Characterization of Escherichia coli MoeB and its involvement in the activation of molybdopterin synthase for the biosynthesis of the molybdenum cofactor

Amino acid sequence comparisons of Escherichia coli MoeB suggested that the MoeB-dependent formation of a C-terminal thiocarboxylate on the MoaD subun it of molybdopterin synthase might resemble the ubiquitin-activating step i n the ubiquitin-targeted degradation of proteins in eukaryotes. To determin e the exact role of MoeB in molybdopterin biosynthesis, the protein was pur ified after homologous overexpression. Using purified proteins, we have dem onstrated the ATP-dependent formation of a complex of MoeB and MoaD adenyla te that is stable to gel filtration. Mass spectrometry of the complex revea led a peak of a molecular mass of 9,073 Da, the expected mass of MoaD adeny late. However, unlike the ubiquitin activation reaction, the formation of a thioester intermediate between MoeB and MoaD could not be observed. There was also no evidence for a MoeB-bound sulfur during the sulfuration of MoaD . Amino acid substitutions were generated in every cysteine residue in MoeB . All of these exhibited activity comparable to the wild type, with the exc eption of mutations in cysteine residues located in putative Zn-binding mot ifs. For these cysteines, loss of activity correlated with loss of metal bi nding.