Flavin Reductase Research Articles

Cryptochromes Mediate Intrinsic Photomechanical Transduction in Avian Iris and Somatic Striated Muscle.

Irises isolated from the eyes of diverse species constrict when exposed to light. Depending on species this intrinsic photomechanical transduction response (PMTR) requires either melanopsin or cryptochrome (CRY) photopigment proteins, generated by their respective association with retinoid or flavin adenine dinucleotide (FAD) chromophores. Although developmentally relevant circadian rhythms are also synchronized and reset by these same proteins, the cell type, mechanism, and specificity of photomechanical transduction (PMT) and its relationship to circadian processes remain poorly understood. Here we show that PMTRs consistent with CRY activation by 430 nm blue light occur in developing chicken iris striated muscle, identify relevant mechanisms, and demonstrate that similar PMTRs occur in striated iris and pectoral muscle fibers, prevented in both cases by knocking down CRY gene transcript levels. Supporting CRY activation, iris PMTRs were reduced by inhibiting flavin reductase, but unaffected by melanopsin antagonism. The largest iris PMTRs paralleled the developmental predominance of striated over smooth muscle fibers, and shared their requirement for extracellular Ca2+ influx and release of intracellular Ca2+. Photo-stimulation of identified striated myotubes maintained in dissociated culture revealed the cellular and molecular bases of PMT. Myotubes in iris cell cultures responded to 435 nm light with increased intracellular Ca2+ and contractions, mimicking iris PMTRs and their spectral sensitivity. Interestingly PMTRs featuring contractions and requiring extracellular Ca2+ influx and release of intracellular Ca2+ were also displayed by striated myotubes derived from pectoral muscle. Consistent with these findings, cytosolic CRY1 and CRY2 proteins were detected in both iris and pectoral myotubes, and knocking down myotube CRY1/CRY2 gene transcript levels specifically blocked PMTRs in both cases. Thus CRY-mediated PMT is not unique to iris, but instead reflects a more general feature of developing striated muscle fibers. Because CRYs are core timing components of circadian clocks and CRY2 is critical for circadian regulation of myogenic differentiation CRY-mediated PMT may interact with cell autonomous clocks to influence the progression of striated muscle development.

Biochemical characterization of biliverdins IXβ/δ generated by a selective heme oxygenase.

The pro-oxidant effect of free heme (Fe2+-protoporphyrin IX) is neutralized by phylogenetically-conserved heme oxygenases (HMOX) that generate carbon monoxide, free ferrous iron, and biliverdin (BV) tetrapyrrole(s), with downstream BV reduction by non-redundant NADPH-dependent BV reductases (BLVRA and BLVRB) that retain isomer-restricted functional activity for bilirubin (BR) generation. Regioselectivity for the heme α-meso carbon resulting in predominant BV IXα generation is a defining characteristic of canonical HMOXs, thereby limiting generation and availability of BVs IXβ, IXδ, and IXγ as BLVRB substrates. We have now exploited the unique capacity of the Pseudomonas aeruginosa (P. aeruginosa) hemO/pigA gene for focused generation of isomeric BVs (IXβ and IXδ). A scalable system followed by isomeric separation yielded highly pure samples with predicted hydrogen-bonded structure(s) as documented by 1H NMR spectroscopy. Detailed kinetic studies established near-identical activity of BV IXβ and BV IXδ as BLVRB-selective substrates, with confirmation of an ordered sequential mechanism of BR/NADP+ dissociation. Halogenated xanthene-based compounds previously identified as BLVRB-targeted flavin reductase inhibitors displayed comparable inhibition parameters using BV IXβ as substrate, documenting common structural features of the cofactor/substrate-binding pocket. These data provide further insights into structure/activity mechanisms of isomeric BVs as BLVRB substrates, with potential applicability to further dissect redox-regulated functions in cytoprotection and hematopoiesis.

Cloning and expression of the flavin reductase LuxG from Photobacterium leiognathi YL and its improvement for NADH detection.

In the present study, we aimed to purify and characterize LuxG obtained from Photobacterium leiognathi YL and examine its improvement for NADH detection. To this end, we cloned and expressed the putative luxG gene of P. leiognathi YL in the Escherichia coli BL21 strain. The product of luxG is a flavin reductase that consists of 206 amino acids, corresponding to a subunit molecular mass of ∼26 kDa. Phylogenetic analysis demonstrated that P. leiognathi YL LuxG has a rather distant evolutionary relationship with Frase I of Aliivibrio fischeri and Frp of Vibrio harveyi, but a close evolutionary relationship with Fre from Escherichia coli, which are all enzymes related to oxido-reductase. Further comparison shows that the changes in the functionally conserved sites may contribute to the functional divergence of LuxG and Fre. LuxG could supply reduced flavin mononucleotide (FMN) for bacterial luminescence by catalyzing the oxidation of nicotinamide adenine dinucleotide hydrogen (NADH). Based on this, a coupled pure enzyme bioluminescent system was established and used for NADH detection. The NADH samples with concentrations of 0.1-1 nM were used to validate the linear relationship, and it was found that the logarithmic deviations were less than 3%, which showed more sensitive and stable results than the NADH detection by recombinant E. coli including the exogenously expressed luciferase and intrinsic Fre. Investigation of P. leiognathi YL LuxG would provide a basic understanding of its evolution, and structural and functional properties, which might contribute to the development of a NADH detection kit in the future.

Increasing the catalytic power of the flavin reductase DszD enzyme using site-directed mutagenesis method in Rhodococcus erythropolis

Increasing the catalytic power of the flavin reductase DszD enzyme using site-directed mutagenesis method in Rhodococcus erythropolis

Identification of the NADH-oxidase gene in Trichomonas vaginalis

The microaerophilic human parasite Trichomonas vaginalis causes infections in the urogenital tract and is one of the most often sexually transmitted pathogens worldwide. Due to its anaerobic metabolism, it has to quickly remove intracellular oxygen in order to avoid deactivation of essential metabolic enzymes such as oxygen-sensitive pyruvate:ferredoxin oxidoreductase (PFOR). Two major enzyme activities which are responsible for the removal, i.e. reduction, of molecular oxygen have been identified in T. vaginalis flavin reductase, formerly designated NADPH oxidase, which indirectly reduces oxygen to hydrogen peroxide via flavin mononucleotide (FMN), and NADH oxidase which reduces oxygen to water. Flavin reductase has been identified and characterized at the gene level as well as enzymatically, but NADH oxidase has so far only been characterized enzymatically with enzyme isolated from T. vaginalis cell extracts. In this study, we identified NADH oxidase by mass spectrometry after isolation of the enzyme from gel bands positively staining for NADH oxidase activity. In strain C1 (ATCC 30001) which is known to lack NADH oxidase activity completely, the NADH oxidase gene has a deletion at position 1540 of the open reading frame leading to a frame shift and, as a consequence, to premature termination of the encoded polypeptide.

PSIII-32 The influence of liver copper concentration on copper homeostatic liver proteins in beef cattle

Abstract The objective of this experiment was to investigate the influence of liver Cu concentrations on the relative abundance of liver Cu homeostatic proteins in beef cattle. Archived liver biopsy samples were selected based on Cu concentrations (n = 4 samples 21.7±1.35 mg Cu/kg DM-deficient; and n = 4 samples 73.3 ±10.7 mg Cu/kg DM-adequate). Liver samples were obtained from a subset of multiparous beef cows receiving a forage-based diet with no supplemental Cu (basal diet 6.25 mg Cu/kg DM) or 10 mg Cu/kg DM total diet (Cu supplemented as CuSO4·5H2O) for 99 d. Liver proteins were identified using mass spectrometry, normalized, and relative abundance determined using Scaffold software. A total of 895 identical proteins were identified in each sample and relative abundance of each Cu specific homeostatic protein (n = 13) was recorded. Data were analyzed as a randomized complete block design using R software. Copper homeostatic liver proteins identified were: aldehyde dehydrogenase, apolipoprotein A-1, betaine homocysteine methyltransferase, carbonic anhydrase II, Cu chaperone for superoxide dismutase, Cu transport protein, cytochrome c oxidase Cu chaperone, extracellular superoxide dismutase, flavin reductase, glutamate dehydrogenase, glutathione synthetase, protein disulphide isomerase A3, and Cu-zinc superoxide dismutase. By design, liver Cu concentrations were greater (P < 0.05) in Cu adequate vs. Cu deficient liver samples. Copper deficient liver samples had greater (P < 0.05) relative abundance of glutathione synthetase compared to Cu adequate liver samples. The relative abundance of all other Cu homeostatic liver proteins identified were similar (P > 0.05) across Cu concentrations. These data suggest that deficient and adequate liver Cu concentrations ranging from 16.0 to 109.0 mg Cu/kg DM have minimal impact on the relative abundance of Cu homeostatic proteins in beef cattle. Further investigation is needed to determine if liver Cu concentration influences Cu homeostatic protein function.

Unveiling Two Consecutive Hydroxylations: Mechanisms of Aromatic Hydroxylations Catalyzed by Flavin-Dependent Monooxygenases for the Biosynthesis of Actinorhodin and Related Antibiotics.

Flavin-dependent monooxygenases are ubiquitous in living systems and are classified into single- or two-component systems. Actinorhodin, produced by Streptomyces coelicolor, is a representative polycyclic polyketide that is hydroxylated through the action of the two-component ActVA-5/ActVB hydroxylase system. These homologous systems are widely distributed in bacteria, but their reaction mechanisms remain unclear. This in vitro investigation has provided chemical proof of two consecutive hydroxylations via hydroxynaphthalene intermediates involved in actinorhodin biosynthesis. The ActVA-5 oxygenase component catalyzed a stepwise dihydroxylation of the substrate, whereas the ActVB flavin reductase not only supplied a reduced cofactor, but also regulated the quinone-hydroquinone interconversion of an intermediate. Our study provides clues for understanding the general biosynthetic mechanisms of highly functionalized aromatic natural products with structural diversity.

Binding of FAD and tryptophan to the tryptophan 6-halogenase Thal is negatively coupled.

Flavin‐dependent halogenases require reduced flavin adenine dinucleotide (FADH2), O2, and halide salts to halogenate their substrates. We describe the crystal structures of the tryptophan 6‐halogenase Thal in complex with FAD or with both tryptophan and FAD. If tryptophan and FAD were soaked simultaneously, both ligands showed impaired binding and in some cases only the adenosine monophosphate or the adenosine moiety of FAD was resolved, suggesting that tryptophan binding increases the mobility mainly of the flavin mononucleotide moiety. This confirms a negative cooperativity between the binding of substrate and cofactor that was previously described for other tryptophan halogenases. Binding of substrate to tryptophan halogenases reduces the affinity for the oxidized cofactor FAD presumably to facilitate the regeneration of FADH2 by flavin reductases.

Conferring the Metabolic Self-Sufficiency of the CAM Plasmid of Pseudomonas putida ATCC 17453: The Key Role of Putidaredoxin Reductase.

The relative importance of camphor (CAM) plasmid-coded putidaredoxin reductase (PdR) and the chromosome-coded flavin reductases Frp1, Frp2 and Fred for supplying reduced FMN (FNR) to the enantiocomplementary 2,5- and 3,6-diketocamphane monooxygenases (DKCMOs) that are essential for the growth of Pseudomonas putida ATCC 17453 on (rac)-camphor was examined. By undertaking studies in the time window prior to the induction of Fred, and selectively inhibiting Frp1 and 2 with Zn2+, it was confirmed that PdR could serve as the sole active supplier of FNR to the DKCMOs. This establishes for the first time that the CAM plasmid can function as an autonomous extrachromosomal genetic element able to express all the enzymes and redox factors necessary to ensure entry of the C10 bicyclic terpene into the central pathways of metabolism via isobutyryl-CoA.

Bromination of L-tryptophan in a Fermentative Process With Corynebacterium glutamicum.

Brominated compounds such as 7-bromo-l-tryptophan (7-Br-Trp) occur in Nature. Many synthetic and natural brominated compounds have applications in the agriculture, food, and pharmaceutical industries, for example, the 20S-proteasome inhibitor TMC-95A that may be derived from 7-Br-Trp. Mild halogenation by cross-linked enzyme aggregates containing FAD-dependent halogenase, NADH-dependent flavin reductase, and alcohol dehydrogenase as well as by fermentation with recombinant Corynebacterium glutamicum expressing the genes for the FAD-dependent halogenase RebH and the NADH-dependent flavin reductase RebF from Lechevalieria aerocolonigenes have recently been developed as green alternatives to more hazardous chemical routes. In this study, the fermentative production of 7-Br-Trp was established. The fermentative process employs an l-tryptophan producing C. glutamicum strain expressing rebH and rebF from L. aerocolonigenes for halogenation and is based on glucose, ammonium and sodium bromide. C. glutamicum tolerated high sodium bromide concentrations, but its growth rate was reduced to half-maximal at 0.09 g L−1 7-bromo-l-tryptophan. This may be, at least in part, due to inhibition of anthranilate phosphoribosyltransferase by 7-Br-Trp since anthranilate phosphoribosyltransferase activity in crude extracts was half-maximal at about 0.03 g L−1 7-Br-Trp. Fermentative production of 7-Br-Trp by recombinant C. glutamicum was scaled up to a working volume of 2 L and operated in batch and fed-batch mode. The titers were increased from batch fermentation in CGXII minimal medium with 0.3 g L−1 7-Br-Trp to fed-batch fermentation in HSG complex medium, where up to 1.2 g L−1 7-Br-Trp were obtained. The product isolated from the culture broth was characterized by NMR and LC-MS and shown to be 7-Br-Trp.

Hyperthermophilic flavin reductase from Sulfolobus solfataricus P2: Production and biochemical characterization.

Nicotinamide adenine dinucleotide phosphate (NAD(P)H)-flavin oxidoreductases (flavin reductases) catalyze the reduction of flavin by NAD(P)H and provide the reduced form of flavin mononucleotide (FMN) to flavin-dependent monooxygenases. Based on bioinformatics analysis, we identified a putative flavin reductase gene, sso2055, in the genome of hyperthermophilic archaeon Sulfolobus solfataricus P2, and further cloned this target sequence into an expression vector. The cloned flavin reductase (EC. 1.5.1.30) was purified to homogeneity and characterized further. The purified enzyme exists as a monomer of 17.8kDa, free of chromogenic cofactors. Homology modeling revealed this enzyme as a TIM barrel, which is also supported by circular dichroism measurements revealing a beta-sheet rich content. The optimal pH for SSO2055 activity was pH 6.5 in phosphate buffer and the highest activity observed was at 120°C within the measurable temperature. We showed that this enzyme can use FMN and flavin adenine dinucleotide (FAD) as a substrate to generate their reduced forms. The purified enzyme is predicted to be a potential flavin reductase of flavin-dependent monooxygenases that could be involved in the biodesulfurization process of S. solfataricus P2.

BILIVERDIN REDUCTASE B/ FLAVIN REDUCTASE (BLVRB/FLR) EXHIBITS HEME-REGULATED, NADPH-DEPENDENT REDUCTASE ACTIVITY AND CONFERS CYTOPROTECTION IN DEVELOPING ERYTHROID CELLS

Biliverdin reductase B (BLVRB), also known as flavin reductase (FLR), is especially expressed in cells having high levels of heme synthesis (e.g. developing erythroid cells, fetal and adult liver). BLVRB exhibits NADPH-dependent reductase activity toward non-alpha isomers of biliverdin, flavins and redox cycling agents, and also has pharmacological importance in the treatment of methemoglobinemia. Nonetheless, the physiological function of BLVRB is obscure. In this work, the functional role of BLVRB in developing erythroid cells in relation to its heme-associated properties is elucidated. Recombinant mouse BLVRB binds ferric heme with a submicromolar affinity in association with inhibition of the NADPH-dependent reductase activity while displaying heme peroxidase activity. The interaction of BLVRB with heme in developing erythroid cells (MEL) was demonstrated by proteomic analysis of heme peroxidase-associated proteins. Upon induction of erythroid differentiation of MEL cells, the BLVRB protein expression is markedly up-regulated. Knockdown of BLVRB in the induced MEL cells aggravates intracellular oxidation and cytotoxicity mediated by thiol depleting agent and heme synthesis inhibitor, suggesting that BLVRB may maintain the cell viability under oxidative stress or heme deficient conditions. Our study suggests that BLVRB/FLR, which is usually regarded as a biliverdin reducing enzyme in the heme degradation pathway, is an erythroid active, heme-regulated NADPH-dependent reductase. It confers cytoprotection in developing erythroid cells under oxidative stress conditions in connection with cellular heme levels. This work was supported by General Research Fund (17160916) and Health and Medical Research Fund (04151846).

Biosynthesis of l-4-Chlorokynurenine, an Antidepressant Prodrug and a Non-Proteinogenic Amino Acid Found in Lipopeptide Antibiotics.

l-4-Chlorokynurenine (l-4-Cl-Kyn) is a neuropharmaceutical drug candidate that is in development for the treatment of major depressive disorder. Recently, this amino acid was naturally found as a residue in the lipopeptide antibiotic taromycin. Herein, we report the unprecedented conversion of l-tryptophan into l-4-Cl-Kyn catalyzed by four enzymes in the taromycin biosynthetic pathway from the marine bacterium Saccharomonospora sp. CNQ-490. We used genetic, biochemical, structural, and analytical techniques to establish l-4-Cl-Kyn biosynthesis, which is initiated by the flavin-dependent tryptophan chlorinase Tar14 and its flavin reductase partner Tar15. This work revealed the first tryptophan 2,3-dioxygenase (Tar13) and kynurenine formamidase (Tar16) enzymes that are selective for chlorinated substrates. The substrate scope of Tar13, Tar14, and Tar16 was examined and revealed intriguing promiscuity, thereby opening doors for the targeted engineering of these enzymes as useful biocatalysts.

Biosynthesis of l‐4‐Chlorokynurenine, an Antidepressant Prodrug and a Non‐Proteinogenic Amino Acid Found in Lipopeptide Antibiotics

Abstractl‐4‐Chlorokynurenine (l‐4‐Cl‐Kyn) is a neuropharmaceutical drug candidate that is in development for the treatment of major depressive disorder. Recently, this amino acid was naturally found as a residue in the lipopeptide antibiotic taromycin. Herein, we report the unprecedented conversion of l‐tryptophan into l‐4‐Cl‐Kyn catalyzed by four enzymes in the taromycin biosynthetic pathway from the marine bacterium Saccharomonospora sp. CNQ‐490. We used genetic, biochemical, structural, and analytical techniques to establish l‐4‐Cl‐Kyn biosynthesis, which is initiated by the flavin‐dependent tryptophan chlorinase Tar14 and its flavin reductase partner Tar15. This work revealed the first tryptophan 2,3‐dioxygenase (Tar13) and kynurenine formamidase (Tar16) enzymes that are selective for chlorinated substrates. The substrate scope of Tar13, Tar14, and Tar16 was examined and revealed intriguing promiscuity, thereby opening doors for the targeted engineering of these enzymes as useful biocatalysts.

Functional and mechanistic characterization of an atypical flavin reductase encoded by the pden_5119 gene in Paracoccus denitrificans.

Pden_5119, annotated as an NADPH-dependent FMN reductase, shows homology to proteins assisting in utilization of alkanesulfonates in other bacteria. Here, we report that inactivation of the pden_5119 gene increased susceptibility to oxidative stress, decreased growth rate and increased growth yield; growth on lower alkanesulfonates as sulfur sources was not specifically influenced. Pden_5119 transcript rose in response to oxidative stressors, respiratory chain inhibitors and terminal oxidase downregulation. Kinetic analysis of a fusion protein suggested a sequential mechanism in which FMN binds first, followed by NADH. The affinity of flavin toward the protein decreased only slightly upon reduction. The observed strong viscosity dependence of kcat demonstrated that reduced FMN formed tends to remain bound to the enzyme where it can be re-oxidized by oxygen or, less efficiently, by various artificial electron acceptors. Stopped flow data were consistent with the enzyme-FMN complex being a functional oxidase that conducts the reduction of oxygen by NADH. Hydrogen peroxide was identified as the main product. As shown by isotope effects, hydride transfer occurs from the pro-S C4 position of the nicotinamide ring and partially limits the overall turnover rate. Collectively, our results point to a role for the Pden_5119 protein in maintaining the cellular redox state.

A two-component monooxygenase initiates a novel 2-bromo-4-nitrophenol catabolic pathway in newly isolated Cupriavidus sp. strain NyZ375

As a vital byproduct of chemical intermediates for pesticides and cytotoxin inhibitors, 2-bromo-4-nitrophenol (2B4NP) is toxic to aquatic life. However, bacterial cultures mineralizing 2B4NP have not been reported. In this study, Cupriavidus sp. strain NyZ375 was isolated from aromatics-polluted soil and identified by 16S rRNA gene sequencing. Via an oxidative pathway, it grows on 120 μM 2B4NP as the only carbon source, releasing approximately equivalent amount of nitrite. Three genes (bnpABC) in a 4.8-kb DNA fragment from a draft genome was found to be responsible for the initial reactions in 2B4NP degradation. BnpAB are the monooxygenase and flavin reductase components, respectively, catalyzing the monooxygenation of 2B4NP to bromohydroquinone (BHQ, 63%) and 1,2,4-benzenetriol (BT, 37%) with concomitant nitrite release, in the presence of NADH and FAD. BnpC is a hydroxyquinol 1,2-dioxygenase catalyzing the ring-cleavage oxidation of BT to maleylacetate. The by-product BHQ was not further degraded by strain NyZ375 but was not toxic to its growth at a concentration of 100 μM. It can be tentatively concluded that BnpAB is evolutionary close to the 2,4,6-trichlorophenol monooxygenase TcpAX from Cupriavidus necator JMP134. The 2B4NP catabolism by this new isolate illustrates a pathway distinct from those of all identified 2-chloro-4-nitrophenol utilizers.

A novel sulfonamide resistance mechanism by two-component flavin-dependent monooxygenase system in sulfonamide-degrading actinobacteria

Sulfonamide-degrading bacteria have been discovered in various environments, suggesting the presence of novel resistance mechanisms via drug inactivation. In this study, Microbacterium sp. CJ77 capable of utilizing various sulfonamides as a sole carbon source was isolated from a composting facility. Genome and proteome analyses revealed that a gene cluster containing a flavin-dependent monooxygenase and a flavin reductase was highly up-regulated in response to sulfonamides. Biochemical analysis showed that the two-component monooxygenase system was key enzymes for the initial cleavage of sulfonamides. Co-expression of the two-component system in Escherichia coli conferred decreased susceptibility to sulfamethoxazole, indicating that the genes encoding drug-inactivating enzymes are potential resistance determinants. Comparative genomic analysis revealed that the gene cluster containing sulfonamide monooxygenase (renamed as sulX) and flavin reductase (sulR) was highly conserved in a genomic island shared among sulfonamide-degrading actinobacteria, all of which also contained sul1-carrying class 1 integrons. These results suggest that the sulfonamide metabolism may have evolved in sulfonamide-resistant bacteria which had already acquired the class 1 integron under sulfonamide selection pressures. Furthermore, the presence of multiple insertion sequence elements and putative composite transposon structures containing the sulX gene cluster indicated potential mobilization. This is the first study to report that sulX responsible for both sulfonamide degradation and resistance is prevalent in sulfonamide-degrading actinobacteria and its genetic signatures indicate horizontal gene transfer of the novel resistance gene.

Kinetics and Catabolic Pathways of the Insecticide Chlorpyrifos, Annotation of the Degradation Genes, and Characterization of Enzymes TcpA and Fre in Cupriavidus nantongensis X1T.

Chlorpyrifos is one of the most used organophosphorus insecticides. It is commonly degraded to 3,5,6-trichloro-2-pyridinol (TCP), which is water-soluble and toxic. Bacteria can degrade chlorpyrifos and TCP, but the biodegradation mechanism has not been well-characterized. Recently isolated Cupriavidus nantongensis X1T can completely degrade 100 mg/L chlorpyrifos and 20 mg/L TCP with half-lives of 6 and 8 h, respectively. We annotated a complete gene cluster responsible for TCP degradation in recently sequenced strain X1T. Two key genes, tcpA and fre, were cloned from X1T and transferred and expressed in Escherichia coli BL21(DE3). Degradation of TCP by X1T whole cell was compared with that by the enzymes 2,4,6-trichlorophenol monooxygenase and NAD(P)H:flavin reductase expressed and purified from E. coli BL21(DE3). Novel metabolites of TCP were isolated and characterized, indicating stepwise dechlorination of TCP, which was confirmed by TCP disappearance, mass balance, and detection and formation kinetics of chloride ion from TCP.

Straightforward Regeneration of Reduced Flavin Adenine Dinucleotide Required for Enzymatic Tryptophan Halogenation.

Flavin-dependent halogenases are known to regioselectively introduce halide substituents into aromatic moieties, for example, the indole ring of tryptophan. The process requires halide salts and oxygen instead of molecular halogen in the chemical halogenation. However, the reduced cofactor flavin adenine dinucleotide (FADH2) has to be regenerated using a flavin reductase. Consequently, coupled biocatalytic steps are usually applied for cofactor regeneration. Nicotinamide adenine dinucleotide (NADH) mimics can be employed stoichiometrically to replace enzymatic cofactor regeneration in biocatalytic halogenation. Chlorination of l-tryptophan is successfully performed using such NADH mimics. The efficiency of this approach has been compared to the previously established enzymatic regeneration system using the two auxiliary enzymes flavin reductase (PrnF) and alcohol dehydrogenase (ADH). The reaction rates of some of the tested mimics were found to exceed that of the enzymatic system. Continuous enzymatic halogenation reaction for reaction scale-up is also possible.

Investigations of two-component flavin-dependent monooxygenase systems.

Bacterial two-component flavin-dependent monooxygenase systems catalyze the oxidation of diverse metabolic reactions. There are several shared mechanistic features in the two-component monooxygenase systems that differ from canonical monooxygenase enzymes. The flavin reductases catalyze the reductive half-reaction, and the reduced flavin is transferred to the monooxygenase enzyme. The oxidative half-reaction catalyzed by the monooxygenase enzyme has been proposed to occur through the formation of a (hydro)peroxyflavin intermediate. In some two-component flavin-dependent systems the mechanism of flavin transfer involves protein-protein interactions between the flavin reductase and monooxygenase enzyme. Methods are presented that provide an alternative approach from flavin-bound monooxygenases to evaluate the kinetic properties and flavin transfer mechanism of the two-component flavin-dependent monooxygenase systems.