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 ofEscherichia 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...

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Bibliographic Details
Published in:The Journal of biological chemistry 2001-09, Vol.276 (37), p.34695-34701
Main Authors: Leimkühler, Silke, Wuebbens, Margot M., Rajagopalan, K.V.
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - pharmacology
Amino Acid Sequence
Bacterial Proteins - chemistry
Bacterial Proteins - isolation & purification
Bacterial Proteins - physiology
Chromatography, Gel
Coenzymes
Enzyme Activation
Escherichia coli
Escherichia coli Proteins
Metalloproteins - biosynthesis
MoaD adenylate
MoeB protein
Molecular Sequence Data
molybdenum
molybdopterin
molybdopterin synthase
Mutagenesis, Site-Directed
Nucleotidyltransferases
Protein Subunits
Pteridines
Structure-Activity Relationship
Sulfurtransferases - metabolism
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Summary:Amino acid sequence comparisons ofEscherichia 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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M102787200