Riboflavin Synthase of Escherichia coli

The S41A mutant of riboflavin synthase from Escherichia coli catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a very low rate. Quenching of presteady-state reaction mixtures with trifluoroacetic acid afforded a compound with an absorption maximum at 412 nm (pH 1.0) that can be converted to a mixture of riboflavin and 6,7-dimethyl-8-ribityllumazine by treatment with wild-type riboflavin synthase. The compound was shown to qualify as a kinetically competent intermediate of the riboflavin synthase-catalyzed reaction. Multinuclear NMR spectroscopy, using various 13 C-and 15 N-labeled samples, revealed a pentacyclic structure arising by dimerization of 6,7-dimethyl-8-ribityllumazine. Enzyme-catalyzed fragmentation of this compound under formation of riboflavin can occur easily by a sequence of two elimination reactions.T he final step in the biosynthesis of riboflavin is the dismutation of 6,7-dimethyl-8-ribityllumazine (1) affording riboflavin (5) and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (6) in stoichiometric amounts (Fig. 1). The reaction involves the transfer of a 4-carbon moiety from a donor substrate molecule to an acceptor molecule.Rowan and Wood (1) showed that the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine can proceed in the absence of any catalyst under relatively mild experimental conditions. Thus, riboflavin is obtained on boiling of neutral or acidic aqueous solutions of 6,7-dimethyl-8-ribityllumazine (2, 3). The enzyme-catalyzed and the uncatalyzed reactions proceed with identical regiochemistry involving a head-to-tail arrangement of the two 4-carbon moieties from which the xylene ring of riboflavin is assembled (4-6).The enzyme-catalyzed reaction requires the simultaneous presence of two substrate molecules with an essentially antiparallel orientation at the active site of the enzyme (4). Ligandbinding studies confirmed that each subunit of the homotrimeric riboflavin synthase can bind two lumazine molecules (7).More recently, it was shown that each riboflavin synthase subunit comprises two tandem domains with similar sequence and folding topology. Each of these domains can accommodate one lumazine molecule, and the active sites are located at the interfaces of the N-and C-terminal domains, respectively (8, 9).The position 7 methyl group of 6,7-dimethyl-8-ribityllumazine (1) is unusually acidic with a pK value of 8.5. The exomethylene type lumazine anion (1a) was proposed to attack the adduct of a second substrate molecule and a nucleophile (2) resulting in the formation of a lumazine dimer (3) as an initial reaction step ( Fig. 1; refs. 4, 10, and 11). A ring-opening reaction was suggested to afford the intermediate 4.The crystal structure of riboflavin synthase is still unknown despite considerable efforts. Recently, mutants of riboflavin synthase from Escherichia coli have been identified with reduced rates of riboflavin formation (12). Specifically, an S41A mutant catalyzed the formation of riboflavin at a rate that was lower by several orde...

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“…[U- (15). The recombinant E. coli strain BL21 (DE3) expressing the E. coli riboflavin synthase S41A mutant is described elsewhere (12).…”

Section: Methodsmentioning

confidence: 99%

“…Recently, mutants of riboflavin synthase from Escherichia coli have been identified with reduced rates of riboflavin formation (12). Specifically, an S41A mutant catalyzed the formation of riboflavin at a rate that was lower by several orders of magnitude than the rate of the wild-type enzyme.…”

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confidence: 99%

A pentacyclic reaction intermediate of riboflavin synthase

Illarionov

Eisenreich

Bacher

2001

Proc. Natl. Acad. Sci. U.S.A.

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“…The enzyme does not require cofactors or metal ions for activity, and systematic mutagenesis failed to identify amino acid residues that could be assigned a specific role in enzyme catalysis (16). Although certain modifications of the N-terminal amino acid sequence are conducive to the loss of catalytic activity, the respective amino acids are not in direct contact with the substrate and must exert their influence by indirect effects on the active site topology.…”

Section: Resultsmentioning

confidence: 99%

“…A recombinant strain of E. coli engineered for overexpression of the ribC gene specifying riboflavin synthase of E. coli has been described elsewhere (16).…”

Section: Methodsmentioning

confidence: 99%

Presteady State Kinetic Analysis of Riboflavin Synthase

Illarionov

Haase

Bacher

et al. 2003

Journal of Biological Chemistry

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Riboflavin synthase catalyzes a mechanistically complex dismutation affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione from 6,7-dimethyl-8-ribityllumazine. The kinetics of the enzyme from Escherichia coli were studied under single turnover conditions. Stopped flow as well as quenched flow experiments documented the transient formation of a pentacyclic reaction intermediate. No other transient species were sufficiently populated to allow detection. The data are best described by a sequence of one second order and one first order reaction.Riboflavin synthase catalyzes a complex dismutation affording riboflavin (8) and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (9) from 6,7-dimethyl-8-ribityllumazine (1) (1). The enzyme-catalyzed dismutation can be described as an exchange of a 4-carbon unit between one substrate molecule acting as donor and a second substrate molecule acting as acceptor (typically, however, dismutation reactions involve the exchange of simple particles, e.g. hydride anions) (1, 2). The regiochemical features of the reaction require an antiparallel arrangement of the two identical substrate molecules at the active site of the enzyme (3-5).Riboflavin synthases of Bacillus subtilis and Escherichia coli are homotrimers of 23.4-kDa subunits (6, 7). Each subunit folds into two closely similar domains (7,8). The homotrimeric enzymes have six substrate-binding sites (one binding site each on each N-terminal and C-terminal domain, respectively, of each subunit) (9 -11). Surprisingly, the crystal structure of riboflavin synthase from E. coli shows an inherently asymmetric molecule where only a single N-terminal domain and a single C-terminal domain are appropriately oriented for catalysis of a two-substrate reaction (8).Recently, we reported the formation of the pentacyclic lumazine dimer 6 from 6,7-dimethyl-8-ribityllumazine by the S41A mutant of E. coli riboflavin synthase (12). The native enzyme can generate 6,7-dimethyl-8-ribityllumazine (backward reaction) as well as a mixture of riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (forward reaction) from that adduct, which fulfills the criteria for a kinetically competent reaction intermediate. The hypothetical reaction mechanism shown in Fig. 1 combines the earlier mechanistic suggestions of Rowan and Wood (13) and Beach and Plaut (14) with the more recent findings.This paper describes presteady state kinetic studies on this mechanistically complex enzyme-catalyzed reaction. and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione were synthesized by published procedures (15). A recombinant strain of E. coli engineered for overexpression of the ribC gene specifying riboflavin synthase of E. coli has been described elsewhere (16). EXPERIMENTAL PROCEDURES MaterialsProtein Purification-All procedures were performed at 4°C unless otherwise stated. Frozen cell mass (5 g) was thawed in 25 ml of 50 mM Tris hydrochloride, pH 7.2, containing 0.5 mM EDTA and 0.5 mM dithiothreitol (buffer A). The suspension was subjecte...

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“…The ribE gene encodes 6-7-dimethyl-8-ribityllumazine synthase, an enzyme that catalyzes the penultimate step in the riboflavin biosynthesis pathway. [16][17][18] Our data suggest that the valine 54 to glycine mutation of RibE is responsible for the temperature sensitivity of the Keio dbpA deletion strain. To further confirm the involvement of this mutation in the growth defect of our strain, RibE was ectopically expressed and growth examined at the nonpermissive temperature.…”

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confidence: 90%