Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

Biochem Biophys Res Commun. 2006 Jun 9;344(3):1008-16. doi: 10.1016/j.bbrc.2006.04.003. Epub 2006 Apr 19.


FAD synthetase (FADS) (EC is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl(2), as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8+/-1.3nmol of FAD synthesized/min/mg protein and exhibited a K(M) value for FMN of 1.5+/-0.3microM. This is the first report on characterization of human FADS, and the first cloning and over-expression of FADS from an organism higher than yeast.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Enzyme Activation
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Gene Expression Regulation, Bacterial / physiology
  • Gene Expression Regulation, Enzymologic / physiology
  • Humans
  • Isoenzymes / biosynthesis
  • Isoenzymes / genetics
  • Molecular Sequence Data
  • Nucleotidyltransferases / analysis
  • Nucleotidyltransferases / biosynthesis*
  • Nucleotidyltransferases / chemistry*
  • Nucleotidyltransferases / genetics
  • Recombinant Proteins / biosynthesis
  • Sequence Homology, Amino Acid


  • Isoenzymes
  • Recombinant Proteins
  • Nucleotidyltransferases
  • FMN adenylyltransferase