Cofactor Analysis of Arabidopsis thaliana Cnx1G Domain

Molybdenum is found in the active sites of a diverse group of enzymes that are known to play crucial roles in both biological and environmental cycles. The mononuclear molybdenum enzymes form a class of enzymes that share the molybdenum cofactor (Moco) as a common structural motif essential for activity. Moco is synthesized by a conserved multi-step pathway starting with GTP, in which the final step involves the adenylylation of the molybdopterin (MPT) to form MPT-AMP before molybdenum can be inserted. In plants, the two-domain protein Cnx1 first adenylylates MPT by its C-terminal G domain (Cnx1G) and subsequently, its N-terminal E domain (Cnx1E) incorporates molybdenum concomitant with cleavage of the adenyl-moiety. The crystal structure of Arabidopsis thaliana Cnx1G demonstrated the existence of MPT-AMP. Furthermore copper was found as novel MPT ligand, which was identified by its anomalous diffraction [1]. It was suggested that the in vivo incorporation of copper into MPT acts to protect the reactive dithiolene group prior to molybdenum insertion. However, the biological function of copper in Moco biosynthesis remains unclear and moreover, nothing is known about the specific function of proximal amino acid residues surrounding the copper-binding site. As a consequence, we conducted Extended X-ray Absorption Fine Structure (EXAFS) experiments on Cu-MPT