Insights into molybdenum cofactor deficiency provided by the crystal structure of the molybdenum cofactor biosynthesis protein MoaC

Background: The molybdenum cofactor (Moco) is an essential component of a l arge family of enzymes involved in important transformations in carbon, nit rogen and sulfur metabolism. The Moco biosynthetic pathway is evolutionaril y conserved and found in archaea, eubacteria and eukaryotes. In humans, gen etic deficiencies of enzymes involved in this pathway trigger an autosomal recessive and usually deadly disease with severe neurological symptoms. The MoaC protein, together with the MoaA protein, is involved in the first ste p of Moco biosynthesis. Results: MoaC from Escherichia coli has been expressed and purified to homo geneity and its crystal structure determined at 2 Angstrom resolution. The enzyme is organized into a tightly packed hexamer with 32 symmetry. The mon omer consists of an antiparallel, four-stranded beta sheet packed against t wo long alpha helices, and its fold belongs to the ferredoxin-like family. Analysis of structural and biochemical data strongly suggests that the acti ve site is located at the interface of two monomers in a pocket that contai ns several strictly conserved residues. Conclusions: Asp128 in the putative active site appears to be important for catalysis as its replacement with alanine almost completely abolishes prot ein activity. The structure of the Asp128-->Ala variant reveals substantial conformational changes in an adjacent loop. In the human MoaC ortholog, su bstitution of Thrl 82 with proline causes Moco deficiency, and the correspo nding substitution in MoaC severely compromises activity. This residue is l ocated near the N-terminal end of helix alpha 4 at an interface between two monomers, The MoaC structure provides a framework for the analysis of addi tional dysiunctional mutations in the corresponding human gene.