TY - JOUR A1 - Schrapers, Peer A1 - Hartmann, Tobias A1 - Kositzki, Ramona A1 - Dau, Holger A1 - Reschke, Stefan A1 - Schulzke, Carola A1 - Leimkühler, Silke A1 - Haumann, Michael T1 - 'Sulfido and Cysteine Ligation Changes at the Molybdenum Cofactor during Substrate Conversion by Formate Dehydrogenase (FDH) from Rhodobacter capsulatus JF - Inorganic chemistry N2 - Formate dehydrogenase (FDH) enzymes are attractive catalysts for potential carbon dioxide conversion applications. The FDH from Rhodobacter capsulatus (RcFDH) binds a bis-molybdopterin-guanine-dinucleotide (bis-MGD) cofactor, facilitating reversible formate (HCOO-) to CO2 oxidation. We characterized the molecular structure of the active site of wildtype RcFDH and protein variants using X-ray absorption spectroscopy (XAS) at the Mo K-edge. This approach has revealed concomitant binding of a sulfido ligand (Mo=S) and a conserved cysteine residue (S(Cys386)) to Mo(VI) in the active oxidized molybdenum cofactor (Moco), retention of such a coordination motif at Mo(V) in a chemically reduced enzyme, and replacement of only the S(Cys386) ligand by an oxygen of formate upon Mo(IV) formation. The lack of a Mo=S bond in RcFDH expressed in the absence of FdsC implies specific metal sulfuration by this bis-MGD binding chaperone. This process still functioned in the Cys386Ser variant, showing no Mo-S(Cys386) ligand, but retaining a Mo=S bond. The C386S variant and the protein expressed without FdsC were inactive in formate oxidation, supporting that both Moligands are essential for catalysis. Low-pH inhibition of RcFDH was attributed to protonation at the conserved His387, supported by the enhanced activity of the His387Met variant at low pH, whereas inactive cofactor species showed sulfido-to-oxo group exchange at the Mo ion. Our results support that the sulfido and S(Cys386) ligands at Mo and a hydrogen-bonded network including His387 are crucial for positioning, deprotonation, and oxidation of formate during the reaction cycle of RcFDH. Y1 - 2015 U6 - https://doi.org/10.1021/ic502880y SN - 0020-1669 SN - 1520-510X VL - 54 IS - 7 SP - 3260 EP - 3271 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Hartmann, Tobias A1 - Schwanhold, Nadine A1 - Leimkühler, Silke T1 - Assembly and catalysis of molybdenum or tungsten-containing formate dehydrogenases from bacteria JF - Biochimica et biophysica acta : Proteins and proteomics N2 - The global carbon cycle depends on the biological transformations of C-1 compounds, which include the reductive incorporation of CO2 into organic molecules (e.g. in photosynthesis and other autotrophic pathways), in addition to the production of CO2 from formate, a reaction that is catalyzed by formate dehydrogenases (FDHs). FDHs catalyze, in general, the oxidation of formate to CO2 and H+. However, selected enzymes were identified to act as CO2 reductases, which are able to reduce CO2 to formate under physiological conditions. This reaction is of interest for the generation of formate as a convenient storage form of H-2 for future applications. Cofactor-containing FDHs are found in anaerobic bacteria and archaea, in addition to facultative anaerobic or aerobic bacteria. These enzymes are highly diverse and employ different cofactors such as the molybdenum cofactor (Moco), FeS clusters and flavins, or cytochromes. Some enzymes include tungsten (W) in place of molybdenum (Mo) at the active site. For catalytic activity, a selenocysteine (SeCys) or cysteine (Cys) ligand at the Mo atom in the active site is essential for the reaction. This review will focus on the characterization of Mo- and W-containing FDHs from bacteria, their active site structure, subunit compositions and its proposed catalytic mechanism. We will give an overview on the different mechanisms of substrate conversion available so far, in addition to providing an outlook on bio-applications of FDHs. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications. (C) 2014 Elsevier B.V. All rights reserved. KW - Molybdenum cofactor KW - L-Cysteine desulfurase KW - Formate dehydrogenase KW - Chaperone KW - Bis-MGD Y1 - 2015 U6 - https://doi.org/10.1016/j.bbapap.2014.12.006 SN - 1570-9639 SN - 0006-3002 VL - 1854 IS - 9 SP - 1090 EP - 1100 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Coelho, Catarina A1 - Foti, Alessandro A1 - Hartmann, Tobias A1 - Santos-Silva, Teresa A1 - Leimkühler, Silke A1 - Romao, Maria Joao T1 - Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase JF - Nature chemical biology N2 - Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-angstrom resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-angstrom resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs. Y1 - 2015 U6 - https://doi.org/10.1038/NCHEMBIO.1895 SN - 1552-4450 SN - 1552-4469 VL - 11 IS - 10 SP - 779 EP - + PB - Nature Publ. Group CY - New York ER -