Evolution of Vitamin B2 Biosynthesis

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...

Section: Discussionmentioning

confidence: 99%

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

Sandoval

Roje

2005

“…Most eubacteria contain a bifunctional protein; for instance Bacillus subtilis RibG (BsRibG) 3 is composed of an N-terminal deaminase domain (D domain) and a C-terminal reductase domain (R domain) (4). In contrast, in fungi, plants and most archaea, these two enzymes are separate (5)(6)(7). In yeast, Rib7 is the corresponding reductase, whereas the C-terminal domain of Rib2 is responsible for deamination, in addition to N-terminal pseudouridine synthase activity.…”

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confidence: 99%

Crystal Structure of a Bifunctional Deaminase and Reductase from Bacillus subtilis Involved in Riboflavin Biosynthesis

Chen¹,

Chang²,

Chao

et al. 2006

Bacterial RibG is an attractive candidate for development of antimicrobial drugs because of its involvement in the riboflavin biosynthesis. The crystal structure of Bacillus subtilis RibG at 2.41-Å resolution displayed a tetrameric ring-like structure with an extensive interface of ϳ2400 Å 2 /monomer. The N-terminal deaminase domain belongs to the cytidine deaminase superfamily. A structurebased sequence alignment of a variety of nucleotide deaminases reveals not only the unique signatures in each family member for gene annotation but also putative substrate-interacting residues for RNA-editing deaminases. The strong structural conservation between the C-terminal reductase domain and the pharmaceutically important dihydrofolate reductase suggests that the two reductases involved in the riboflavin and folate biosyntheses evolved from a single ancestral gene. Together with the binding of the essential cofactors, zinc ion and NADPH, the structural comparison assists substrate modeling into the active-site cavities allowing identification of specific substrate recognition. Finally, the present structure reveals that the deaminase and the reductase are separate functional domains and that domain fusion is crucial for the enzyme activities through formation of a stable tetrameric structure.Flavin coenzymes are ubiquitous in all organisms because of their involvements in central metabolic pathways. Plants and many microorganisms obtain the precursor riboflavin by biosynthesis, whereas animals depend on nutritional sources. Numerous pathogenic microorganisms are unable to take up flavins from the environment and hence are absolutely dependent on their endogenous production. Therefore, the enzymes involved in riboflavin biosynthesis have the potential to become attractive candidates for the design of new defenses against antibiotic-resistant pathogens.During riboflavin biosynthesis (1), GTP cyclohydrolase II first catalyzes the hydrolytic C-8 release of GTP to yield formate and pyrophosphate as side products. The product, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5Ј-phosphate (compound 1) is converted into 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5Ј-phosphate (compound 4) by deamination of the pyrimidine ring and NAD(P)Hdependent reduction of the ribose (Fig. 1). The deamination and reduction steps have been shown to proceed in the opposite order in yeast and Escherichia coli (2, 3). Most eubacteria contain a bifunctional protein; for instance Bacillus subtilis RibG (BsRibG) 3 is composed of an N-terminal deaminase domain (D domain) and a C-terminal reductase domain (R domain) (4). In contrast, in fungi, plants and most archaea, these two enzymes are separate (5-7). In yeast, Rib7 is the corresponding reductase, whereas the C-terminal domain of Rib2 is responsible for deamination, in addition to N-terminal pseudouridine synthase activity.The D domain of bacterial RibG was expected to belong to the cytidine deaminase (CDA) superfamily because of the two conserved signatures, H(C)XE and PCX 8 -9 C. The CDA s...

“…1) (4,5). In most eubacteria, the responsible enzyme is a bifunctional protein such as Bacillus subtilis RibG (BsRibG), which is composed of an N-terminal deaminase domain (D domain) and a C-terminal reductase domain (R domain).…”

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confidence: 99%

“…2 In eubacteria and plants, DAROPP is deaminated into 5-diamino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5Ј-phosphate (AROPP) and subsequently reduced into 5-amino-6-ribitylamino-2,4 (1H,3H)-pyrimidinedione 5Ј-phosphate (ARIPP) (Fig. 1) (4,5). In most eubacteria, the responsible enzyme is a bifunctional protein such as Bacillus subtilis RibG (BsRibG), which is composed of an N-terminal deaminase domain (D domain) and a C-terminal reductase domain (R domain).…”

mentioning

confidence: 99%

Complex Structure of Bacillus subtilis RibG

Chen¹,

Lin²,

Yu³

et al. 2009

Bacterial RibG is a potent target for antimicrobial agents, because it catalyzes consecutive deamination and reduction steps in the riboflavin biosynthesis. In the N-terminal deaminase domain of Bacillus subtilis RibG, structure-based mutational analyses demonstrated that Glu 51 and Lys 79 are essential for the deaminase activity. In the C-terminal reductase domain, the complex structure with the substrate at 2.56-Å resolution unexpectedly showed a ribitylimino intermediate bound at the active site, and hence suggested that the ribosyl reduction occurs through a Schiff base pathway. Lys 151 seems to have evolved to ensure specific recognition of the deaminase product rather than the substrate. Glu 290 , instead of the previously proposed Asp 199 , would seem to assist in the proton transfer in the reduction reaction. A detailed comparison reveals that the reductase and the pharmaceutically important enzyme, dihydrofolate reductase involved in the riboflavin and folate biosyntheses, share strong conservation of the core structure, cofactor binding, catalytic mechanism, even the substrate binding architecture.Flavin coenzymes are essential for a wide variety of physiological processes particularly the redox reactions and hence are ubiquitously found in all organisms (1). The precursor riboflavin is biosynthesized in plants and many microorganisms (2, 3). The imidazole ring of GTP is first hydrolytically opened with elimination of formate by GTP cyclohydrolase II to yield 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5Ј-phosphate (DAROPP).2 In eubacteria and plants, DAROPP is deaminated into 5-diamino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5Ј-phosphate (AROPP) and subsequently reduced into 5-amino-6-ribitylamino-2,4 (1H,3H)-pyrimidinedione 5Ј-phosphate (ARIPP) ( Fig. 1) (4,5). In most eubacteria, the responsible enzyme is a bifunctional protein such as Bacillus subtilis RibG (BsRibG), which is composed of an N-terminal deaminase domain (D domain) and a C-terminal reductase domain (R domain). In contrast, in fungi and some archaea, DAROPP is first reduced into 2, 5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5Ј-phosphate (DARIPP) and then deaminated into ARIPP. The responsible enzymes are two separate proteins, Rib7 and Rib2 (6, 7). Furthermore, animals lack this biosynthetic pathway and hence must obtain riboflavin from dietary sources. Thus the enzymes of the riboflavin biosynthesis pathway have a strong possibility of becoming novel antimicrobial targets, particularly for the development of new chemotherapeutic agents to help defend against antibiotic-resistant pathogens (8, 9).Recently, we have solved the tetrameric ring-like structure of BsRibG (10). The D domain belongs to the cytidine deaminase superfamily, in which the members catalyze the hydrolytic deamination of the base moiety of a variety of nucleotides including RNA, DNA, mononucleotides, and several therapeutically useful analogues (11). Despite no significant sequence similarity, the R domain is the only protein so far to share high structural homol...