title: Function and structure of the molybdenum cofactor carrier protein from Chlamydomonas reinhardtii.
abstract: The molybdenum cofactor (Moco) forms the catalytic site in all eukaryotic molybdenum enzymes and is synthesized by a multistep biosynthetic pathway. The mechanism of transfer, storage, and insertion of Moco into the appropriate apo-enzyme is poorly understood. In Chlamydomonas reinhardtii, a Moco carrier protein (MCP) has been identified and characterized recently. Here we show biochemical evidence that MCP binds Moco as well as the tungstate-substituted form of the cofactor (Wco) with high affinity, whereas molybdopterin, the ultimate cofactor precursor, is not bound. This binding selectivity points to a specific metal-mediated interaction with MCP, which protects Moco and Wco from oxidation with t((1/2)) of 24 and 96 h, respectively. UV-visible spectroscopy showed defined absorption bands at 393, 470, and 570 nm pointing to ene-diothiolate and protein side-chain charge transfer bonds with molybdenum. We have determined the crystal structure of MCP at 1.6 Angstrom resolution using seleno-methionated and native protein. The monomer constitutes a Rossmann fold with two homodimers forming a symmetrical tetramer in solution. Based on conserved surface residues, charge distribution, shape, in silico docking studies, structural comparisons, and identification of an anionbinding site, a prominent surface depression was proposed as a Moco-binding site, which was confirmed by structure-guided mutagenesis coupled to substrate binding studies.
authors.affiliation: Institute of Plant Biology, Technical University Braunschweig, 38106 Braunschweig, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Fischer
authors.forename: Angel
authors.initials: A
authors.lastname: Llamas
authors.forename: Manuel
authors.initials: M
authors.lastname: Tejada-Jimenez
authors.forename: Nils
authors.initials: N
authors.lastname: Schrader
authors.forename: Jochen
authors.initials: J
authors.lastname: Kuper
authors.forename: Farid S
authors.initials: FS
authors.lastname: Ataya
authors.forename: Aurora
authors.initials: A
authors.lastname: Galvan
authors.forename: Ralf R
authors.initials: RR
authors.lastname: Mendel
authors.forename: Emilio
authors.initials: E
authors.lastname: Fernandez
authors.forename: Guenter
authors.initials: G
authors.lastname: Schwarz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D016825:Chlamydomonas reinhardtii / Q000737:chemistry* / Q000201:enzymology / Q000502:physiology*; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000378:metabolism; D000090006:Molybdenum Cofactors; D011485:Protein Binding; D011621:Pteridines / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014414:Tungsten / Q000737:chemistry
chemical_list: D002352:Carrier Proteins; D003067:Coenzymes; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D011621:Pteridines; D014414:Tungsten; C022261:molybdopterin