Studies on flavinadenine dinucleotide-synthesizing enzyme in plants

Giri, K. V.; Rao, N. Appaji; Cama, H. R.; Kumar, Sathish

doi:10.1042/bj0750381

Cited by 32 publications

(10 citation statements)

References 5 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…FADSs from sources other than rat [31], were experimentally proven to catalyze the reverse reaction, i.e. FMN formation starting from FAD [33,34]. The rate of FMN formation from FAD by 6‐His–hFADS2 was measured by monitoring continuously the increase in flavin fluorescence (450 nm excitation, 520 nm emission) in the presence of 0.5 μ m FAD, 5 m m MgCl 2 and 0.025 or 1 m m NaPPi (Fig.…”

Section: Resultsmentioning

confidence: 99%

Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo‐flavoproteins

et al. 2011

View full text Add to dashboard Cite

A soluble form of human FAD synthase (isoform 2; hFADS2) was produced and purified to homogeneity as a recombinant His-tagged protein.The enzyme binds 1 mole of the FAD product very tightly, although noncovalently. Complete release of FAD from the 'as isolated' protein requires extensive denaturation. A 75 : 25 mixture of apo ⁄ holoprotein could be prepared by treatment with mild chaotropes, allowing estimatation of the contribution made by bound FAD to the protein stability and evaluatation of whether structural rearrangements may be required for FAD release. Under turnover conditions, the enzyme catalyzes FAD assembly from ATP and FMN and, at a much lower rate, the pyrophosphorolytic hydrolysis of FAD. Several mechanistic features of both reactions were investigated in detail, along with their dependence on environmental conditions (pH, temperature, dependence on metals). Our data indicate that FAD release may represent the rate-limiting step of the whole catalytic cycle and that the process leading to FAD synthesis, and delivery to client apoproteins may be tightly controlled.

show abstract

Section: Resultsmentioning

confidence: 99%

Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo‐flavoproteins

et al. 2011

View full text Add to dashboard Cite

show abstract

“…Using ruptured mitochondria, functional characterization of the mitochondrial RK and FADS was performed (Figs 5-7). Both of the TBY-2 mitochondrial FAD-forming enzymes are activated by MgCl 2 , a feature common to other RK(s) and FADS(s) previously characterized from prokaryotic and eukaryotic sources [20][21][22]27,28,[33][34][35][36][37][38][39][40][41][42].…”

Section: Discussionmentioning

confidence: 99%

The occurrence of riboflavin kinase and FAD synthetase ensures FAD synthesis in tobacco mitochondria and maintenance of cellular redox status

et al. 2008

View full text Add to dashboard Cite

Whereas mammals must obtain riboflavin (Rf, vitamin B 2 ) from food, plants, along with fungi and bacteria, can synthesize Rf de novo. The primary role of Rf in cell metabolism derives from its conversion into FMN and FAD, the redox cofactors of a large number of dehydrogenases, reductases and oxidases [1].Most flavoenzymes are compartmented in the cellular organelles, where they ensure the functionality of mitochondrial electron transport, photosynthesis, metabolism of fatty acids, some amino acids, choline and betaine, and synthesis of vitamin B 6 , vitamin B 12 , folate, and protoporphyrin. FAD is also the coenzyme of glutathione reductase, which mediates regeneration of reduced glutathione (GSH), a scavenger of free radicals and reactive oxygen species and a modulator of protein function by S-glutathionylation [2]. Ero1p-and sulfhydryl oxidase-dependent folding of secretory proteins also depend on FAD [3][4][5].In plants, FAD is involved in ascorbate (ASC) biosynthesis and recycling, thus playing a crucial role in cell defence against oxidative stress and in programmed cell death [6][7][8][9][10]. Interestingly, the last enzyme in the ASC biosynthetic pathway, l-galactonolactone dehydrogenase (EC 1.3.2.3), is a mitochondrial flavoenzyme [11][12][13][14][15] Intact mitochondria isolated from Nicotiana tabacum cv. Bright Yellow 2 (TBY-2) cells can take up riboflavin via carrier-mediated systems that operate at different concentration ranges and have different uptake efficiencies. Once inside mitochondria, riboflavin is converted into catalytically active cofactors, FMN and FAD, due to the existence of a mitochondrial riboflavin kinase (EC 2.7.1.26) and an FAD synthetase (EC 2.7.7.2). Newly synthesized FAD can be exported from intact mitochondria via a putative FAD exporter. The dependence of FMN synthesis rate on riboflavin concentration shows saturation kinetics with a sigmoidal shape (S 0.5 , V max and Hill coefficient values 0.32 ± 0.12 lm, 1.4 nmolAEmin )1 AEmg )1 protein and 3.1, respectively). The FAD-forming enzymes are both activated by MgCl 2 , and reside in two distinct monofunctional enzymes, which can be physically separated in mitochondrial soluble and membrane-enriched fractions, respectively.

show abstract

“…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.…”

mentioning

confidence: 99%

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

Sandoval

Roje

2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

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...

show abstract

Studies on flavinadenine dinucleotide-synthesizing enzyme in plants

Cited by 32 publications

References 5 publications

Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo‐flavoproteins

Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo‐flavoproteins

The occurrence of riboflavin kinase and FAD synthetase ensures FAD synthesis in tobacco mitochondria and maintenance of cellular redox status

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

Contact Info

Product

Resources

About