TY - JOUR A1 - Reschke, Stefan A1 - Sigfridsson, Kajsa G. V. A1 - Kaufmann, Paul A1 - Leidel, Nils A1 - Horn, Sebastian A1 - Gast, Klaus A1 - Schulzke, Carola A1 - Haumann, Michael A1 - Leimkühler, Silke T1 - Identification of a bis-molybdopterin intermediate in molybdenum cofactor biosynthesis in escherichia coli JF - The journal of biological chemistry N2 - The molybdenum cofactor is an important cofactor, and its biosynthesis is essential for many organisms, including humans. Its basic form comprises a single molybdopterin (MPT) unit, which binds a molybdenum ion bearing three oxygen ligands via a dithiolene function, thus forming Mo-MPT. In bacteria, this form is modified to form the bis-MPT guanine dinucleotide cofactor with two MPT units coordinated at one molybdenum atom, which additionally contains GMPs bound to the terminal phosphate group of the MPTs (bis-MGD). The MobA protein catalyzes the nucleotide addition to MPT, but the mechanism of the biosynthesis of the bis-MGD cofactor has remained enigmatic. We have established an in vitro system for studying bis-MGD assembly using purified compounds. Quantification of the MPT/molybdenum and molybdenum/phosphorus ratios, time-dependent assays for MPT and MGD detection, and determination of the numbers and lengths of Mo-S and Mo-O bonds by X-ray absorption spectroscopy enabled identification of a novel bis-Mo-MPT intermediate on MobA prior to nucleotide attachment. The addition of Mg-GTP to MobA loaded with bis-Mo-MPT resulted in formation and release of the final bis-MGD product. This cofactor was fully functional and reconstituted the catalytic activity of apo-TMAO reductase (TorA). We propose a reaction sequence for bis-MGD formation, which involves 1) the formation of bis-Mo-MPT, 2) the addition of two GMP units to form bis-MGD on MobA, and 3) the release and transfer of the mature cofactor to the target protein TorA, in a reaction that is supported by the specific chaperone TorD, resulting in an active molybdoenzyme. Y1 - 2013 U6 - https://doi.org/10.1074/jbc.M113.497453 SN - 0021-9258 SN - 1083-351X VL - 288 IS - 41 SP - 29736 EP - 29745 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - JOUR A1 - Havelius, Kajsa G. V. A1 - Reschke, Stefan A1 - Horn, Sebastian A1 - Doerlng, Alexander A1 - Niks, Dimitri A1 - Hille, Russ A1 - Schulzke, Carola A1 - Leimkühler, Silke A1 - Haumann, Michael T1 - Structure of the molybdenum site in YedY, a sulfite oxidase homologue from escherichia coli JF - Inorganic chemistry N2 - YedY from Escherichia coil is a new member of the sulfite oxidase family of molybdenum cofactor (Moco)-containing oxidoreductases. We investigated the atomic structure of the molybdenum site in YedY by X-ray absorption spectroscopy, in comparison to human sulfite oxidase (hSO) and to a Mo(IV) model complex. The K-edge energy was indicative of Mo(V) in YedY, in agreement with X- and Q-band electron paramagnetic resonance results, whereas the hSO protein contained Mo(VI). In YedY and hSO, molybdenum is coordinated by two sulfur ligands from the molybdopterin ligand of the Moco, one thiolate sulfur of a cysteine (average Mo-S bond length of similar to 2.4 angstrom), and one (axial) oxo ligand (Mo=O, similar to 1.7 angstrom). hSO contained a second oxo group at Mo as expected, but in YedY, two species in about a 1:1 ratio were found at the active site, corresponding to an equatorial Mo-OH bond (similar to 2.1 angstrom) or possibly to a shorter M-O(-) bond. Yet another oxygen (or nitrogen) at a similar to 2.6 angstrom distance to Mo in YedY was identified, which could originate from a water molecule in the substrate binding cavity or from an amino acid residue close to the molybdenum site, i.e., Glu104, that is replaced by a glycine in hSO, or Asn45. The addition of the poor substrate dimethyl sulfoxide to YedY left the molybdenum coordination unchanged at high pH. In contrast, we found indications that the better substrate trimethylamine N-oxide and the substrate analogue acetone were bound at a similar to 2.6 angstrom distance to the molybdenum, presumably replacing the equatorial oxygen ligand. These findings were used to interpret the recent crystal structure of YedY and bear implications for its catalytic mechanism. Y1 - 2011 U6 - https://doi.org/10.1021/ic101291j SN - 0020-1669 VL - 50 IS - 3 SP - 741 EP - 748 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Samuel, Prinson P. A1 - Horn, Sebastian A1 - Döring, Alexander A1 - Havelius, Kajsa G. V. A1 - Reschke, Stefan A1 - Leimkühler, Silke A1 - Haumann, Michael A1 - Schulzke, Carola T1 - A Crystallographic and Mo K-Edge XAS Study of Molybdenum Oxo Bis-,Mono-, and Non-Dithiolene Complexes - First-Sphere Coordination Geometry and Noninnocence of Ligands JF - European journal of inorganic chemistry : a journal of ChemPubSoc Europe N2 - Ten square-based pyramidal molybdenum complexes with different sulfur donor ligands, that is, a variety of dithiolenes and sulfides, were prepared, which mimic coordination motifs of the molybdenum cofactors of molybdenum-dependent oxidoreductases. The model compounds were investigated by Mo K-edge X-ray absorption spectroscopy (XAS) and (with one exception) their molecular structures were analyzed by X-ray diffraction to derive detailed information on bond lengths and geometries of the first coordination shell of molybdenum. Only small variations in Mo=O and Mo-S bond lengths and their respective coordination angles were observed for all complexes including those containing Mo(CO)(2) or Mo(mu-S)(2)Mo motifs. XAS analysis (edge energy) revealed higher relative oxidation levels in the molybdenum ion in compounds with innocent sulfur-based ligands relative to those in dithiolene complexes, which are known to exhibit noninnocence, that is, donation of substantial electron density from ligand to metal. In addition, longer average Mo-S and Mo=O bonds and consequently lower.(Mo=O) stretching frequencies in the IR spectra were observed for complexes with dithiolene-derived ligands. The results emphasize that the noninnocent character of the dithiolene ligand influences the electronic structure of the model compounds, but does not significantly affect their metal coordination geometry, which is largely determined by the Mo(IV) or (V) ion itself. The latter conclusion also holds for the molybdenum site geometries in the oxidized Mo-VI cofactor of DMSO reductase and the reduced Mo-IV cofactor of arsenite oxidase. The innocent behavior of the dithiolene molybdopterin ligands observed in the enzymes is likely to be related to cofactor-protein interactions. KW - Molybdenum KW - Enzyme models KW - X-ray absorption spectroscopy KW - Noninnocence KW - Bioinorganic chemistry Y1 - 2011 U6 - https://doi.org/10.1002/ejic.201100331 SN - 1434-1948 IS - 28 SP - 4387 EP - 4399 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Horn, Sebastian A1 - Hempel, Stefan A1 - Ristow, Michael A1 - Rillig, Matthias C. A1 - Kowarik, Ingo A1 - Caruso, Tancredi T1 - Plant community assembly at small scales: Spatial vs. environmental factors in a European grassland JF - Acta oecologica : international journal of ecology N2 - Dispersal limitation and environmental conditions are crucial drivers of plant species distribution and establishment. As these factors operate at different spatial scales, we asked: Do the environmental factors known to determine community assembly at broad scales operate at fine scales (few meters)? How much do these factors account for community variation at fine scales? In which way do biotic and abiotic interactions drive changes in species composition? We surveyed the plant community within a dry grassland along a very steep gradient of soil characteristics like pH and nutrients. We used a spatially explicit sampling design, based on three replicated macroplots of 15 x 15, 12 x 12 and 12 x 12 m in extent. Soil samples were taken to quantify several soil properties (carbon, nitrogen, plant available phosphorus, pH, water content and dehydrogenase activity as a proxy for overall microbial activity). We performed variance partitioning to assess the effect of these variables on plant composition and statistically controlled for spatial autocorrelation via eigenvector mapping. We also applied null model analysis to test for non-random patterns in species co-occurrence using randomization schemes that account for patterns expected under species interactions. At a fine spatial scale, environmental factors explained 18% of variation when controlling for spatial autocorrelation in the distribution of plant species, whereas purely spatial processes accounted for 14% variation. Null model analysis showed that species spatially segregated in a non-random way and these spatial patterns could be due to a combination of environmental filtering and biotic interactions. Our grassland study suggests that environmental factors found to be directly relevant in broad scale studies are present also at small scales, but are supplemented by spatial processes and more direct interactions like competition. (C) 2015 Elsevier Masson SAS. All rights reserved. KW - Assembly pattern KW - Dispersal limitation KW - Festuca brevipila KW - Niche partitioning KW - Null model KW - Plant community ecology KW - Variance partitioning Y1 - 2015 U6 - https://doi.org/10.1016/j.actao.2015.01.004 SN - 1146-609X SN - 1873-6238 VL - 63 SP - 56 EP - 62 PB - Elsevier CY - Paris ER -