The Mechanism of nucleotide-assisted molybdenum insertion into molybdopterin. A novel route toward metal cofactor assembly.
The molybdenum cofactor (Moco) is synthesized by an ancient and conserved biosynthetic pathway. In plants, the two-domain protein Cnx1 catalyzes the insertion of molybdenum into molybdopterin (MPT), a metal-free phosphorylated pyranopterin carrying an ene-dithiolate. Recently, we identified a novel biosynthetic intermediate, adenylated molybdopterin (MPT-AMP), which is synthesized by the C-terminal G domain of Cnx1. Here, we show that MPT-AMP and molybdate bind in an equimolar and cooperative way to the other N-terminal E domain (Cnx1E). Tungstate and sulfate compete for molybdate, which demonstrates the presence of an anion-binding site for molybdate. Cnx1E catalyzes the Zn(2+)-/Mg(2+)-dependent hydrolysis of MPT-AMP but only when molybdate is bound as co-substrate. MPT-AMP hydrolysis resulted in stoichiometric release of Moco that was quantitatively incorporated into plant apo-sulfite oxidase. Upon Moco formation AMP is release as second product of the reaction. When comparing MPT-AMP hydrolysis with the formation of Moco and AMP a 1.5-fold difference in reaction rates were observed. Together with the strict dependence of the reaction on molybdate the formation of adenylated molybdate as reaction intermediate in the nucleotide-assisted metal transfer reaction to molybdopterin is proposed.
Llamas
Angel
A
Institute of Plant Biology, Technical University Braunschweig, 38106 Braunschweig, Germany.
Otte
Tanja
T
Multhaup
Gerd
G
Mendel
Ralf R
RR
Schwarz
Guenter
G
D000249:Adenosine Monophosphate / Q000737:chemistry / Q000378:metabolism; D017360:Arabidopsis / Q000737:chemistry / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000378:metabolism; D037281:Calnexin / Q000378:metabolism; D002384:Catalysis; D003067:Coenzymes / Q000096:biosynthesis* / Q000737:chemistry; D006868:Hydrolysis; D008667:Metalloproteins / Q000096:biosynthesis* / Q000737:chemistry; D008958:Models, Molecular; D000090006:Molybdenum Cofactors; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000378:metabolism; D011485:Protein Binding; D011621:Pteridines / Q000737:chemistry; D013379:Substrate Specificity
D000249:Adenosine Monophosphate; D029681:Arabidopsis Proteins; D037281:Calnexin; D003067:Coenzymes; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D050862:Oxidoreductases Acting on Sulfur Group Donors; D011621:Pteridines; C080351:CNX1 protein, Arabidopsis; C022261:molybdopterin