Reference "Flavin nucleotide metabolism in plants: monofunctional enzymes synthesize fad in plastids"

Reference ID

54976

Title

Flavin nucleotide metabolism in plants: monofunctional enzymes synthesize fad in plastids

Source

J Biol Chem, 2008, vol. 283, pp. 30890-30900

Authors (3)

Sandoval-F-J	Zhang-Y
Roje-S

Abstract

FAD synthetases (EC 2.7.7.2) catalyze biosynthesis of FAD from FMN and ATP.
Monofunctional FAD synthetases are known to exist in mammals and yeast;
bifunctional enzymes also catalyzing phosphorylation of riboflavin to FMN are
known to exist in bacteria. Previously known eukaryotic enzymes with FAD
synthetase activity have no sequence similarity to prokaryotic enzymes with
riboflavin kinase and FAD synthetase activities. Proteins homologous to
bacterial bifunctional FAD synthetases, yet shorter and lacking amino acid
motifs at the C terminus, were found by bioinformatic analyses in vascular plant
genomes, suggesting that plants contain a type of FAD synthetase previously
known to exist only in prokaryotes. The Arabidopsis thaliana genome encodes two
of such proteins. Both proteins, which we named AtRibF1 and AtRibF2, carry N-
terminal extensions with characteristics of organellar targeting peptides.
AtRibF1 and AtRibF2 cDNAs were cloned by reverse transcription-PCR. Only FAD
synthetase activity was detected in the recombinant enzymes produced in
Escherichia coli. FMN and ATP inhibited both enzymes. Kinetic parameters of
AtRibF1 and AtRibF2 for the two substrates were similar. Confocal microscopy of
protoplasts transformed with enhanced green fluorescence protein-fused proteins
showed that AtRibF1 and AtRibF2 are targeted to plastids. In agreement with
subcellular localization to plastids, Percoll-isolated chloroplasts from pea
(Pisum sativum) synthesized FAD from imported riboflavin. Riboflavin kinase, FMN
hydrolase, and FAD pyrophosphatase activities were detected in Percoll-isolated
chloroplasts and mitochondria from pea. We propose from these new findings a
model for subcellular distribution of enzymes that synthesize and hydrolyze
flavin nucleotides in plants.