Biocatalytic in Vitro and in Vivo FMN Prenylation and (De)carboxylase Activation.

Reversible UbiD-like (de)carboxylases represent a large family of mostly uncharacterized enzymes, which require the recently discovered prenylated FMN (prFMN) cofactor for activity. Functional characterization of novel UbiDs is hampered by a lack of robust protocols for prFMN generation and UbiD activation. Here, we report two systems for in vitro and in vivo FMN prenylation and UbiD activation under aerobic conditions. The in vitro one-pot prFMN cascade includes five enzymes: FMN prenyltransferase (UbiX), prenol kinase, polyphosphate kinase, formate dehydrogenase, and FMN reductase, which use prenol, polyphosphate, formate, ATP, NAD+, and FMN as substrates and cofactors. Under aerobic conditions, this cascade produced prFMN from FMN with over 98% conversion and activated purified ferulic acid decarboxylase Fdc1 from Aspergillus niger and protocatechuic acid decarboxylase ENC0058 from Enterobacter cloaceae. The in vivo system for FMN prenylation and UbiD activation is based on the coexpression of Fdc1 and UbiX in Escherichia coli cells under aerobic conditions in the presence of prenol. The in vitro and in vivo FMN prenylation cascades will facilitate functional characterization of novel UbiDs and their applications.

Structure-Activity Relationship of Flavin Analogues That Target the Flavin Mononucleotide Riboswitch.

The flavin mononucleotide (FMN) riboswitch is an emerging target for the development of novel RNA-targeting antibiotics. We previously discovered an FMN derivative, 5FDQD, that protects mice against diarrhea-causing Clostridium difficile bacteria. Here, we present the structure-based drug design strategy that led to the discovery of this fluoro-phenyl derivative with antibacterial properties. This approach involved the following stages: (1) structural analysis of all available free and bound FMN riboswitch structures; (2) design, synthesis, and purification of derivatives; (3) in vitro testing for productive binding using two chemical probing methods; (4) in vitro transcription termination assays; and (5) resolution of the crystal structures of the FMN riboswitch in complex with the most mature candidates. In the process, we delineated principles for productive binding to this riboswitch, thereby demonstrating the effectiveness of a coordinated structure-guided approach to designing drugs against RNA.

Site-Selective Synthesis of (15)N- and (13)C-Enriched Flavin Mononucleotide Coenzyme Isotopologues.

Flavin mononucleotide (FMN) is a coenzyme for numerous proteins involved in key cellular and physiological processes. Isotopically labeled flavin is a powerful tool for studying the structure and mechanism of flavoenzyme-catalyzed reactions by a variety of techniques, including NMR, IR, Raman, and mass spectrometry. In this report, we describe the preparation of labeled FMN isotopologues enriched with (15)N and (13)C isotopes at various sites in the pyrazine and pyrimidine rings of the isoalloxazine core of the cofactor from readily available precursors by a five-step chemo-enzymatic synthesis.

Modified-release capsules containing sodium riboflavin 5'-phosphate.

INTRODUCTION: The focus of this work was to produce delayed-release capsules containing riboflavin (vitamin B2, as API) layered pellets. Riboflavin therapy is indicated in patients with a riboflavin deficiency, which usually occurs in conjunction with malabsorption, alcoholism or a protein-calorie deficiency and rarely as the sole vitamin deficiency. Riboflavin is readily absorbed from the upper gastrointestinal tract by a specific transport mechanism. The dissolution rate of coated capsules was controlled through the coating of the capsules and the thickness of the coating layer. METHODS: The core pellets (Cellet 300) were loaded with a 10% aqueous solution of sodium riboflavin 5'-phosphate by a layering technique in a coating pan. Hard capsules were filled with riboflavin layered pellets and coated with Eudragit NE polymer with different coating layer thicknesses. The dissolution was tested in gastric and intestinal fluids with the half-change method. The dissolution profiles were analyzed with the use of different mathematical models and an attempt was made to predict the optimum coating film thickness that ensures the required degree and rate of dissolution. RESULTS: A new solid dosage form was developed which can enhance the bioavailability of riboflavin. RRSBW distribution and the Chapman-Richards growth function were used to fit the dissolution profiles. Statistical analysis indicated that the best products were described by the Chapman-Richards equation. The results were utilized to create a theoretical model suitable for prediction of the optimum film thickness that ensures the required release of riboflavin.

Rapid one-pot synthesis of riboflavin isotopomers.

Flavocoenzymes labeled with stable isotopes are important reagents for the study of flavoproteins using isotope-sensitive methods such as NMR, ENDOR, infrared, and Raman spectroscopy. We describe highly versatile one-pot methods for the preparation of riboflavin isotopomers labeled with (13)C in every desired position of the xylene moiety. The starting materials are commercially available (13)C-labeled glucose samples, which are converted into riboflavin using enzymes of the oxidative pentose phosphate pathway in combination with recombinant enzymes of the riboflavin biosynthetic pathway. The overall reaction comprises six enzyme-catalyzed reaction steps for the synthesis of the vitamin and two auxiliary enzymes for in situ recycling of cofactors. The overall yields of riboflavin based on isotope-labeled glucose are 35-50%.

Influence of 8 alpha-imidazole substitution of the FMN cofactor on the rate of electron transfer from the neutral semiquinones of two flavodoxins to cytochrome c.

The effects of substituting an imidazole ring onto the 8 alpha-position of the FMN cofactor on the kinetics of electron transfer from the neutral semiquinone forms of Azotobacter and Clostridium flavodoxins to oxidized horse heart cytochrome c have been investigated by stopped-flow methods. Although 8 alpha-substitution does not alter the mechanistic pathway of the reaction, the rate constants are decreased by factors of 10-30, without significant changes in the equilibrium association constants of the intermediate electron-transfer complexes. Protonation of the imidazole ring further decreases the observed second-order rate constants for the electron-transfer reaction by factors of 20-50. The pKa values for the 8 alpha-imidazole ring in both flavodoxin semiquinones were determined to be approximately 7. In contrast, the reactions of the native flavodoxins with cytochrome c are pH independent. The results are consistent with a structural model of the intermediate complex [Simondsen, R. P., Weber, P. C., Salemme, F. R., & Tollin, G. (1982) Biochemistry 21, 6366-6375], which postulates a close fit between the exposed dimethylbenzene ring of the FMN and the heme edge within a nonpolar interface region. The results further indicate that electron transfer is uncoupled from proton transfer, that it is the rate-limiting step, and that it occurs prior to proton transfer at all pH values. Finally, the results do not provide support for a direct role of the imidazole ring in the facilitation of one-electron transfer in those enzymes containing 8 alpha-N-histidylflavin coenzymes.

Enzymatic synthesis of riboflavin and FMN specifically labeled with 13C in the xylene ring.

The condensation of 3-hydroximino-2-butanone (1) with 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (2) yields 6,7-dimethyl-8-ribityllumazine (3). At slightly alkaline pH, the carbonyl group of 1 reacts preferentially with the 5-amino group of 2 (regioselectivity, 4:1). Under acidic conditions, the reaction occurs with higher yield and marginal regioselectivity of opposite direction (1:1.4). Appropriately 13C-labeled samples of 1 afford 3 labeled at C-6 alpha, C-6, C-7 or C-7 alpha. [6 alpha, 7 alpha-13C2]-3 was prepared by condensation of 2 with [1,4-13C2]diacetyl. The lumazines 3 were converted to riboflavin by the enzyme, riboflavin synthase, with almost quantitative yield. By this procedure, any C-atom of the carbocyclic moiety of riboflavin can be selectively labeled with 13C at high abundance. Phosphorylation yields the respectively 13C-labeled FMN samples.

Absolute stereochemistry of flavins in enzyme-catalyzed reactions.

The 8-demethyl-8-hydroxy-5-deaza-5-carba analogues of FMN and FAD have been synthesized. Several apoproteins of flavoenzymes were successfully reconstituted with these analogues. This and further tests established that these analogues could serve as general probes for flavin stereospecificity in enzyme-catalyzed reactions. The method used by us involved stereoselective introduction of label on one enzyme combined with transfer to and analysis on a second enzyme. Using as a reference glutathione reductase from human erythrocytes for which the absolute stereochemistry of catalysis is known from X-ray studies [Pai, E. F., & Schulz, G. E. (1983) J. Biol. Chem. 258, 1752-1758], we were able to determine the absolute stereospecificities of other flavoenzymes. We found that glutathione reductase (NADPH), general acyl-CoA dehydrogenase (acyl-CoA), mercuric reductase (NADPH), thioredoxin reductase (NADPH), p-hydroxybenzoate hydroxylase (NADPH), melilotate hydroxylase (NADH), anthranilate hydroxylase (NADPH), and glucose oxidase (glucose) all use the re face of the flavin ring when interacting with the substrates given in parentheses.

8alpha-hydroxyflavinmononucleotide and related compounds.

2', 3', 4'-Triacetyl-FMN has been transformed by selective radical bromination into 2', 3', 4'-triacetyl-8alpha-bromo-FMN, and the following hydrolysis of the latter has afforded 8alpha-hydroxy-FMN. The presence of the hydroxy group in the 8alpha position of 8alpha-hydroxy-FMN is confirmed by its acetylation into 2', 3'-diacetyl-8alpha-acetoxyriboflavin-4', 5'-cyclophosphate. The absorption spectra of the synthesized compounds have shown the reduction of the extinction ratios of the first and second absorption maxima in comparison with the extinction of the same maxima for 8alpha-hydroxyriboflavin. Unlike FMN, fluorescence quenching for 8alpha-hydroxy-FMN has been found.