The Diverse Roles of Flavin Coenzymes Nature's Most Versatile Thespians (original) (raw)
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Unusual non-enzymatic flavin catalysis enhances understanding of flavoenzymes
FEBS Letters, 2015
Flavin cofactors are central to many biochemical transformations and are typically tightly bound as part of a catalytically active flavoenzyme. This work indicates that naturally occurring flavins can act as stand-alone catalysts to promote the oxidation of biosynthetically inspired heterocycles in aqueous buffers. Flavin activity was compared with that of oxidases important in non-ribosomal peptide synthesis, providing a rare direct comparison between the catalytic efficacy of flavins alone and in the context of a full flavoenzyme. This study suggests that such oxidases are likely to possess an active site base, as oxidase activity was greater than that of flavins alone, particularly for less acidic substrates. These findings offer perspective on the development of robust and catalytically effective, designed miniature flavoenzymes.
Current Opinion in Chemical Biology, 2007
Flavoenzymes are colourful oxidoreductases that catalyze a large variety of different types of reactions. Flavoenzymes have been extensively studied for their structural and mechanistic properties and are gaining momentum in industrial biocatalytic applications. Some of these enzymes catalyze the oxidative modification of protein substrates. New insights in oxidative flavoenzymes and in particular in novel family members point towards their potential application in the pharmaceutical, finechemical and food industries. 23. Ko HS, Yokoyama Y, Ohno N, Okadome M, Amachi S, Shinoyama H, Fujii T: Purification and characterization of intracellular and extracellular, thermostable and alkali-tolerant alcohol oxidases produced by a thermophilic fungus, Thermoascus aurantiacus NBRC 31693. J Biosci Bioeng 2005, 99:348-353. 24. Ferreira P, Ruiz-Duenas FJ, Martinez MJ, van Berkel WJH, Martinez AT: Site-directed mutagenesis of selected residues at the active site of aryl-alcohol oxidase, an H 2 O 2 -producing ligninolytic enzyme. FEBS J 2006, 273:4878-4888. 25. van den Heuvel RH, van den Berg WA, Rovida S, van Berkel WJH: Laboratory-evolved vanillyl-alcohol oxidase produces natural vanillin.
Same Substrate, Many Reactions: Oxygen Activation in Flavoenzymes
Chemical Reviews, 2018
Over time, organisms have evolved strategies to cope with the abundance of dioxygen on Earth. Oxygen-utilizing enzymes tightly control the reactions involving O 2 mostly by modulating the reactivity of their cofactors. Flavins are extremely versatile cofactors that are capable of undergoing redox reactions by accepting either one electron or two electrons, alternating between the oxidized and the reduced states. The physical and chemical principles of flavin-based chemistry have been investigated widely. In the following pages we summarize the state of the art on a key area of research in flavin enzymology: the molecular basis for the activation of O 2 by flavin-dependent oxidases and monooxygenases. In general terms, oxidases use O 2 as an electron acceptor to produce H 2 O 2 , while monooxygenases activate O 2 by forming a flavin intermediate and insert an oxygen atom into the substrate. First, we analyze how O 2 reaches the flavin cofactor embedded in the protein matrix through dedicated access pathways. Then we approach O 2 activation from the perspective of the monooxygenases, their preferred intermediate, the C(4a)−(hydro)peroxyflavin, and the cases in which other intermediates have been described. Finally, we focus on understanding how the architectures developed in the active sites of oxidases promote O 2 activation and which other factors operate in its reactivity.
Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis
Frontiers in Chemistry, 2015
The primary role of the water-soluble vitamin B 2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in a broad spectrum of biological activities, among which energetic metabolism and chromatin remodeling. Subcellular localisation of FAD synthase (EC 2.7.7.2, FADS), the second enzyme in the FAD forming pathway, is addressed here in HepG2 cells by confocal microscopy, in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalyzed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesizing activity, hFADS is able to operate as a FAD "chaperone." The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear lysine-specific demethylase 1 (LSD1) or a mitochondrial dimethylglycine dehydrogenase (Me 2 GlyDH, EC 1.5.8.4). Both enzymes carry out similar reactions of oxidative demethylation, in which tetrahydrofolate is converted into 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells.
Flavoprotein oxidases: classification and applications
Applied Microbiology and Biotechnology, 2013
This review provides an overview of oxidases that utilise a flavin cofactor for catalysis. This class of oxidative flavoenzymes has shown to harbour a large number of biotechnologically interesting enzymes. Applications range from their use as biocatalysts for the synthesis of pharmaceutical compounds to the integration in biosensors. Through the recent developments in genome sequencing, the number of newly discovered oxidases is steadily growing. Recent progress in the field of flavoprotein oxidase discovery and the obtained biochemical knowledge on these enzymes are reviewed. Except for a structure-based classification of known flavoprotein oxidases, also their potential in recent biotechnological applications is discussed.
A novel two-protein component flavoprotein hydroxylase
European Journal of Biochemistry, 2001
p-Hydroxyphenylacetate (HPA) hydroxylase (HPAH) was purified from Acinetobacter baumannii and shown to be a two-protein component enzyme. The small component (C 1 ) is the reductase enzyme with a subunit molecular mass of 32 kDa. C 1 alone catalyses HPA-stimulated NADH oxidation without hydroxylation of HPA. C 1 is a flavoprotein with FMN as a native cofactor but can also bind to FAD. The large component (C 2 ) is the hydroxylase component that hydroxylates HPA in the presence of C 1 . C 2 is a tetrameric enzyme with a subunit molecular mass of 50 kDa and apparently contains no redox centre. FMN, FAD, or riboflavin could be used as coenzymes for hydroxylase activity with FMN showing the highest activity. Our data demonstrated that C 2 alone was capable of utilizing reduced FMN to form the product 3,4-dihydroxyphenylacetate. Mixing reduced flavin with C 2 also resulted in the formation of a flavin intermediate that resembled a C(4a)-substituted flavin species indicating that the reaction mechanism of the enzyme proceeded via C(4a)substituted flavin intermediates. Based on the available evidence, we conclude that the reaction mechanism of HPAH from A. baumannii is similar to that of bacterial luciferase. The enzyme uses a luciferase-like mechanism and reduced flavin (FMNH 2 , FADH 2 , or reduced riboflavin) to catalyse the hydroxylation of aromatic compounds, which are usually catalysed by FAD-associated aromatic hydroxylases. Abbreviations: HPA, p-hydroxyphenylacetate; HPAH, p-hydroxyphenylacetate hydroxylase; DHPA, 3,4-dihydroxyphenylacetate; DHPAO, 3,4-dihydroxyphenylacetate dioxygenase; CHS, 5-carboxymethylmuconate semialdehyde; C 1 , the small component of Acinetobacter baumannii HPAH; C 2 , the large component of Acinetobacter baumannii HPAH; hpaC, the small component of Escherishia coli HPAH. Eur. J. Biochem. 268, 5550-5561 (2001) q FEBS 2001 q FEBS 2001 p-Hydroxyphenylacetate hydroxylase from A. baumannii (Eur. J. Biochem. 268) 5551 q FEBS 2001 p-Hydroxyphenylacetate hydroxylase from A. baumannii (Eur. J. Biochem. 268) 5557
Flavin oxidation in flavin-dependent N-monooxygenases
Protein science : a publication of the Protein Society, 2018
Siderophore A (SidA) from Aspergillus fumigatus is a flavin-containing monooxygenase that hydroxylates ornithine (Orn) at the amino group of the side chain. Lysine (Lys) also binds to the active site of SidA; however, hydroxylation is not efficient and H O is the main product. The effect of pH on steady-state kinetic parameters were measured and the results were consistent with Orn binding with the side chain amino group in the neutral form. From the pH dependence on flavin oxidation in the absence of Orn, a pK value >9 was observed and assigned to the FAD-N5 atom. In the presence of Orn, the pH dependence displayed a pK value of 6.7 ± 0.1 and of 7.70 ± 0.10 in the presence of Lys. Q102 interacts with NADPH and, upon mutation to alanine, leads to destabilization of the C4a-hydroperoxyflavin (FAD ). Flavin oxidation with Q102A showed a pK value of ~8.0. The data are consistent with the pK of the FAD N5-atom being modulated to a value > 9 in the absence of Orn, which aids in the...
Stereochemistry and accessibility of prosthetic groups in flavoproteins
Biochemistry, 1988
Using 8-demethyl-8-hydroxy-5-deaza-5-carba analogues of the appropriate flavin nucleotides, we determined the stereochemistry of interaction between coenzyme and substrate for several flavoproteins. The enzymes were D-amino acid oxidase, L-lactate oxidase, and D-lactate dehydrogenase, all three of which interact with pyruvate, as well as cyclohexanone monooxygenase and 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase, which were both probed with nicotinamide nucleotides. L-Lactate oxidase and D-lactate dehydrogenase used the si face of the modified flavin ring while the other three enzymes showed re-side specificity. This selection of flavoenzymes includes FAD-and FMN-dependent enzymes, enzymes that follow a carbanion mechanism, and others that have hydride transfer as an integral part of their reaction pathway. is a flavin analogue whose specific features make it well suited for probing stereospecificities of flavin coenzymes in enzymic reactions. Its shape and charge usually do not interfere with binding at active sites of enzymes; it can be transformed to the FMN and FAD levels, respectively, by the well-characterized riboflavin kinase/FAD synthetase from Brevibacterium ammoniagenes