Archaeal RibL: a new FAD synthetase that is air sensitive

Biochemistry. 2010 Oct 12;49(40):8748-55. doi: 10.1021/bi100817q. Epub 2010 Sep 20.

Abstract

FAD synthetases catalyze the transfer of the AMP portion of ATP to FMN to produce FAD and pyrophosphate (PP(i)). Monofunctional FAD synthetases exist in eukaryotes, while bacteria have bifunctional enzymes that catalyze both the phosphorylation of riboflavin and adenylation of FMN to produce FAD. Analyses of archaeal genomes did not reveal the presence of genes encoding either group, yet the archaea contain FAD. Our recent identification of a CTP-dependent archaeal riboflavin kinase strongly indicated the presence of a monofunctional FAD synthetase. Here we report the identification and characterization of an archaeal FAD synthetase. Methanocaldococcus jannaschii gene MJ1179 encodes a protein that is classified in the nucleotidyl transferase protein family and was previously annotated as glycerol-3-phosphate cytidylyltransferase (GCT). The MJ1179 gene was cloned and its protein product heterologously expressed in Escherichia coli. The resulting enzyme catalyzes the adenylation of FMN with ATP to produce FAD and PP(i). The MJ1179-derived protein has been designated RibL to indicate that it follows the riboflavin kinase (RibK) step in the archaeal FAD biosynthetic pathway. Aerobically isolated RibL is active only under reducing conditions. RibL was found to require divalent metals for activity, the best activity being observed with Co(2+), where the activity was 4 times greater than that with Mg(2+). Alkylation of the two conserved cysteines in the C-terminus of the protein resulted in complete inactivation. RibL was also found to catalyze cytidylation of FMN with CTP, making the modified FAD, flavin cytidine dinucleotide (FCD). Unlike other FAD synthetases, RibL does not catalyze the reverse reaction to produce FMN and ATP from FAD and PP(i). Also in contrast to other FAD synthetases, PP(i) inhibits the activity of RibL.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Cloning, Molecular
  • Diphosphates / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Flavin Mononucleotide / metabolism
  • Flavin-Adenine Dinucleotide / metabolism
  • Flavins / metabolism
  • Gene Expression
  • Genes, Archaeal
  • Methanococcus / enzymology*
  • Mutation
  • Nucleotidyltransferases / genetics
  • Nucleotidyltransferases / isolation & purification
  • Nucleotidyltransferases / metabolism*
  • Oxidation-Reduction
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Substrate Specificity

Substances

  • Diphosphates
  • Flavins
  • Recombinant Proteins
  • Flavin-Adenine Dinucleotide
  • Flavin Mononucleotide
  • Adenosine Triphosphate
  • Nucleotidyltransferases
  • FMN adenylyltransferase