurospora crassa, is a member of the mononuclear molybdenum enzyme family and contains a Mo, heme and FAD cofactor which are involved in electron transfer from NAD(P)H to the (Mo) active site where reduction of nitrate to nitrite takes place. NR was adsorbed on an edge plane pyrolytic graphite (EPG) working electrode. Non-turnover redox responses were observed in the absence of nitrate from holo NR and three variants lacking the FAD, heme or Mo cofactor. The FAD response is due to dissociated cofactor in all cases. In the presence of nitrate, NR shows a pronounced cathodic catalytic wave with an apparent Michaelis constant (KM) of 39µM (pH7). The catalytic cathodic current increases with temperature from 5 to 35°C and an activation enthalpy of 26kJmol(-1) was determined. In spite of dissociation of the FAD cofactor, catalytically activity is maintained.

Aminopyrazine Pathway to the Moco Metabolite Dephospho Form A.

An efficient synthesis of the molybdopterin/molybdenum cofactor (Moco) oxidation product dephospho Form A is described that assembles the pteridinone system starting from an iodinated aminopyrazine. The sodium salt of dephospho Form A could be purified by precipitation from methanol, which paved the way to the title compound in the 100 mg range. By HPLC, the synthetic material was compared with a sample isolated from a recombinant Moco containing protein. Analysis of dephospho Form A is the only method that allows the quantification of the Moco content of crude cell extracts and recombinant protein preparations.

Linear Discriminant Analysis Identifies Mitochondrially Localized Proteins in Neurospora crassa.

Besides their role as powerhouses, mitochondria play a pivotal role in the spatial organization of numerous enzymatic functions. They are connected to the ER, and many pathways are organized through the mitochondrial membranes. Thus, the precise definition of mitochondrial proteomes remains a challenging task. Here, we have established a proteomic strategy to accurately determine the mitochondrial localization of proteins from the fungal model organism Neurospora crassa. This strategy relies on both highly pure mitochondria as well as the quantitative monitoring of mitochondrial components along their consecutive enrichment. Pure intact mitochondria were obtained by a multistep approach combining differential and density Percoll (ultra) centrifugations. When compared with three other intermediate enrichment stages, peptide sequencing and quantitative profiling of pure mitochondrial fractions revealed prototypic regulatory profiles of per se mitochondrial components. These regulatory profiles constitute a distinct cluster defining the mitochondrial compartment and support linear discriminant analyses, which rationalized the annotation process. In total, this approach experimentally validated the mitochondrial localization of 512 proteins including 57 proteins that had not been reported for N. crassa before.

Enzymatic characterization of recombinant nitrate reductase expressed and purified from Neurospora crassa.

We established an expression and purification procedure for recombinant protein production in Neurospora crassa (N. crassa). This Strep-tag® based system was successfully used for purifying recombinant N. crassa nitrate reductase (NR), whose enzymatic activity was compared to recombinant N. crassa NR purified from Escherichia coli. The purity of the two different NR preparations was similar but NR purified from N. crassa showed a significantly higher nitrate turnover rate. Two phosphorylation sites were identified for NR purified from the endogenous expression system. We conclude that homologous expression of N. crassa NR yields a higher active enzyme and propose that NR phosphorylation causes enhanced enzymatic activity.

Biochemical characterization of molybdenum cofactor-free nitrate reductase from Neurospora crassa.

Nitrate reductase (NR) is a complex molybdenum cofactor (Moco)-dependent homodimeric metalloenzyme that is vitally important for autotrophic organism as it catalyzes the first and rate-limiting step of nitrate assimilation. Beside Moco, eukaryotic NR also binds FAD and heme as additional redox active cofactors, and these are involved in electron transfer from NAD(P)H to the enzyme molybdenum center where reduction of nitrate to nitrite takes place. We report the first biochemical characterization of a Moco-free eukaryotic NR from the fungus Neurospora crassa, documenting that Moco is necessary and sufficient to induce dimer formation. The molybdenum center of NR reconstituted in vitro from apo-NR and Moco showed an EPR spectrum identical to holo-NR. Analysis of mutants unable to bind heme or FAD revealed that insertion of Moco into NR occurs independent from the insertion of any other NR redox cofactor. Furthermore, we showed that at least in vitro the active site formation of NR is an autonomous process.

Genetic characterization of the Neurospora crassa molybdenum cofactor biosynthesis.

Molybdenum (Mo) is a trace element that is essential for important cellular processes. To gain biological activity, Mo must be complexed in the molybdenum cofactor (Moco), a pterin derivative of low molecular weight. Moco synthesis is a multi-step pathway that involves a variable number of genes in eukaryotes, which are assigned to four steps of eukaryotic Moco biosynthesis. Moco biosynthesis mutants lack any Moco-dependent enzymatic activities, including assimilation of nitrate (plants and fungi), detoxification of sulfite (humans and plants) and utilization of hypoxanthine as sole N-source (fungi). We report the first comprehensive genetic characterization of the Neurospora crassa (N. crassa) Moco biosynthesis pathway, annotating five genes which encode all pathway enzymes, and compare it with the characterized Aspergillus nidulans pathway. Biochemical characterization of the corresponding knock-out mutants confirms our annotation model, documenting the N. crassa/A. nidulans (fungal) Moco biosynthesis as unique, combining the organizational structure of both plant and human Moco biosynthesis genes.

Cell biology of molybdenum in plants and humans.

The transition element molybdenum (Mo) needs to be complexed by a special cofactor in order to gain catalytic activity. With the exception of bacterial Mo-nitrogenase, where Mo is a constituent of the FeMo-cofactor, Mo is bound to a pterin, thus forming the molybdenum cofactor Moco, which in different variants is the active compound at the catalytic site of all other Mo-containing enzymes. In eukaryotes, the most prominent Mo-enzymes are nitrate reductase, sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase, and the mitochondrial amidoxime reductase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also requires iron, ATP and copper. After its synthesis, Moco is distributed to the apoproteins of Mo-enzymes by Moco-carrier/binding proteins. A deficiency in the biosynthesis of Moco has lethal consequences for the respective organisms. In humans, Moco deficiency is a severe inherited inborn error in metabolism resulting in severe neurodegeneration in newborns and causing early childhood death. This article is part of a Special Issue entitled: Cell Biology of Metals.

The structural principles underlying molybdenum insertase complex assembly.

Within the cell, the trace element molybdenum (Mo) is only biologically active when complexed either within the nitrogenase-specific FeMo cofactor or within the molybdenum cofactor (Moco). Moco consists of an organic part, called molybdopterin (MPT) and an inorganic part, that is, the Mo-center. The enzyme which catalyzes the Mo-center formation is the molybdenum insertase (Mo-insertase). Mo-insertases consist of two functional domains called G- and E-domain. The G-domain catalyzes the formation of adenylated MPT (MPT-AMP), which is the substrate for the E-domain, that catalyzes the actual molybdate insertion reaction. Though the functions of E- and G-domain have been elucidated to great structural and mechanistic detail, their combined function is poorly characterized. In this work, we describe a structural model of the eukaryotic Mo-insertase Cnx1 complex that was generated based on cross-linking mass spectrometry combined with computational modeling. We revealed Cnx1 to form an asymmetric hexameric complex which allows the E- and G-domain active sites to align in a catalytic productive orientation toward each other.

Catalytic electrochemistry of the bacterial Molybdoenzyme YcbX.

Molybdenum-dependent enzymes that can reduce N-hydroxylated substrates (e.g. N-hydroxyl-purines, amidoximes) are found in bacteria, plants and vertebrates. They are involved in the conversion of a wide range of N-hydroxylated organic compounds into their corresponding amines, and utilize various redox proteins (cytochrome b5, cyt b5 reductase, flavin reductase) to deliver reducing equivalents to the catalytic centre. Here we present catalytic electrochemistry of the bacterial enzyme YcbX from Escherichia coli utilizing the synthetic electron transfer mediator methyl viologen (MV2+). The electrochemically reduced form (MV+.) acts as an effective electron donor for YcbX. To immobilize YcbX on a glassy carbon electrode, a facile protein crosslinking approach was used with the crosslinker glutaraldehyde (GTA). The YcbX-modified electrode showed a catalytic response for the reduction of a broad range of N-hydroxylated substrates. The catalytic activity of YcbX was examined at different pH values exhibiting an optimum at pH 7.5 and a bell-shaped pH profile with deactivation through deprotonation (pKa1 9.1) or protonation (pKa2 6.1). Electrochemical simulation was employed to obtain new biochemical data for YcbX, in its reaction with methyl viologen and the organic substrates 6-N-hydroxylaminopurine (6-HAP) and benzamidoxime (BA).

Electrochemically driven catalysis of the bacterial molybdenum enzyme YiiM.

The Mo-dependent enzyme YiiM enzyme from Escherichia coli is a member of the sulfite oxidase family and shares many similarities with the well-studied human mitochondrial amidoxime reducing component (mARC). We have investigated YiiM catalysis using electrochemical and spectroscopic methods. EPR monitored redox potentiometry found the active site redox potentials to be MoVI/V -0.02 V and MoV/IV -0.12 V vs NHE at pH 7.2. In the presence of methyl viologen as an electrochemically reduced electron donor, YiiM catalysis was studied with a range of potential substrates. YiiM preferentially reduces N-hydroxylated compounds such as hydroxylamines, amidoximes, N-hydroxypurines and N-hydroxyureas but shows little or no activity against amine-oxides or sulfoxides. The pH optimum for catalysis was 7.1 and a bell-shaped pH profile was found with pKa values of 6.2 and 8.1 either side of this optimum that are associated with protonation/deprotonations that modulate activity. Simulation of the experimental voltammetry elucidated kinetic parameters associated with YiiM catalysis with the substrates 6-hydroxyaminopurine and benzamidoxime.

Identification and biochemical characterization of molybdenum cofactor-binding proteins from Arabidopsis thaliana.

The molybdenum cofactor (Moco) forms part of the catalytic center in all eukaryotic molybdenum enzymes and is synthesized in a highly conserved pathway. Among eukaryotes, very little is known about the processes taking place subsequent to Moco biosynthesis, i.e. Moco transfer, allocation, and insertion into molybdenum enzymes. In the model plant Arabidopsis thaliana, we identified a novel protein family consisting of nine members that after recombinant expression are able to bind Moco with K(D) values in the low micromolar range and are therefore named Moco-binding proteins (MoBP). For two of the nine proteins atomic structures are available in the Protein Data Bank. Surprisingly, both crystal structures lack electron density for the C terminus, which may indicate a high flexibility of this part of the protein. C-terminal truncated MoBPs showed significantly decreased Moco binding stoichiometries. Experiments where the MoBP C termini were exchanged among MoBPs converted a weak Moco-binding MoBP into a strong binding MoBP, thus indicating that the MoBP C terminus, which is encoded by a separate exon, is involved in Moco binding. MoBPs were able to enhance Moco transfer to apo-nitrate reductase in the Moco-free Neurospora crassa mutant nit-1. Furthermore, we show that the MoBPs are localized in the cytosol and undergo protein-protein contact with both the Moco donor protein Cnx1 and the Moco acceptor protein nitrate reductase under in vivo conditions, thus indicating for the MoBPs a function in Arabidopsis cellular Moco distribution.

Deconstructing the electron transfer chain in a complex molybdoenzyme: Assimilatory nitrate reductase from Neurospora crassa.

Nitrate reductase (NR) from the fungus Neurospora crassa is a complex homodimeric metallo-flavoenzyme, where each protomer contains three distinct domains; the catalytically active terminal molybdopterin cofactor, a central heme-containing domain, and an FAD domain which binds with the natural electron donor NADPH. Here, we demonstrate the catalytic voltammetry of variants of N. crassa NRs on a modified Au electrode with the electrochemically reduced forms of benzyl viologen (BV2+) and anthraquinone sulfonate (AQS-) acting as artificial electron donors. The biopolymer chitosan used to entrap NR on the electrode non-covalently and the enzyme film was both stable and highly active. Electrochemistry was conducted on two distinct forms; one lacking the FAD cofactor and the other lacking both the FAD and heme cofactors. While both enzymes showed catalytic nitrate reductase activity, removal of the heme cofactor resulted in a more significant effect on the rate of nitrate reduction. Electrochemical simulation was carried out to enable kinetic characterisation of both the NR:nitrate and NR:mediator reactions.

Mechanism of molybdate insertion into pterin-based molybdenum cofactors.

The molybdenum cofactor (Moco) is found in the active site of numerous important enzymes that are critical to biological processes. The bidentate ligand that chelates molybdenum in Moco is the pyranopterin dithiolene (molybdopterin, MPT). However, neither the mechanism of molybdate insertion into MPT nor the structure of Moco prior to its insertion into pyranopterin molybdenum enzymes is known. Here, we report this final maturation step, where adenylated MPT (MPT-AMP) and molybdate are the substrates. X-ray crystallography of the Arabidopsis thaliana Mo-insertase variant Cnx1E S269D D274S identified adenylated Moco (Moco-AMP) as an unexpected intermediate in this reaction sequence. X-ray absorption spectroscopy revealed the first coordination sphere geometry of Moco trapped in the Cnx1E active site. We have used this structural information to deduce a mechanism for molybdate insertion into MPT-AMP. Given their high degree of structural and sequence similarity, we suggest that this mechanism is employed by all eukaryotic Mo-insertases.

The structure of the Moco carrier protein from Rippkaea orientalis.

The molybdenum cofactor (Moco) is the prosthetic group of all molybdenum-dependent enzymes except for nitrogenase. The multistep biosynthesis pathway of Moco and its function in molybdenum-dependent enzymes are already well understood. The mechanisms of Moco transfer, storage and insertion, on the other hand, are not. In the cell, Moco is usually not found in its free form and remains bound to proteins because of its sensitivity to oxidation. The green alga Chlamydomonas reinhardtii harbors a Moco carrier protein (MCP) that binds and protects Moco but is devoid of enzymatic function. It has been speculated that this MCP acts as a means of Moco storage and transport. Here, the search for potential MCPs has been extended to the prokaryotes, and many MCPs were found in cyanobacteria. A putative MCP from Rippkaea orientalis (RoMCP) was selected for recombinant production, crystallization and structure determination. RoMCP has a Rossmann-fold topology that is characteristic of nucleotide-binding proteins and a homotetrameric quaternary structure similar to that of the MCP from C. reinhardtii. In each protomer, a positively charged crevice was identified that accommodates up to three chloride ions, hinting at a potential Moco-binding site. Computational docking experiments supported this notion and gave an impression of the RoMCP-Moco complex.

Identification and characterisation of the Volvox carteri Moco carrier protein.

The molybdenum cofactor (Moco) is a redox active prosthetic group found in the active site of Moco-dependent enzymes (Mo-enzymes). As Moco and its intermediates are highly sensitive towards oxidative damage, these are believed to be permanently protein bound during synthesis and upon maturation. As a major component of the plant Moco transfer and storage system, proteins have been identified that are capable of Moco binding and release but do not possess Moco-dependent enzymatic activities. The first protein found to possess these properties was the Moco carrier protein (MCP) from the green alga Chlamydomonas reinhardtii. Here, we describe the identification and biochemical characterisation of the Volvox carteri (V. carteri) MCP and, for the first time, employ a comparative analysis to elucidate the principles behind MCP Moco binding. Doing so identified a sequence region of low homology amongst the existing MCPs, which we showed to be essential for Moco binding to V. carteri MCP.