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

LeimkУМhler S, Wuebbens MM, Rajagopalan KV.

Department of Biochemistry, Duke University Medical Center, Durham, North
Carolina 27710, USA.

Amino acid sequence comparisons of Escherichia coli MoeB suggested that the
MoeB-dependent formation of a C-terminal thiocarboxylate on the MoaD subunit of
molybdopterin synthase might resemble the ubiquitin-activating step in the
ubiquitin-targeted degradation of proteins in eukaryotes. To determine the exact
role of MoeB in molybdopterin biosynthesis, the protein was purified after
homologous overexpression. Using purified proteins, we have demonstrated the
ATP-dependent formation of a complex of MoeB and MoaD adenylate that is stable to
gel filtration. Mass spectrometry of the complex revealed a peak of a molecular
mass of 9,073 Da, the expected mass of MoaD adenylate. 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 exception of mutations in cysteine residues located in
putative Zn-binding motifs. For these cysteines, loss of activity correlated with
loss of metal binding.