A Molecular Basis for Tungstate Selectivity in Prokaryotic ABC Transport Systems ▿

The essential trace compounds tungstate and molybdate are taken up by cells via ABC transporters. Despite their similar ionic radii and chemical properties, the WtpA protein selectively binds tungstate in the presence of molybdate. Using site-directed mutagenesis of conserved binding pocket residues, we established a molecular basis for tungstate selectivity

Oxidative power: Tools for assessing lpmo activity on cellulose

Lytic polysaccharide monooxygenases (LPMOs) have sparked a lot of research regarding their fascinating mode-of-action. Particularly, their boosting effect on top of the well-known cel-lulolytic enzymes in lignocellulosic hydrolysis makes them industrially relevant targets. As more characteristics of LPMO and its key role have been elucidated, the need for fast and reliable methods to assess its activity have become clear. Several aspects such as its co-substrates, electron donors, inhibiting factors, and the inhomogeneity of lignocellulose had to be considered during experimental design and data interpretation, as they can impact and often hamper outcomes. This review provides an overview of the currently available methods to measure LPMO activity, including their potential and limitations, and it is illustrated with practical examples

WOR5, a Novel Tungsten-Containing Aldehyde Oxidoreductase from Pyrococcus furiosus with a Broad Substrate Specificity

WOR5 is the fifth and last member of the family of tungsten-containing oxidoreductases purified from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimeric protein (subunit, 65 kDa) that contains one [4Fe-4S] cluster and one tungstobispterin cofactor per subunit. It has a broad substrate specificity with a high affinity for several substituted and nonsubstituted aliphatic and aromatic aldehydes with various chain lengths. The highest catalytic efficiency of WOR5 is found for the oxidation of hexanal (V(max) = 15.6 U/mg, K(m) = 0.18 mM at 60°C). Hexanal-incubated enzyme exhibits S = 1/2 electron paramagnetic resonance signals from [4Fe-4S](1+) (g values of 2.08, 1.93, and 1.87) and W(5+) (g values of 1.977, 1.906, and 1.855). Cyclic voltammetry of ferredoxin and WOR5 on an activated glassy carbon electrode shows a catalytic wave upon addition of hexanal, suggesting that ferredoxin can be a physiological redox partner. The combination of WOR5, formaldehyde oxidoreductase, and aldehyde oxidoreductase forms an efficient catalyst for the oxidation of a broad range of aldehydes in P. furiosus

Tungsten Transport Protein A (WtpA) in Pyrococcus furiosus: the First Member of a New Class of Tungstate and Molybdate Transporters

A novel tungstate and molybdate binding protein has been discovered from the hyperthermophilic archaeon Pyrococcus furiosus. This tungstate transport protein A (WtpA) is part of a new ABC transporter system selective for tungstate and molybdate. WtpA has very low sequence similarity with the earlier-characterized transport proteins ModA for molybdate and TupA for tungstate. Its structural gene is present in the genome of numerous archaea and some bacteria. The identification of this new tungstate and molybdate binding protein clarifies the mechanism of tungstate and molybdate transport in organisms that lack the known uptake systems associated with the ModA and TupA proteins, like many archaea. The periplasmic protein of this ABC transporter, WtpA (PF0080), was cloned and expressed in Escherichia coli. Using isothermal titration calorimetry, WtpA was observed to bind tungstate (dissociation constant [K(D)] of 17 ± 7 pM) and molybdate (K(D) of 11 ± 5 nM) with a stoichiometry of 1.0 mol oxoanion per mole of protein. These low K(D) values indicate that WtpA has a higher affinity for tungstate than do ModA and TupA and an affinity for molybdate similar to that of ModA. A displacement titration of molybdate-saturated WtpA with tungstate showed that the tungstate effectively replaced the molybdate in the binding site of the protein

One- and two-electron reduction of molybdate reversibly bound to the archaeal tungstate/molybdate transporter WtpA

Reversible binding of the tetrahedral oxoanions MoO4 2- and WO4 2- to two carboxylato ligands of the soluble scavenger protein WtpA from the hyperthermophilic archaeon Pyrococcus furiosus enforces a quasi-octahedral MO6 coordination in which the +VI oxidation state is destabilized.BiotechnologyApplied Science

Oleate Hydratase Catalyzes the Hydration of a Nonactivated Carbon-Carbon Bond▿ †

The hydration of oleic acid into 10-hydroxystearic acid was originally described for a Pseudomonas cell extract almost half a century ago. In the intervening years, the enzyme has never been characterized in any detail. We report here the isolation and characterization of oleate hydratase (EC 4.2.1.53) from Elizabethkingia meningoseptica

Distorted octahedral coordination of tungstate in a subfamily of specific binding proteins

Bacteria and archaea import molybdenum andtungsten from the environment in the form of theoxyanions molybdate (MoO42-) and tungstate (WO42-).These substrates are captured by an external, high-affinitybinding protein, and delivered to ATP binding cassettetransporters, which move them across the cell membrane.We have recently reported a crystal structure of themolybdate/tungstate binding protein ModA/WtpA fromArchaeoglobus fulgidus, which revealed an octahedrallycoordinated central metal atom. By contrast, the previouslydetermined structures of three bacterial homologs showedtetracoordinate molybdenum and tungsten atoms in theirbinding pockets. Until then, coordination numbers abovefour had only been found for molybdenum/tungsten inmetalloenzymes where these metal atoms are part of thecatalytic cofactors and coordinated by mostly non-oxygenligands. We now report a high-resolution structure ofA. fulgidus ModA/WtpA, as well as crystal structures offour additional homologs, all bound to tungstate. Thesecrystal structures match X-ray absorption spectroscopymeasurements from soluble, tungstate-bound protein, andreveal the details of the distorted octahedral coordination.Our results demonstrate that the distorted octahedralgeometry is not an exclusive feature of the A. fulgidusprotein, and suggest distinct binding modes of the bindingproteins from archaea and bacteria

Anion binding in biological systems

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