Biosynthesis of riboflavin: characterization of the bifunctional deaminase-reductase of Escherichia coli and Bacillus subtilis

Richter, Gerald; Fischer, Markus; Krieger, Cornelia; Eberhardt, Sabine; Lüttgen, Holger; Gerstenschläger, Ine; Bacher, Adelbert

doi:10.1128/jb.179.6.2022-2028.1997

Cited by 105 publications

(103 citation statements)

References 20 publications

(8 reference statements)

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“…The results also show that the PyrR enzyme parallels bacterial pyrimidine reductases in accepting either NADPH or NADH but differs in preferring NADPH (Richter et al, 1997) and that, like other plant riboflavin synthesis enzymes (Gerdes et al, 2012), both PyrR and PyrD are targeted to plastids.…”

Section: Discussionmentioning

confidence: 72%

“…Reactants and products were analyzed as their diacetyl derivatives and detected by fluorometric HPLC ( Fig. 4; Richter et al, 1997).…”

Section: Bioinformatic Analysis Of E Coli Ribd Homologs Pyrd and Pyrrmentioning

confidence: 99%

“…In E. coli, the deamination and reduction steps are catalyzed by a single bifunctional enzyme, RibD, which has N-terminal deaminase and C-terminal reductase domains that retain their respective activities when expressed separately (Richter et al, 1997;Magalhães et al, 2008). The situation in plants seems superficially similar but is in fact more complex (Gerdes et al, 2012).…”

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confidence: 99%

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Identification and Characterization of the Missing Pyrimidine Reductase in the Plant Riboflavin Biosynthesis Pathway

et al. 2012

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Riboflavin (vitamin B2) is the precursor of the flavin coenzymes flavin mononucleotide and flavin adenine dinucleotide. In Escherichia coli and other bacteria, sequential deamination and reduction steps in riboflavin biosynthesis are catalyzed by RibD, a bifunctional protein with distinct pyrimidine deaminase and reductase domains. Plants have two diverged RibD homologs, PyrD and PyrR; PyrR proteins have an extra carboxyl-terminal domain (COG3236) of unknown function. Arabidopsis (Arabidopsis thaliana) PyrD (encoded by At4g20960) is known to be a monofunctional pyrimidine deaminase, but no pyrimidine reductase has been identified. Bioinformatic analyses indicated that plant PyrR proteins have a catalytically competent reductase domain but lack essential zinc-binding residues in the deaminase domain, and that the Arabidopsis PyrR gene (At3g47390) is coexpressed with riboflavin synthesis genes. These observations imply that PyrR is a pyrimidine reductase without deaminase activity. Consistent with this inference, Arabidopsis or maize (Zea mays) PyrR (At3g47390 or GRMZM2G090068) restored riboflavin prototrophy to an E. coli ribD deletant strain when coexpressed with the corresponding PyrD protein (At4g20960 or GRMZM2G320099) but not when expressed alone; the COG3236 domain was unnecessary for complementing activity. Furthermore, recombinant maize PyrR mediated NAD(P)H-dependent pyrimidine reduction in vitro. Import assays with pea (Pisum sativum) chloroplasts showed that PyrR and PyrD are taken up and proteolytically processed. Ablation of the maize PyrR gene caused early seed lethality. These data argue that PyrR is the missing plant pyrimidine reductase, that it is plastid localized, and that it is essential. The role of the COG3236 domain remains mysterious; no evidence was obtained for the possibility that it catalyzes the dephosphorylation that follows pyrimidine reduction.

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Section: Discussionmentioning

confidence: 72%

“…Reactants and products were analyzed as their diacetyl derivatives and detected by fluorometric HPLC ( Fig. 4; Richter et al, 1997).…”

Section: Bioinformatic Analysis Of E Coli Ribd Homologs Pyrd and Pyrrmentioning

confidence: 99%

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confidence: 99%

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Identification and Characterization of the Missing Pyrimidine Reductase in the Plant Riboflavin Biosynthesis Pathway

et al. 2012

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“…In fact, all eight carbon atoms of the xylene moiety are derived from the product of the enzyme. In the biosynthetic pathway, 3,4-dihydroxy-2-butanone 4-phosphate is condensed with 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione derived from GTP (14,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). The reaction product, 6,7-dimethyl-8-ribityllumazine, undergoes a highly unusual dismutation catalyzed by riboflavin synthase, which involves the exchange of a 4-carbon fragment between two identical substrate molecules.…”

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confidence: 99%

Structure of 3,4-Dihydroxy-2-butanone 4-Phosphate Synthase from Methanococcus jannaschii in Complex with Divalent Metal Ions and the Substrate Ribulose 5-Phosphate

Steinbacher

Schiffmann

Richter

et al. 2003

Journal of Biological Chemistry

107

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Skeletal rearrangements of carbohydrates are crucial for many biosynthetic pathways. In riboflavin biosynthesis ribulose 5-phosphate is converted into 3,4-dihydroxy-2-butanone 4-phosphate while its C4 atom is released as formate in a sequence of metal-dependent reactions. Here, we present the crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal center presumably consisting of non-catalytic zinc and calcium ions at 1.7-Å resolution. The carbonyl group (O2) and two out of three free hydroxyl groups (OH3 and OH4) of the substrate are metal-coordinated. We correlate previous mutational studies on this enzyme with the present structural results. Residues of the first coordination sphere involved in metal binding are indispensable for catalytic activity. Only Glu-185 of the second coordination sphere cannot be replaced without complete loss of activity. It contacts the C3 hydrogen atom directly and probably initiates enediol formation in concert with both metal ions to start the reaction sequence. Mechanistic similarities to Rubisco acting on the similar substrate ribulose 1,5-diphosphate in carbon dioxide fixation as well as other carbohydrate (reducto-) isomerases are discussed.Riboflavin (vitamin B 2 ) is biosynthesized in plants and numerous microorganisms but not in animals, which depend on nutritional sources. Its derivatives, flavin mononucleotide (FMN) and flavinadenine dinucleotide (FAD), are indispensable in all cells where they serve a variety of redox reactions (1). They have also been shown to serve a variety of other functions in cells such as DNA photorepair (2), light sensing (3, 4), and bioluminescence (5-8) and in reactions without net-redox change (9).Gram-negative bacteria are absolutely dependent on endogenous synthesis of riboflavin, because they are devoid of an uptake system for flavins or flavocoenzymes. Hence, riboflavindeficient mutants e.g. of Escherichia coli and Salmonella sp. require extremely high concentrations of exogenous riboflavin for growth. The same is true for yeasts such as Saccharomyces cerevisiae and Candida guilliermondii (for review see Refs. 1 and 10 -13). Thus, these organisms should be vulnerable to inhibitors of the riboflavin biosynthesis, which could therefore qualify as novel anti-infective agents. The absence of the riboflavin pathway in the human host appears advantageous in this context, because host/parasite selectivity of inhibitory agents would not constitute a problem. The mechanistic and structural analysis of the riboflavin pathway could serve as the basis for the rational design of riboflavin pathway inhibitors.3,4-Dihydroxy-2-butanone 4-phosphate synthase supplies the building blocks for the assembly of the xylene ring of the vitamin (14 -17). In fact, all eight carbon atoms of the xylene moiety are derived from the product of the enzyme. In the biosynthetic pathway, 3,4-dihydroxy-2-butanone 4-phosphate is condensed with 5-amino-6-ribitylamino-2,4(1H,3H)-...

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“…Construction of Expression Plasmids for the folX and folB GenesThe expression plasmids for the folX and folB genes were constructed as described by Richter et al (16). The folX and folB genes of E. coli were amplified by PCR with chromosomal DNA from E. coli XL1-Blue as template.…”

Section: Materials-6-hydroxymethylpterinmentioning

confidence: 99%

Biosynthesis of Pteridines in Escherichia coli

Haussmann

Rohdich

Schmidt

et al. 1998

Journal of Biological Chemistry

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An open reading frame located at 69.0 kilobases on the Escherichia coli chromosome was shown to code for dihydroneopterin aldolase, catalyzing the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin in the biosynthetic pathway of tetrahydrofolate. The gene was subsequently designated folB. The FolB protein shows 30% identity to the paralogous dihydroneopterin-triphosphate epimerase, which is specified by the folX gene located at 2427 kilobases on the E. coli chromosome. The folX and folB gene products were both expressed to high yield in recombinant E. coli strains, and the recombinant proteins were purified to homogeneity. Both enzymes form homo-octamers. Aldolase can use L-threo-dihydroneopterin and D-erythro-dihydroneopterin as substrates for the formation of 6-hydroxymethyldihydropterin, but it can also catalyze the epimerization of carbon 2 of dihydroneopterin and dihydromonapterin at appreciable velocity. Epimerase catalyzes the epimerization of carbon 2 in the triphosphates of dihydroneopterin and dihydromonapterin. However, the enzyme can also catalyze the cleavage of the position 6 side chain of several pteridine derivatives at a slow rate. Steady-state kinetic parameters are reported for the various enzyme-catalyzed reactions. We propose that the polarization of the 2-hydroxy group of the substrate could serve as the initial reaction step for the aldolase as well as for the epimerase activity. A deletion mutant obtained by targeting the folX gene of E. coli has normal growth properties on complete medium as well as on minimal medium. Thus, the physiological role of the E. coli epimerase remains unknown. The open reading frame ygiG of Hemophilus influenzae specifies a protein with the catalytic properties of an aldolase. However, the genome of H. influenzae does not specify a dihydroneopterin-triphosphate epimerase.

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Biosynthesis of riboflavin: characterization of the bifunctional deaminase-reductase of Escherichia coli and Bacillus subtilis

Cited by 105 publications

References 20 publications

Identification and Characterization of the Missing Pyrimidine Reductase in the Plant Riboflavin Biosynthesis Pathway

Identification and Characterization of the Missing Pyrimidine Reductase in the Plant Riboflavin Biosynthesis Pathway

Structure of 3,4-Dihydroxy-2-butanone 4-Phosphate Synthase from Methanococcus jannaschii in Complex with Divalent Metal Ions and the Substrate Ribulose 5-Phosphate

Biosynthesis of Pteridines in Escherichia coli

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