Plant Riboflavin Biosynthesis

The Arabidopsis thaliana open reading frame At4g20960 predicts a protein whose N-terminal part is similar to the eubacterial 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5-phosphate deaminase domain. A synthetic open reading frame specifying a pseudomature form of the plant enzyme directed the synthesis of a recombinant protein which was purified to apparent homogeneity and was shown by NMR spectroscopy to convert 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5-phosphate into 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5-phosphate at a rate of 0.9 mol mg ؊1 min ؊1 . The substrate and product of the enzyme are both subject to spontaneous anomerization of the ribosyl side chain as shown by 13 C NMR spectroscopy. The protein contains 1 eq of Zn 2؉ /subunit. The deaminase activity could be assigned to the N-terminal section of the plant protein. The deaminase domains of plants and eubacteria share a high degree of similarity, in contrast to deaminases from fungi. These data show that the riboflavin biosynthesis in plants proceeds by the same reaction steps as in eubacteria, whereas fungi use a different pathway.

Section: Resultssupporting

confidence: 76%

Evolution of Vitamin B2 Biosynthesis

Fischer

Römisch

Saller

et al. 2004

“…Presence of N-terminal extensions with characteristics of chloroplast targeting peptides in plant enzymes of the riboflavin biosynthesis pathway (60 -62), and efficient import of lumazine synthase from spinach by pea chloroplasts (62), suggest that riboflavin is synthesized only in plastids. This raises the questions whether flavin nucleotides are synthesized from riboflavin in one or multiple subcellular compartments, and whether riboflavin or flavin nucleotides are transported across organellar membranes in plants.…”

Section: Discussionmentioning

confidence: 99%

Flavin Nucleotide Metabolism in Plants

Sandoval¹,

Zhang²,

Roje³

2008

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.FMN and FAD are essential cofactors for a variety of enzymes that participate in many metabolic processes in all organisms. In plants, these cofactors are required for photosynthesis, mitochondrial electron transport, fatty acid oxidation, photoreception, DNA repair, metabolism of other cofactors, and biosynthesis of many secondary metabolites. Metabolism of FMN and FAD includes reactions that synthesize and hydrolyze these flavin nucleotides. Enzymes catalyzing flavin nucleotide biosynthesis and hydrolysis, and their subcellular localization, are incompletely understood in plants and in other organisms.Phosphorylation of riboflavin to FMN and adenylylation of FMN to FAD are, respectively, catalyzed by the enzymes riboflavin kinase (EC 2.7.1.26) and FAD synthetase (EC 2.7.7.2) in the presence of ATP and Mg 2ϩ . These enzymes have been found in prokaryotes and eukaryotes.In prokaryotes, bifunctional enzymes with riboflavin kinase and FAD synthetase activities (1-5), and monofunctional enzymes with only riboflavin kinase activity (6, 7) have been described. No monofunctional FAD synthetases have yet been found in prokaryotes. Bioinformatic evidence suggests that the bifunctional enzymes are prevalent among the prokaryotic species sequenced to date (8).In ma...

“…Three of the plant enzymes involved in the biosynthesis of the precursor riboflavin (lumazine synthase, bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase, and 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5Ј-phosphate deaminase) have recently been cloned and characterized (57)(58)(59). Notably, all three contain N-terminal extensions with characteristics of chloroplast targeting peptides.…”

Section: Discussionmentioning

confidence: 99%

An FMN Hydrolase Is Fused to a Riboflavin Kinase Homolog in Plants

Sandoval

Roje

2005

The cofactors FMN and FAD participate in numerous vital processes in all organisms. Some of these processes are mitochondrial electron transport, photosynthesis, fatty acid oxidation, and metabolism of vitamins B6, B12, and folates. FMN and FAD are respectively synthesized by the enzymes riboflavin kinase (EC 2.7.1.26) and FAD synthetase (EC 2.7.7.2) in the presence of ATP and Mg 2ϩ . Bifunctional enzymes with riboflavin kinase and FAD synthetase activities have been characterized and cloned from bacteria (1-4). Biochemical characterization of the Corynebacterium ammoniagenes enzyme has established that phosphorylation of riboflavin to FMN is essentially irreversible, and that adenylylation of FMN to FAD is readily reversible (1). Homologs of bifunctional enzymes with riboflavin kinase and FAD synthetase activities from many other bacterial species exist in GenBank TM , which suggests that this type of enzyme may be ubiquitous among bacteria. Yet, this is not the only type of bacterial riboflavin kinase; monofunctional enzymes from Bacillus subtilis and Streptococcus agalactiae have been cloned and characterized (5, 6).Only monofunctional enzymes with riboflavin kinase or FAD synthetase activity have so far been studied and cloned in eukaryotes. Both enzymes have been purified from rat tissues and biochemically characterized (7-11). Three monofunctional riboflavin kinases (one from mammals and two from yeast), with sequence homology to the C-terminal domains of the bacterial bifunctional enzymes, have been cloned (12-14). The Saccharomyces cerevisiae enzyme resides in microsomes and the inner mitochondrial membrane (12). Crystal structures have been determined for riboflavin kinases from humans and Schizosaccharomyces pombe (13-15). Fad1p from S. cerevisiae is the only eukaryotic FAD synthetase that has been cloned (16). This enzyme shares little or no sequence similarity to the bacterial bifunctional enzymes with riboflavin kinase and FAD synthetase activities and probably resides in the cytosol.Although FMN and FAD play vital roles in metabolism, little is known about the enzymes that synthesize these cofactors in plants. Monofunctional riboflavin kinases or FAD synthetases have been assayed in various plant species (17)(18)(19)(20), and a monofunctional riboflavin kinase has been purified from mung bean (17). However, no plant riboflavin kinases or FAD synthetases have been cloned and fully characterized. Subcellular localization of these enzymes has not been investigated, except for a single study showing riboflavin kinase activity in the cytosol and in an organellar fraction containing chloroplasts and mitochondria in spinach (21).Enzymes that catalyze hydrolysis of FAD to FMN and AMP have been investigated in various organisms (21-30), but none have been cloned to date. Those enzymes are not specific for FAD, as they hydrolyze at least one metabolite of the group comprising NAD, NADP, ATP, ADP, CoA, and nucleotide sugars (22, 23, 26 -28). Acid phosphatases that hydrolyze FMN to riboflavin and inorganic pho...