Function and structure of the molybdenum cofactor carrier protein from Chlamydomonas reinhardtii.
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.
Fischer
Katrin
K
Institute of Plant Biology, Technical University Braunschweig, 38106 Braunschweig, Germany.
Llamas
Angel
A
Tejada-Jimenez
Manuel
M
Schrader
Nils
N
Kuper
Jochen
J
Ataya
Farid S
FS
Galvan
Aurora
A
Mendel
Ralf R
RR
Fernandez
Emilio
E
Schwarz
Guenter
G
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
D002352:Carrier Proteins; D003067:Coenzymes; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D011621:Pteridines; D014414:Tungsten; C022261:molybdopterin