Determination of acid dissociation constants of flavin analogues by capillary zone electrophoresis.

Acid dissociation constants (pKa ) of nine kinds of flavin analogues as molecular catalyst candidates were determined by CZE. Although some of the analogues are instable and degradable under the light exposure or in alkaline aqueous solutions, the effective electrophoretic mobility of the flavin analogue of interest has been measured with the residual substance. The pKa values of the flavin analogues were analyzed through the changes in the effective electrophoretic mobility with varying pH of the separation buffer. One or two steps pKa values were determined by the analysis. One of the degraded species from the flavin analogues, lumichrome, was also detected in the CZE analysis, and its pKa values were also determined. While coexisting impurities generated over the storage conditions were found in some analogues, the pKa values of the target analogues were successfully determined with the help of the CZE separations. A pressure-assisted CZE was utilized for the determination or the estimation of the pKa values of such analogues as possessing carboxylic acid moiety.

Multicomponent spectrofluorimetric method for the assay of carboxymethylflavin and its hydrolytic products: kinetic applications.

The simultaneous assay of carboxymethylflavin (CMF), an intermediate in the photolysis of riboflavin, and its hydrolytic side-chain cleavage products, lumichrome (LC) (acid solution) and LC and lumiflavin (LF) as well as isoalloxazine ring cleavage products, 1,2-dihydro-1-methyl-2-keto-3-quinoxaline carboxylic acid (KA) and 1,2,3,4-tetrahydro-1-methyl-2,3-dioxo-quinoxaline (DQ) (alkaline solution) has been carried out by a multicomponent spectrofluorimetric method. The method is based on the adjustment of pH of the degraded solutions to 2.0 and extraction of LC and LF with chloroform. The chloroform extract is evaporated to dryness under reduced pressure, the residue dissolved in pH 6.5 citro-phosphate buffer and LC and LF determined at their fluorescence maxima at 478 and 530 nm, respectively. The pH of the aqueous phase is re-adjusted to 6.5 and the solution used for the determination of CMF, KA and DQ at the wavelengths of 530, 443 and 420 nm, respectively. The proposed method has been validated according to ICH guidelines. The calibration curves for CMF and its hydrolytic products are linear in the concentration range of 0.5-5.0 × 10-6  M. The mean recovery ranges from 99.0-102.0% with relative standard deviation (RSD) of 0.19-0.99%. The limit of detection (LOD) and the limit of quantification (LOQ) are in the range of 1.17-1.78 × 10-7  M and 3.55-5.40 × 10-7  M, respectively. The uniformity of molar balance of CMF and degradation products during hydrolytic reactions indicates the accuracy of the proposed method for the spectrofluorimetric assay of the compounds. It has been applied to study the kinetics of hydrolytic reactions of CMF.

Stability-indicating spectrofluorimetric method for the assay of riboflavin and photoproducts: Kinetic applications.

A stability-indicating spectrofluorimetric method has been developed for the simultaneous assay of riboflavin (RF) and photoproducts, formylmethylflavin (FMF), lumichrome (LC) and lumiflavin (LF) in aqueous solution. The method is based on the extraction of LC formed in acid solution and LC and LF formed in alkaline solution with chloroform at pH 2.0 and their assay by fluorescence measurements at 478 and 530 nm, respectively. The aqueous phase, on readjustment of the pH to 6.5, is used to extract FMF with chloroform and its assay is carried out at 530 nm. The aqueous phase is then used for the assay of RF at 530 nm. The proposed method gives more accurate results for the assay of RF compared to those of the United States Pharmacopeia (USP) spectrofluorimetric method which does not take into account the presence of RF photoproducts having similar fluorescence characteristics. The proposed method along with the USP method has been applied to the study of the kinetics of photolysis of RF, assay of stored commercial vitamin preparations and their radiated samples. The results show that the USP method does not distinguish between the fluorescence of RF and its photoproducts, and, therefore, gives erroneous results with about 11% excess in the quantity of the vitamin compared to that of the proposed method. This is due to the interference of the fluorescence of photoproducts in the assay of RF. The method has been validated for various analytical parameters according to the guideline of the International Council for Harmonization (ICH).

Electrostatic Modification for Promotion of Flavin-Mediated Oxidation of a Probe for Flavin Detection.

Electrostatic effects on the redox photochemistry of synthetic probes (1, 2, and 1-Zn) are examined by adjusting the thermodynamic driving force of their oxidation reactions. The redox photochemistry was simply controlled by introducing a zinc binding site (2,2'-dipicolylamine (DPA)) on the coumarin moiety of probe 2. Zinc complexation produced a positively charged environment on the coumarin (1-Zn), which lowered the electron density of a nearby 9 H-xanthene ring, attenuating the auto-oxidation of 1-Zn by 45 % compared with that of probe 1 at 298 K. The positive net charge of 1-Zn also provided an attractive Coulombic force toward the phosphate of flavin mononucleotide and flavin adenine dinucleotide, which lowered the reduction potential of the electron acceptor (isoalloxazine) and improved intermolecular electron transfer from the 9 H-xanthene ring to isoalloxazine. The flavin-mediated oxidation rate of 1-Zn was increased to 1.5 times that of probe 2. Probe 1-Zn showed highly selective sensing behaviour toward flavins, producing an intense brightness (ϵΦF =2.80×103 m-1  cm-1 ) in the long-wavelength regions (λmax =588 nm) upon flavin-mediated oxidation. Furthermore, probes 1-Zn and 2 were successfully applied to eosinophil imaging and the differential diagnosis of eosinophilia; this demonstrates their use as diagnostic tools.

Effect of oxygen on the per-cell extracellular electron transfer rate of Shewanella oneidensis MR-1 explored in bioelectrochemical systems.

Extracellular electron transfer (EET) is a mechanism that enables microbes to respire solid-phase electron acceptors. These EET reactions most often occur in the absence of oxygen, since oxygen can act as a competitive electron acceptor for many facultative microbes. However, for Shewanella oneidensis MR-1, oxygen may increase biomass development, which could result in an overall increase in EET activity. Here, we studied the effect of oxygen on S. oneidensis MR-1 EET rates using bioelectrochemical systems (BESs). We utilized optically accessible BESs to monitor real-time biomass growth, and studied the per-cell EET rate as a function of oxygen and riboflavin concentrations in BESs of different design and operational conditions. Our results show that oxygen exposure promotes biomass development on the electrode, but significantly impairs per-cell EET rates even though current production does not always decrease with oxygen exposure. Additionally, our results indicated that oxygen can affect the role of riboflavin in EET. Under anaerobic conditions, both current density and per-cell EET rate increase with the riboflavin concentration. However, as the dissolved oxygen (DO) value increased to 0.42 mg/L, riboflavin showed very limited enhancement on per-cell EET rate and current generation. Since it is known that oxygen can promote flavins secretion in S. oneidensis, the role of riboflavin may change under anaerobic and aerobic conditions. Biotechnol. Bioeng. 2017;114: 96-105. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

First Report on Rare Unifloral Honey of Endemic Moltkia petraea (Tratt.) Griseb. from Croatia: Detailed Chemical Screening and Antioxidant Capacity.

Rare Moltkia petraea (Tratt.) Griseb. honey from Croatia was first time characterised. The spectrophotometric assays on CIE L*a*b*Cab *hab ° colour coordinates, total phenol content and antioxidant capacity (FRAP, CUPRAC, DPPH• and ABTS•+ assays) determined higher honey values generally close to dark honeys ranges. Headspace solid-phase microextraction (HS-SPME) on two fibres after GC-FID and GC/MS revealed the major compounds 2-phenylacetaldehyde (12.8%; 15.6%), benzaldehyde (11.1%; 10.0%), octane (9.3%; 7.6%), nonane, propan-2-one, pentan-2-one, pentanal and nonanal (4.9%; 14.5%). Ultrasonic solvent extraction (USE) mainly isolated non-specific higher molecular compounds characteristic of the comb environment. Targeted HLPC-DAD analysis of the honey determined higher concentration of phenylalanine (212.08 mg/kg) and lumichrome (16.25 mg/kg) along with tyrosine and kojic acid. The headspace composition (chemical fingerprint) and high concentration of lumichrome can be considered particular for M. petraea honey.

Autofluorescence of eccrine sweat glands.

BACKGROUND/PURPOSE: The monitoring of autofluorescence in skin tissue samples can have diagnostic and therapy significance. In this study, we are the first to describe autofluorescence of eccrine sweat glands, which is important and helpful for the diagnosis and therapy of diseases that involve the eccrine sweat glands. METHODS: Eccrine sweat gland autofluorescence in haematoxylin-eosin (HE) stained skin tissue sections was observed under a fluorescence microscope, which was compared to the immunofluorescence of keratin 19 and 15 in the skin tissue sections. The single eccrine sweat glands from five volunteers including three males and two females were isolated and also observed under a fluorescence microscope. The autofluorescence intensity of the single eccrine sweat gland was measured using a laser confocal scanning microscope system. RESULTS: Eccrine sweat gland autofluorescence in HE stained skin tissue sections appears green under GFP fliter system (470/40 nm) and red under N2.1 fliter system (515-560 nm). Furthermore, the single eccrine sweat gland showed various autofluorescence colours, including green under wide blue and red under wide green. The autofluorescence intensity of the single eccrine sweat gland was measured. The spectrum excited at 488 nm exhibited two peaks located at approximately 530 nm (11.54 ± 4.66) and 590 nm (10.38 ± 4.33). The results suggest flavin and lipopigment as the endogenous fluorophores. CONCLUSION: The autofluorescence of the HE stained eccrine sweat gland sections is simple and helpful for easily determining the structure of eccrine sweat glands. The autofluorescence of the single eccrine sweat gland may be due to the existence of flavin and lipopigment.

Characterization of flavins in roots of Fe-deficient strategy I plants, with a focus on Medicago truncatula.

The root accumulation and excretion of riboflavin (Rbfl) and Rbfl derivatives have been studied in the model legume species Medicago truncatula, grown in hydroponics in two different Fe deficiency conditions, with and without CaCO(3). Using high resolution mass spectrometry techniques coupled to liquid chromatography, three different flavin derivatives not previously reported in plants, putatively identified as 7-hydroxy-Rbfl, 7α-hydroxy-Rbfl and 7-carboxy-Rbfl, were found along with Rbfl in Fe-deficient M. truncatula roots. In the presence of CaCO(3) most of the flavins were accumulated in the roots, whereas in the absence of CaCO(3) there was partial export to the nutrient solution. The major flavins in roots and nutrient solution were Rbfl and 7-hydroxy-Rbfl, respectively. Flavins were located in the root cortex and epidermal cells, preferentially in a root region near the apex that also exhibited increased ferric chelate reductase (FCR) activity. Six out of 15 different species of horticultural interest showed root increases in both Rbfl (four of them also having Rbfl derivatives) and FCR. No significant correlation was found between Rbfl and either phosphoenolpyruvate carboxylase or FCR activities, whereas the latter two showed a good correlation between them. The possible roles of Rbfl and Rbfl derivatives in roots and nutrient solutions are discussed. Medicago truncatula is proposed as a model system for flavin studies.

Synchronous high-performance liquid chromatography with a photodiode array detector and mass spectrometry for the determination of citrinin, monascin, ankaflavin, and the lactone and acid forms of monacolin K in red mold rice.

The Monascus fermentation product red mold rice (RMR) has been found to contain the cholesterol-lowering agent monacolin K (MK) in both its lactone (MKL) and acid (MKA) forms and the mycotoxin citrinin (CT). The yellow pigments in RMR, namely, monascin (MS) and ankaflavin (AK), have been reported to exhibit antimetastatic and antiangiogenic activities. Currently, MK and these yellow pigments are usually detected in RMR by different analytical methods that are inconvenient, expensive, and time-consuming. The goal of this study was to establish a rapid, synchronous analytical method for determination of the MKA, MKL, MS, AK, and CT levels in RMR. MKA, MKL, MS, AK, and CT were extracted by the same extraction method, then separated by RP-HPLC with a C18 column. The effluent from the column was passed through a photodiode array detector and then introduced directly into a fluorescence detector. The results showed that high recovery rates of MKA, MKL, MS, AK, and CT are possible if RMR powder is extracted with 75% ethanol (10 mL) at 80 degrees C for 30 min. With regard to the optimal conditions of the HPLC, the peaks of MKA, MKL, MS, AK, and CT can be clearly separated from any noise peaks by isocratic elution with a mobile phase comprising 0.05% trifluoroacetic acid in acetonitrile-water (62.5 + 37.5, v/v).

C-Terminal Extension of a Plant Cryptochrome Dissociates from the ß-Sheet of the Flavin-Binding Domain.

Plant cryptochromes are central blue light receptors in land plants and algae. Photoreduction of the flavin bound to the photolyase homology region (PHR) causes a dissociation of the C-terminal extension (CCT) as effector via an unclear pathway. We applied the recently developed in-cell infrared difference (ICIRD) spectroscopy to study the response of the full-length pCRY from Chlamydomonas reinhardtii in living bacterial cells, because the receptor degraded upon isolation. We demonstrate a stabilization of the flavin neutral radical as photoproduct and of the resulting ß-sheet reorganization by binding of cellular ATP. Comparison between light-induced structural responses of full-length pCRY and PHR reveals a downshift in frequency of the ß-sheet signal, implying an association of the CCT close to the only ß-sheet of the PHR in the dark. We provide a missing link in activation of plant cryptochromes after flavin photoreduction by indicating that ß-sheet reorganization causes the CCT release and restructuring.

Free radical imaging of endogenous redox molecules using dynamic nuclear polarization magnetic resonance imaging.

Redox reactions accompanied by the oxidation-reduction of endogenous molecules play important roles in maintaining homeostasis in living organisms. In humans, numerous endogenous molecules that contribute toward maintaining physiological conditions form free radicals via electron transfer. A typical example of this is the mitochondrial electron transport chain, which is involved in energy production. If free radicals derived from endogenous molecules could be visualized and exploited as biological and functional probes, redox reactions mediated by endogenous molecules could be detected non-invasively. We succeeded in visualizing the free radicals derived from endogenous molecules using an in vivo dynamic nuclear polarization (DNP) magnetic resonance imaging (MRI) system. In this review, we describe the visualization of endogenous redox molecules, such as flavins and ubiquinones, which are mitochondrial electron carriers, as well as vitamin E and vitamin C (ascorbate). In addition, we describe the application of melanin free radicals for the in vivo visualization of metabola without using probes via in vivo DNP-MRI.

Mosquito NADPH-cytochrome P450 oxidoreductase: kinetics and role of phenylalanine amino acid substitutions at leu86 and leu219 in CYP6AA3-mediated deltamethrin metabolism.

The NADPH-cytochrome P450 oxidoreductase (CYPOR) enzyme is a membrane-bound protein and contains both FAD and FMN cofactors. The enzyme transfers two electrons, one at a time, from NADPH to cytochrome P450 enzymes to function in the enzymatic reactions. We previously expressed in Escherichia coli the membrane-bound CYPOR (flAnCYPOR) from Anopheles minimus mosquito. We demonstrated the ability of flAnCYPOR to support the An. minimus CYP6AA3 enzyme activity in deltamethrin degradation in vitro. The present study revealed that the flAnCYPOR purified enzyme, analyzed by a fluorometric method, readily lost its flavin cofactors. When supplemented with exogenous flavin cofactors, the activity of flAnCYPOR-mediated cytochrome c reduction was increased. Mutant enzymes containing phenylalanine substitutions at leucine residues 86 and 219 were constructed and found to increase retention of FMN cofactor in the flAnCYPOR enzymes. Kinetic study by measuring cytochrome c-reducing activity indicated that the wild-type and mutant flAnCYPORs followed a non-classical two-site Ping-Pong mechanism, similar to rat CYPOR. The single mutant (L86F or L219F) and double mutant (L86F/L219F) flAnCYPOR enzymes, upon reconstitution with the An. minimus cytochrome P450 CYP6AA3 and a NADPH-regenerating system, increased CYP6AA3-mediated deltamethrin degradation compared to the wild-type flAnCYPOR enzyme. The increased enzyme activity could illustrate a more efficient electron transfer of AnCYPOR to CYP6AA3 cytochrome P450 enzyme. Addition of extra flavin cofactors could increase CYP6AA3-mediated activity supported by wild-type and mutant flAnCYPOR enzymes. Thus, both leucine to phenylalanine substitutions are essential for flAnCYPOR enzyme in supporting CYP6AA3-mediated metabolism.

Structural transition dynamics of biologically active flavins in alkylglucoside surfactants aggregates.

The photophysics and structural transition dynamics of a bio-active flavin lumichrome (LM) entrapped in two sugars based neutral surfactants were reported. Sugar-based surfactants, which were used for research purpose are potential environmentally friendly, green alternative amphiphilic surface active substance compared to other kinds of common surfactants. Here, two alkyl glucoside surfactants n-octyl-ß-D-glucopyranoside (OBG) and n-octyl-ß-D-thioglucopyranoside (OBTG) were used. This work is carried out by using steady-state absorption and fluorescence emission spectroscopy along with time-resolved fluorescence emission techniques. Photophysics of LM was modulated several folds in these two sugar-based neutral micelles. LM exhibits excitation and emission wavelength dependent fluorescence properties in these two sugars based neutral micelles. LM confined in the micellar environments exhibited structural transition dynamism, i.e. different kinds of conformers are equilibrated. Herein, different conformers of LM are identified and explained with the help of spectroscopic methods. From the fluorescence anisotropy measurement, it was found that the rotational relaxation time of LM in OBG micelle was more compared to that in OBTG micelle, which indicates that the LM molecule faced much more constrained environment in OBG micellar media.

Crystallographic, spectroscopic, and computational analysis of a flavin C4a-oxygen adduct in choline oxidase.

Flavin C4a-OO(H) and C4a-OH adducts are critical intermediates proposed in many flavoenzyme reaction mechanisms, but they are rarely detected even by rapid transient kinetics methods. We observe a trapped flavin C4a-OH or C4a-OO(H) adduct by single-crystal spectroscopic methods and in the 1.86 A resolution X-ray crystal structure of choline oxidase. The microspectrophotometry results show that the adduct forms rapidly in situ at 100 K upon exposure to X-rays. Density functional theory calculations establish the electronic structures for the flavin C4a-OH and C4a-OO(H) adducts and estimate the stabilization energy of several active site hydrogen bonds deduced from the crystal structure. We propose that the enzyme-bound FAD is reduced in the X-ray beam. The aerobic crystals then form either a C4a-OH or C4a-OO(H) adduct, but an insufficient proton inventory prevents their decay at cryogenic temperatures.

A bifunctional molecule as an artificial flavin mononucleotide cyclase and a chemosensor for selective fluorescent detection of flavins.

Flavins, comprising flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin (RF, vitamin B(2)), play important roles in numerous redox reactions such as those taking place in the electron-transfer chains of mitochondria in all eukaryotes and of plastids in plants. A selective chemosensor for flavins would be useful not only in the investigation of metabolic processes but also in the diagnosis of diseases related to flavins; such a sensor is presently unavailable. Herein, we report the first bifunctional chemosensor (PTZ-DPA) for flavins. PTZ-DPA consists of bis(Zn(2+)-dipicolylamine) and phenothiazine. Bis(Zn(2+)-dipicolylamine) (referred to here as XyDPA) was found to be an excellent catalyst in the conversion of FAD into cyclic FMN (riboflavin 4',5'-cyclic phosphate, cFMN) under physiological conditions, even at pH 7.4 and 27 degrees C, with less than 1 mol % of substrate. Utilizing XyDPA's superior function as an artificial FMN cyclase and phenothiazine as an electron donor able to quench the fluorescence of an isoalloxazine ring, PTZ-DPA enabled selective fluorescent discrimination of flavins (FMN, FAD, and RF): FAD shows ON(+), FMN shows OFF(-), and RF shows NO(0) fluorescence changes upon the addition of PTZ-DPA. With this selective sensing property, PTZ-DPA is applicable to real-time fluorescent monitoring of riboflavin kinase (RF to FMN), alkaline phosphatase (FMN to RF), and FAD synthetase (FMN to FAD).

Light-emitting-diode-induced chemiluminescence detection for capillary electrophoresis.

In this work, an LED-induced-chemiluminescence (LED-CL) system was developed to extend the application of CL detection in CE. In the LED-CL, the analyte photooxidizes luminol under the irradiation of LEDs and generates CL. Taking the advantage of the small size nature of LEDs, the constructed photoreactor is greatly miniaturized, and especially suitable as a CE detector. The feasibility of the proposed detector was evaluated by detection of riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) after CE separation. Under the optimized conditions, the LODs for RF, FMN and FAD were 0.007, 0.02 and 0.1 microg/mL, respectively, better than those by UV detection. The RSDs were 3.4, 3.6 and 4.1% for 0.5 microg/mL RF, 2 microg/mL FMN and 5 microg/mL FAD, respectively. The LED-CL detector features low cost, miniaturization, fast response, high sensitivity and good reproducibility.

Microspectrophotometry and the photoreceptor of Phycomyces I.

By applying microspectrophotometry to the sporangiophore of Phycomyces blakesleeanus wild-type and the albino car-10(-) type II, absorption spectra were obtained for 1- to 5-day cultures. Spectra in the growing-zone of the wild-type during Stage IVb, taken from 0.1 to 3 mm below the base of the sporangium, show two distinctly different spectra: one is more characteristic of a carotene, the other of a flavin. Combined, these absorption spectra reproduce closely the action spectrum. For the albino car-10(-), which is deficient in carotenes, only the spectrum characteristic of lumichrome or a reduced flavin was found. A c-type cytochrome was isolated from both strains which, if coupled with a flavin, could permit a photoreversible oxidation-reduction system. Birefringent crystals were observed to be aligned in the growing zone in which the photoreceptor is believed to lie. Micro-spectrophotometry of these crystals shows absorption peaks similar to those of riboflavin crystals.

Redox constituents in milk fat globule membranes and rough endoplasmic reticulum from lactating mammary gland.

Milk fat globule membranes (MFGM) and rough endoplasmic reticulum (RER) membranes were isolated from milk and lactating mammary gland from the cow and were characterized by biochemical and electron microscope methods in terms of gross composition (proteins, phospholipids, neutral lipids, cholesterol, RNA, and DNA) and purity. Both fractions contained significant amounts of a b-type cytochrome with several properties similar to those of cytochrome b5 from liver, as well as a rotenone-insensitive NADH- and NADPH-cytochrome c reductase. The b-type cytochrome content in the apical plasma membrane-derived MFGM was of the same order of magnitude as it was in RER membranes. It was characterized by a high resistance to extraction by low- and high-salt concentrations and nonionic detergents. MFGM contained much more flavin and much higher activities of xanthine oxidase than the RER membranes. The same redox components were found in MFGM and mammary RER from women, rats, mice, and goats, but in absolute contents great differences between the species were noted. The cytochromes described here differed from liver cytochrome b5 in some spectral properties. The alpha-band of the reduced hepatic cytochrome b5 is asymmetric with a maximum at 555 nm that is split into two distinct peaks at low temperatures. The alpha-band of the b-type cytochromes from MFGM and mammary RER appears as one symmetrical peak at about 560 nm that is not split at low temperatures. When treated with cyanide, MFGM and mammary microsomes showed difference spectra of a reduced b-type cytochrome. Under the same conditions, liver microsomes gave a completely different spectrum. These findings demonstrate the presence of a b-type cytochrome and associated redox enzymes in MFGM, i.e., a derivative of the apical cell surface membrane that is regularly used for envelopment of the milk fat globule during secretion.

Determination of alloxan by fluorometric high-performance liquid chromatography.

A fluorometric, reversed-phase high-performance liquid chromatography (RP-HPLC) method that allows quantitation of low levels of alloxan has been described. The method involved derivatization of alloxan with 500-200,000-fold excess of 1, 2-phenylenediamine (PD) in 0.1 M acetate buffer, pH 4.5 for 15 min at room temperature. The fluorescent product alloxazine (excitation: 382 nm; emission: 435 nm) was then analyzed by RP-HPLC using an Eclipse XDB-C18 (4.6 x 150 mm) column and a mobile phase consisting of 0.1% trifluoroacetic acid in 15/85 (v/v) acetonitrile/water at a flow of 1 mL/min (injection volume: 20 microL). The method is robust, and as low as 0.1 pmol of the analyte could be successfully detected and quantified. Following a minimal pre-treatment such as ultrafiltration (molecular weight cut-off 5000 Da) or protein precipitation using perchloric acid, acetonitrile, or phosphotungstic acid, the method is suitable for analysis of alloxan in complex physiological fluids (e.g. fetal bovine serum) and tissue homogenates (e.g. heart and kidney). The method has been rigorously evaluated and adapted in the laboratory for routine analysis and determination of alloxan added to cell cultures.

Characterization of MymA protein as a flavin-containing monooxygenase and as a target of isoniazid.

Tuberculosis is a global health problem especially with the emergence of drug-resistant Mycobacterium tuberculosis strains, creating an urgent need to identify new drug targets. The mycobacterial cell wall is an attractive target for chemotherapeutic agents. Gene products of mymA operon are known to be required for the maintenance of cell wall and play an important role in persistence, thus making them important drug targets. This study was undertaken to biochemically characterize the MymA as a flavin-containing monooxygenase (FMO). Our results established its enzymatic activity in vitro and found that the mycobacterial FMO requires NADPH and FAD as cofactors, similar to other characterized bacterial FMOs. The enzyme follows Michaelis-Menten kinetics to catalyze substrates such as trimethylamine and thiourea. We also propose that MymA could be one of the targets of the antituberculosis drug, isoniazid (INH), which is a cell wall inhibitor. Molecular docking studies revealed that INH targeted NADPH-binding site of the MymA. Further, experimental validation revealed that INH inhibits MymA with the IC50 value of 4.9 µm. Thus, this study characterizes for the first time that MymA is a mycobacterial FMO, which may be a target of INH.