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).

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.

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.

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.

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 Mo -0.

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.

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 (BV) and anthraquinone sulfonate (AQS) acting as artificial electron donors.

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 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.

The First Step of Molybdenum Cofactor Biosynthesis: Regulatory Aspects under N-Derepressing and Nitrate-Inducing Conditions.

Molybdenum cofactor (Moco) is the active site prosthetic group found in all Moco dependent enzymes, except for nitrogenase. Mo-enzymes are crucial for viability throughout all kingdoms of life as they catalyze a diverse set of two electron transfer reactions. The highly conserved Moco biosynthesis pathway consists of four different steps in which guanosine triphosphate is converted into cyclic pyranopterin monophosphate, molybdopterin (MPT), and subsequently adenylated MPT and Moco.

Insights into the Cnx1E catalyzed MPT-AMP hydrolysis.

Molybdenum insertases (Mo-insertases) catalyze the final step of molybdenum cofactor (Moco) biosynthesis, an evolutionary old and highly conserved multi-step pathway. In the first step of the pathway, GTP serves as substrate for the formation of cyclic pyranopterin monophosphate, which is subsequently converted into molybdopterin (MPT) in the second pathway step. In the following synthesis steps, MPT is adenylated yielding MPT-AMP that is subsequently used as substrate for enzyme catalyzed molybdate insertion.

A mass spectrometry guided approach for the identification of novel vaccine candidates in gram-negative pathogens.

Vaccination is the most effective method to prevent infectious diseases. However, approaches to identify novel vaccine candidates are commonly laborious and protracted. While surface proteins are suitable vaccine candidates and can elicit antibacterial antibody responses, systematic approaches to define surfomes from gram-negatives have rarely been successful.

S-Sulfocysteine Induces Seizure-Like Behaviors in Zebrafish.

Sulfite is a neurotoxin, which is detoxified by the molybdenum cofactor (Moco)-dependent enzyme sulfite oxidase (SOX). In humans, SOX deficiency causes the formation of the glutamate analog S-Sulfocysteine (SSC) resulting in a constant overstimulation of ionotropic glutamatergic receptors. Overstimulation leads to seizures, severe brain damage, and early childhood death.

Broad application and optimization of a single wash-step for integrated endotoxin depletion during protein purification.

Endotoxins contaminate proteins that are produced in E. coli. High levels of endotoxins can influence cellular assays and cause severe adverse effects when administered to humans.

adhesin HopQ disrupts dimerization in human CEACAMs.

The human gastric pathogen 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.

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.

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.

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).

Helicobacter pylori adhesin HopQ engages in a virulence-enhancing interaction with human CEACAMs.

Helicobacter pylori specifically colonizes the human gastric epithelium and is the major causative agent for ulcer disease and gastric cancer development. Here, we identify members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family as receptors of H. pylori and show that HopQ is the surface-exposed adhesin that specifically binds human CEACAM1, CEACAM3, CEACAM5 and CEACAM6.

Helicobacter pylori HP0231 Influences Bacterial Virulence and Is Essential for Gastric Colonization.

The Dsb protein family is responsible for introducing disulfide bonds into nascent proteins in prokaryotes, stabilizing the structure of many proteins. Helicobacter pylori HP0231 is a Dsb-like protein, shown to catalyze disulfide bond formation and to participate in redox homeostasis. Notably, many H.

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.

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.

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.

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.