An FMN hydrolase is fused to a riboflavin kinase homolog in plants

J Biol Chem. 2005 Nov 18;280(46):38337-45. doi: 10.1074/jbc.M500350200. Epub 2005 Sep 23.

Abstract

Riboflavin kinases catalyze synthesis of FMN from riboflavin and ATP. These enzymes have to date been cloned from bacteria, yeast, and mammals, but not from plants. Bioinformatic approaches suggested that diverse plant species, including many angiosperms, two gymnosperms, a moss (Physcomitrella patens), and a unicellular green alga (Chlamydomonas reinhardtii), encode proteins that are homologous to riboflavin kinases of yeast and mammals, but contain an N-terminal domain that belongs to the haloacid dehalogenase superfamily of enzymes. The Arabidopsis homolog of these proteins was cloned by RT-PCR, and was shown to have riboflavin kinase and FMN hydrolase activities by characterizing the recombinant enzyme produced in Escherichia coli. Both activities of the purified recombinant Arabidopsis enzyme (AtFMN/FHy) increased when the enzyme assays contained 0.02% Tween 20. The FMN hydrolase activity of AtFMN/FHy greatly decreased when EDTA replaced Mg(2+) in the assays, as expected for a member of the Mg(2+)-dependent haloacid dehalogenase family. The functional overexpression of the individual domains in E. coli establishes that the riboflavin kinase and FMN hydrolase activities reside, respectively, in the C-terminal (AtFMN) and N-terminal (AtFHy) domains of AtFMN/FHy. Biochemical characterization of AtFMN/FHy, AtFMN, and AtFHy shows that the riboflavin kinase and FMN hydrolase domains of AtFMN/FHy can be physically separated, with little change in their kinetic properties.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Arabidopsis / enzymology*
  • Arabidopsis / genetics
  • Catalysis
  • Chromatography
  • Chromatography, High Pressure Liquid
  • Cloning, Molecular
  • Computational Biology
  • Cytosol / metabolism
  • DNA, Complementary / metabolism
  • Edetic Acid / chemistry
  • Escherichia coli / metabolism
  • Expressed Sequence Tags
  • Flavin Mononucleotide / chemistry*
  • Gene Expression Regulation, Plant*
  • Humans
  • Hydrogen-Ion Concentration
  • Hydrolases / chemistry*
  • Hydrolases / metabolism
  • Kinetics
  • Magnesium / chemistry
  • Molecular Sequence Data
  • Molecular Weight
  • Phosphotransferases (Alcohol Group Acceptor) / chemistry*
  • Phosphotransferases (Alcohol Group Acceptor) / metabolism
  • Phylogeny
  • Plant Proteins / chemistry*
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Homology, Amino Acid
  • Temperature
  • Time Factors

Substances

  • DNA, Complementary
  • Plant Proteins
  • Recombinant Proteins
  • Flavin Mononucleotide
  • Edetic Acid
  • Phosphotransferases (Alcohol Group Acceptor)
  • riboflavin kinase
  • Hydrolases
  • Magnesium

Associated data

  • GENBANK/AY878327