MocA is a specific cytidylyltransferase involved in molybdopterin cytosine dinucleotide biosynthesis in Escherichia coli

J Biol Chem. 2009 Aug 14;284(33):21891-21898. doi: 10.1074/jbc.M109.008565. Epub 2009 Jun 19.

Abstract

We have purified and characterized a specific CTP:molybdopterin cytidylyltransferase for the biosynthesis of the molybdopterin (MPT) cytosine dinucleotide (MCD) cofactor in Escherichia coli. The protein, named MocA, shows 22% amino acid sequence identity to E. coli MobA, the specific GTP:molybdopterin guanylyltransferase for molybdopterin guanine dinucleotide biosynthesis. MocA is essential for the activity of the MCD-containing enzymes aldehyde oxidoreductase YagTSR and the xanthine dehydrogenases XdhABC and XdhD. Using a fully defined in vitro assay, we showed that MocA, Mo-MPT, CTP, and MgCl2 are required and sufficient for MCD biosynthesis in vitro. The activity of MocA is specific for CTP; other nucleotides such as ATP and GTP were not utilized. In the defined in vitro system a turnover number of 0.37+/-0.01 min(-1) was obtained. A 1:1 binding ratio of MocA to Mo-MPT and CTP was determined to monomeric MocA with dissociation constants of 0.23+/-0.02 microm for CTP and 1.17+/-0.18 microm for Mo-MPT. We showed that MocA was also able to convert MPT to MCD in the absence of molybdate, however, with only one catalytic turnover. The addition of molybdate after one turnover gave rise to a higher MCD production, revealing that MCD remains bound to MocA in the absence of molybdate. This work presents the first characterization of a specific enzyme involved in MCD biosynthesis in bacteria.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Aldehyde Oxidoreductases / metabolism
  • Cytosine Nucleotides / metabolism*
  • Dose-Response Relationship, Drug
  • Escherichia coli / enzymology*
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / metabolism*
  • Gene Expression Regulation, Bacterial*
  • Gene Expression Regulation, Enzymologic*
  • Guanosine Triphosphate / metabolism
  • Kinetics
  • Models, Biological
  • Mutation
  • Protein Binding
  • Pterins / metabolism*
  • Time Factors
  • Xanthine Dehydrogenase / metabolism

Substances

  • Cytosine Nucleotides
  • Escherichia coli Proteins
  • Pterins
  • molybdopterin cytosine dinucleotide
  • Guanosine Triphosphate
  • Adenosine Triphosphate
  • Xanthine Dehydrogenase
  • Aldehyde Oxidoreductases