Gene identification and enzymatic characterization of the initial enzyme in pyrimidine oxidative metabolism, uracil-thymine dehydrogenase.

Uracil-thymine dehydrogenase (UTDH), which catalyzes the irreversible oxidation of uracil to barbituric acid in oxidative pyrimidine metabolism, was purified from Rhodococcus erythropolis JCM 3132. The finding of unusual stabilizing conditions (pH 11, in the presence of NADP+ or NADPH) enabled the enzyme purification. The purified enzyme was a heteromer consisting of three different subunits. The enzyme catalyzed oxidation of uracil to barbituric acid with artificial electron acceptors such as methylene blue, phenazine methosulfate, benzoquinone, and α-naphthoquinone; however, NAD+, NADP+, flavin adenine dinucleotide, and flavin mononucleotide did not serve as electron acceptors. The enzyme acted not only on uracil and thymine but also on 5-halogen-substituted uracil and hydroxypyrimidine (pyrimidone), while dihydropyrimidine, which is an intermediate in reductive pyrimidine metabolism, and purine did not serve as substrates. The activity of UTDH was enhanced by cerium ions, and this activation was observed with all combinations of substrates and electron acceptors.

Optimization of a Colorimetric Assay to Determine Lactate Dehydrogenase B Activity Using Design of Experiments.

Lactate dehydrogenase B (LDH-B) is overexpressed in lung and breast cancer, and it has been considered as a potential target to treat these types of cancer. Herein, we propose a straightforward incomplete factorial (IF) design composed of 12 combinations of two reaction buffers, three pH values, three salt (NaCl) concentrations, and three incubation times, which we called IF-BPST (Buffer/pH/Salt/Time), for the optimization of a colorimetric LDH-B assay in a final volume of 100 µL using 96-well plates. The assay is based on the absorbance change at ~570 nm and the color change of the reaction mixture due to the release of NADH that reacts with nitroblue tetrazolium (NBT) and phenazine methosulfate (PMS), resulting in the formation of a blue-purple formazan. The results obtained using the IF-BPST were comparable with those obtained by response surface methodology. Our work revealed that the NBT/PMS assay with some modifications can be used to measure the activity of LDH-B and other dehydrogenases in a high-throughput screening format at the early stages of drug discovery. LDH-B containing lysates cannot be assayed directly, however, due to the sensitivity of the method toward detergents. Thus, we suggest precipitating the proteins in the lysates to remove the interfering detergents, and then to dissolve the protein pellet in a suitable buffer and carry out the assay.

High-pressure tuning of primary photochemistry in bacterial photosynthesis: membrane-bound versus detergent-isolated reaction centers.

While photosynthesis thrives at close to normal pressures and temperatures, it is presently well known that life is similarly commonplace in the hostile environments of the deep seas as well as around hydrothermal vents. It is thus imperative to understand how key biological processes perform under extreme conditions of high pressures and temperatures. Herein, comparative steady-state and picosecond time-resolved spectroscopic studies were performed on membrane-bound and detergent-purified forms of a YM210W mutant reaction center (RC) from Rhodobacter sphaeroides under modulating conditions of high hydrostatic pressure applied at ambient temperature. A previously established breakage of the lone hydrogen bond formed between the RC primary donor and the protein scaffold was shown to take place in the membrane-bound RC at an almost 3 kbar higher pressure than in the purified RC, confirming the stabilizing role of the lipid environment for membrane proteins. The main change in the multi-exponential decay of excited primary donor emission across the experimental 10 kbar pressure range involved an over two-fold continuous acceleration, the kinetics becoming increasingly mono-exponential. The fastest component of the emission decay, thought to be largely governed by the rate of primary charge separation, was distinctly slower in the membrane-bound RC than in the purified RC. The change in character of the emission decay with pressure was explained by the contribution of charge recombination to emission decreasing with pressure as a result of an increasing free energy gap between the charge-separated and excited primary donor states. Finally, it was demonstrated that, in contrast to a long-term experimental paradigm, adding a combination of sodium ascorbate and phenazine methosulfate to the protein solution potentially distorts natural photochemistry in bacterial RCs.

Understanding the chemistry of the artificial electron acceptors PES, PMS, DCPIP and Wurster's Blue in methanol dehydrogenase assays.

Methanol dehydrogenases (MDH) have recently taken the spotlight with the discovery that a large portion of these enzymes in nature utilize lanthanides in their active sites. The kinetic parameters of these enzymes are determined with a spectrophotometric assay first described by Anthony and Zatman 55 years ago. This artificial assay uses alkylated phenazines, such as phenazine ethosulfate (PES) or phenazine methosulfate (PMS), as primary electron acceptors (EAs) and the electron transfer is further coupled to a dye. However, many groups have reported problems concerning the bleaching of the assay mixture in the absence of MDH and the reproducibility of those assays. Hence, the comparison of kinetic data among MDH enzymes of different species is often cumbersome. Using mass spectrometry, UV-Vis and electron paramagnetic resonance (EPR) spectroscopy, we show that the side reactions of the assay mixture are mainly due to the degradation of assay components. Light-induced demethylation (yielding formaldehyde and phenazine in the case of PMS) or oxidation of PES or PMS as well as a reaction with assay components (ammonia, cyanide) can occur. We suggest here a protocol to avoid these side reactions. Further, we describe a modified synthesis protocol for obtaining the alternative electron acceptor, Wurster's blue (WB), which serves both as EA and dye. The investigation of two lanthanide-dependent methanol dehydrogenases from Methylorubrum extorquens AM1 and Methylacidiphilum fumariolicum SolV with WB, along with handling recommendations, is presented. Lanthanide-dependent methanol dehydrogenases. Understanding the chemistry of artificial electron acceptors and redox dyes can yield more reproducible results.

A fast, sensitive, single-step colorimetric dipstick assay for quantifying ascorbic acid in urine.

The presence of ascorbate in human urine has been shown to be a useful dietary, fruit or vitamin C intake biomarker. More recently it has been discovered that ascorbate levels in urine can be used to facilitate the detection of precancerous colorectal polyps. While there are a number elaborate HPLC, MS or multi-step enzymatic "kit" methods to detect and quantify urinary ascorbate, these are time consuming and expensive. There are also a number of low-cost paper-based ascorbate detection dipsticks. However, the limits of detection and quantification accuracy for these dipsticks are not adequate for applications with human urine. To address these limitations, we have developed a fast, sensitive, single-step colorimetric assay that can be used to quantify ascorbate in urine and other biological fluids. The assay uses the tetrazolium salt, methylthiazolyldiphenyl-tetrazolium bromide (MTT), with the electron carrier phenazine methosulfate (PMS), in a chelated acidic phosphate-buffer to produce a vivid purple color in the presence of ascorbate. Confirmation of the performance of the assay and of its standard curve in human urine was also done using independent LC-MS/MS and NMR analyses. The lower limit of detection of the ascorbate dipstick assay described here was found to be 3.2 µM. The paper dipsticks are stable over a wide range of temperatures and can be stored for up to 150-days.

A salting-out assisted liquid-liquid microextraction procedure for determination of cysteine followed by spectrophotometric detection.

A new analytical method for sensitive determination of cysteine based on its interaction with phenazine methosulfate was developed using salting-out liquid-liquid microextraction followed by spectrophotometric detection. The mechanism of the reaction was studied and confirmed by Fourier transform infrared and mass spectroscopy. Experimental parameters affecting the extraction efficiency were investigated and under the optimal conditions, good linearity was observed in the range 0.2 - 6.0 µg mL-1 with a correlation coefficient of 0.9972. The limit of detection and limit of quantification were found to be 0.07 and 0.21 µg mL -1, respectively. The enrichment factor was 25. The developed methodology was applied for analysis of cysteine in food supplements. The obtained data were in good agreement with LC-MS/MS analysis.

A novel histochemistry assay to assess and quantify focal cytochrome c oxidase deficiency.

Defects in the respiratory chain, interfering with energy production in the cell, are major underlying causes of mitochondrial diseases. In spite of this, the surprising variety of clinical symptoms, disparity between ages of onset, as well as the involvement of mitochondrial impairment in ageing and age-related diseases continue to challenge our understanding of the pathogenic processes. This complexity can be in part attributed to the unique metabolic needs of organs or of various cell types. In this view, it remains essential to investigate mitochondrial dysfunction at the cellular level. For this purpose, we developed a novel enzyme histochemical method that enables precise quantification in fresh-frozen tissues using competing redox reactions which ultimately lead to the reduction of tetrazolium salts and formazan deposition in cytochrome c oxidase-deficient mitochondria. We demonstrate that the loss of oxidative activity is detected at very low levels - this achievement is unequalled by previous techniques and opens up new opportunities for the study of early disease processes or comparative investigations. Moreover, human biopsy samples of mitochondrial disease patients of diverse genotypic origins were used and the successful detection of COX-deficient cells suggests a broad application for this new method. Lastly, the assay can be adapted to a wide range of tissues in the mouse and extends to other animal models, which we show here with the fruit fly, Drosophila melanogaster. Overall, the new assay provides the means to quantify and map, on a cell-by-cell basis, the full extent of COX deficiency in tissues, thereby expending new possibilities for future investigation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Menadione-mediated WST1 reduction assay for the determination of metabolic activity of cultured neural cells.

Cellular reduction of tetrazolium salts to their respective formazans is frequently used to determine the metabolic activity of cultured cells as an indicator of cell viability. For membrane-impermeable tetrazolium salts such as WST1 the application of a membrane-permeable electron cycler is usually required to mediate the transfer of intracellular electrons for extracellular WST1 reduction. Here we demonstrate that in addition to the commonly used electron cycler M-PMS, menadione can also serve as an efficient electron cycler for extracellular WST1 reduction in cultured neural cells. The increase in formazan absorbance in glial cell cultures for the WST1 reduction by menadione involves enzymatic menadione reduction and was twice that recorded for the cytosolic enzyme-independent WST1 reduction in the presence of M-PMS. The optimized WST1 reduction assay allowed within 30 min of incubation a highly reliable detection of compromised cell metabolism caused by 3-bromopyruvate and impaired membrane integrity caused by Triton X-100, with a sensitivity as good as that of spectrophotometric assays which determine cellular MTT reduction or lactate dehydrogenase release. The short incubation period of 30 min and the observed good sensitivity make this optimized menadione-mediated WST1 reduction assay a quick and reliable alternative to other viability and toxicity assays.

Addressed immobilization of biofunctionalized diatoms on electrodes by gold electrodeposition.

Diatoms are single cell microalgae with a silica shell (frustule), which possess a micro/nanoporous pattern of unparalleled diversity far beyond the possibilities of current micro- and nanofabrication techniques. To explore diatoms as natural three-dimensional nanostructured supports in sensing and biosensing devices, a simple, rapid and stable method to immobilize diatoms via gold electrodeposition is described. In this process, gold microstructures are formed, immobilizing diatoms by entrapment or crossing their nanopores. Varying the applied potential, time and HAuCl4 concentration, gold deposits of different morphologies and roughness are obtained, thereby determining the diatom immobilization process. Optical and scanning electron microscopy have been used to characterize diatom immobilization yields, the morphology of the gold microstructures, and the morphological integrity of diatoms. Cyclic voltammetry has been performed to characterize the gold deposits and to demonstrate the enhanced electrocatalytic activity of the gold-diatom electrodes. Electro-addressed immobilization of different diatoms on specific bands of interdigitated electrode arrays has been achieved, highlighting the potential application of diatoms for site-specific immobilization on microarrays. The feasibility to combine tailored immobilization with diatom biofunctionalization has also been demonstrated. Antibody-functionalized diatoms were immobilized on electrodes retaining their ability to detect its cognate antigen. The reported method exploits the natural three-dimensional nanostructures of diatoms together with their easy modification with biomolecules and the simplicity of gold electrodeposition to produce micro/nanostructured and highly electrocatalytic electrodes, providing low-cost and eco-friendly platforms and arrays with potential application in biosensing devices.

Catalytic photoinduced electron transport across a lipid bilayer mediated by a membrane-soluble electron relay.

Unidirectional photocatalytic electron transfer from a hydrophilic electron donor encapsulated in the interior of a liposome, to a hydrophilic electron acceptor on the other side of the membrane, has been achieved using the simple membrane-soluble electron relay 1-methoxy-N-methylphenazinium (MMP(+)). The total amount of photoproduct (>140 nmol) exceeds the number of moles of MMP(+) present (125 nmol), thus showing that the transport of electrons is catalytic.

Magnetic Resonance Imaging of Atherosclerosis Using CD81-Targeted Microparticles of Iron Oxide in Mice.

The goal of this study is to investigate the feasibility of using CD81- (Cluster of Differentiation 81 protein-) targeted microparticles of iron oxide (CD81-MPIO) for magnetic resonance imaging (MRI) of the murine atherosclerosis. CD81-MPIO and IgG- (Immunoglobulin G-) MPIO were prepared by covalently conjugating, respectively, with anti-CD81 monoclonal and IgG antibodies to the surface of the tosyl activated MPIO. The relevant binding capability of the MPIO was examined by incubating them with murine bEnd.3 cells stimulated with phenazine methosulfate (PMS) and its effect in shortening T2 relaxation time was also examined. MRI in apolipoprotein E-deficient mice was studied in vivo. Our results show that CD81-MPIO, but not IgG-MPIO, can bind to the PMS-stimulated bEnd.3 cells. The T2 relaxation time was significantly shortened for stimulated bEnd.3 cells when compared with IgG-MPIO. In vivo MRI in apolipoprotein E-deficient mice showed highly conspicuous areas of low signal after CD81-MPIO injection. Quantitative analysis of the area of CD81-MPIO contrast effects showed 8.96- and 6.98-fold increase in comparison with IgG-MPIO or plain MPIO, respectively (P < 0.01). Histological assay confirmed the expression of CD81 and CD81-MPIO binding onto atherosclerotic lesions. In conclusion, CD81-MPIO allows molecular assessment of murine atherosclerotic lesions by magnetic resonance imaging.

Colorimetric and electrochemical genosensors for the detection of Escherichia coli DNA without amplification in seawater.

Monitoring seawater, particularly recreational water, for indicator bacteria presence is required to protect the public from exposure to fecal pollution and to guarantee the safety of the swimming areas. Two methods for the detection and quantification of Escherichia coli DNA were developed: a colorimetric assay in a microplate and an electrochemical biosensor. These assays were based on the double hybridization recognition of a single-strand DNA capture probe immobilized onto the microplate or the screen-printed carbon electrode to its complementary ssDNA, which is hybridized with an ssDNA signal probe labeled with horseradish peroxidase enzyme. The hybridization recognition step used the colorimetric monitoring of the oxidation state of the 3,3',5,5'-tetramethylbenzidine. The electrochemical monitoring of the oxidation state of 5 methyl-phenazinium methyl sulfate was allowed when the horseradish-peroxidase was in the presence of the mediator (5 methyl-phenazinium methyl sulfate and hydrogen peroxide). These approaches allow for the detection and quantification of 10(2) to 10(3) cells of E. coli in 5l of seawater samples in less than 5h. Detection was achieved without a nucleic acid amplification step. The specificity of the two methods against E. coli was demonstrated by testing a panel of bacteria. The two methods can be used for on-site monitoring of seawater quality.

Aerobic denitration of 2,4,6-trinitrotoluene in the presence of phenazine compounds and reduced pyridine nucleotides.

Phenazine-containing spent culture supernatants of Pseudomonas aeruginosa concentrated with a C18 solid-phase extraction cartridge initiate NAD(P)H-dependent denitration of 2,4,6-trinitrotoluene (TNT). In this study, TNT denitration was investigated under aerobic conditions using two phenazine secondary metabolites excreted by P. aeruginosa, pyocyanin (Py) and its precursor phenazine-1- carboxylic acid (PCA), and two chemically synthesized pyocyanin analogs, phenazine methosulfate (PMS+) and phenazine ethosulfate (PES+). The biomimetic Py/NAD(P)H/O2 system was characterized and found to extensively denitrate TNT in unbuffered aqueous solution with minor production of toxic amino aromatic derivatives. To a much lesser extent, TNT denitration was also observed with PMS+ and PES+ in the presence of NAD(P)H. No TNT denitration was detected with the biomimetic PCA/NAD(P)H/O2 system. Electron paramagnetic resonance (EPR) spectroscopy analysis of the biomimetic Py/NAD(P)H/O2 system revealed the generation of superoxide radical anions (O2 •−). In vitro TNT degradation experiments in the presence of specific inhibitors of reactive oxygen species suggest a nucleophilic attack of superoxide radical anion followed by TNT denitration through an as yet unknown mechanism. The results of this research confirm the high functional versatility of the redox-active metabolite pyocyanin and the susceptibility of aromatic compounds bearing electron withdrawing substituents, such as nitro groups, to superoxide-driven nucleophilic attack.

Ultrasensitive detection of the reduced form of nicotinamide adenine dinucleotide based on carbon nanotube field effect transistor.

We developed a simple, ultrasensitive, and quantitative detection method for the reduced form of nicotinamide adenine dinucleotide (NADH), based on carbon nanotube field effect transistors (CNTFETs). Following the injection of NADH at different concentrations, we obtained different electrical signals from a semiconductor characterization system mimicking biological catalysis of NADH dehydrogenase (CoI). Here, FET was fabricated via photolithography, attaching silicon wells, as the detection chamber, on the channel area of the single wall carbon nanotube (SWCNT). SWCNTs were functionalized with phenazine derivant, a counterpart of the key functional prosthetic group of CoI enzyme. In the presence of NADH, electrons transferred to phenazine derivant through SWCNT, by analogous means of the electron transport chain formed by a series of iron-sulfur (FeS) clusters in CoI. Using this method, the limit of detection was as low as 1 pM, and the range of linear response was 10 pM to 500 nM. Significantly, this approach possesses great potential for applications in real-time detection of NADH at extremely low concentrations, and rigorous analysis for NADH in electrochemical fields.

A blue native polyacrylamide gel electrophoretic technology to probe the functional proteomics mediating nitrogen homeostasis in Pseudomonas fluorescens.

As glutamate and ammonia play a pivotal role in nitrogen homeostasis, their production is mediated by various enzymes that are widespread in living organisms. Here, we report on an effective electrophoretic method to monitor these enzymes. The in gel activity visualization is based on the interaction of the products, glutamate and ammonia, with glutamate dehydrogenase (GDH, EC: 1.4.1.2) in the presence of either phenazine methosulfate (PMS) or 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium (INT). The intensity of the activity bands was dependent on the amount of proteins loaded, the incubation time and the concentration of the respective substrates. The following enzymes were readily identified: glutaminase (EC: 3.5.1.2), alanine transaminase (EC: 2.6.1.2), aspartate transaminase (EC: 2.6.1.1), glycine transaminase (EC: 2.6.1.4), ornithine oxoacid aminotransferase (EC: 2.6.1.13), and carbamoyl phosphate synthase I (EC: 6.3.4.16). The specificity of the activity band was confirmed by high pressure liquid chromatography (HPLC) following incubation of the excised band with the corresponding substrates. These bands are amenable to further molecular characterization by a variety of analytical methods. This electrophoretic technology provides a powerful tool to screen these enzymes that contribute to nitrogen homeostasis in Pseudomonas fluorescens and possibly in other microbial systems.

Oxygen-independent alkane formation by non-heme iron-dependent cyanobacterial aldehyde decarbonylase: investigation of kinetics and requirement for an external electron donor.

Cyanobacterial aldehyde decarbonylase (cAD) is, structurally, a member of the di-iron carboxylate family of oxygenases. We previously reported that cAD from Prochlorococcus marinus catalyzes the unusual hydrolysis of aldehydes to produce alkanes and formate in a reaction that requires an external reducing system but does not require oxygen [Das et al. (2011) Angew. Chem. 50, 7148-7152]. Here we demonstrate that cADs from divergent cyanobacterial classes, including the enzyme from N. puntiformes that was reported to be oxygen dependent, catalyze aldehyde decarbonylation at a much faster rate under anaerobic conditions and that the oxygen in formate derives from water. The very low activity (<1 turnover/h) of cAD appears to result from inhibition by the ferredoxin reducing system used in the assay and the low solubility of the substrate. Replacing ferredoxin with the electron mediator phenazine methosulfate allowed the enzyme to function with various chemical reductants, with NADH giving the highest activity. NADH is not consumed during turnover, in accord with the proposed catalytic role for the reducing system in the reaction. With octadecanal, a burst phase of product formation, k(prod) = 3.4 ± 0.5 min(-1), is observed, indicating that chemistry is not rate-determining under the conditions of the assay. With the more soluble substrate, heptanal, k(cat) = 0.17 ± 0.01 min(-1) and no burst phase is observed, suggesting that a chemical step is limiting in the reaction of this substrate.

Visible wavelength spectrophotometric assays of L-aspartate and D-aspartate using hyperthermophilic enzyme systems.

Methods with which to simply and rapidly assay L-aspartate (L-Asp) and D-aspartate (D-Asp) would be highly useful for physiological research and for nutritional and clinical analyses. Levels of L- and D-Asp in food and cell extracts are currently determined using high-performance liquid chromatography. However, this method is time-consuming and expensive. Here we describe a simple and specific method for using an L-aspartate dehydrogenase (L-AspDH) system to colorimetrically assay L-Asp and a system of three hyperthermophilic enzymes--aspartate racemase (AspR), L-AspDH, and L-aspartate oxidase (L-AO)--to assay D-Asp. In the former, the reaction rate of nicotinamide adenine dinucleotide (NAD(+))-dependent L-AspDH was measured based on increases in the absorbance at 438 nm, reflecting formation of formazan from water-soluble tetrazolium-1 (WST-1), using 1-methoxy-5-methylphenazinum methyl sulfate (mPMS) as a redox mediator. In the latter, D-Asp was measured after first removing L-Asp in the sample solution with L-AO. The remaining D-Asp was then changed to L-Asp using racemase, and the newly formed L-Asp was assayed calorimetrically using NAD(+)-dependent aspartate dehydrogenase as described above. This method enables simple and rapid spectrophotometric determination of 1 to 100 µM L- and D-Asp in the assay systems. In addition, methods were applicable to the L- and D-Asp determinations in some living cells and foods.

Evaluation and optimization of high-throughput enzymatic assays for fast l-ascorbic acid quantification in fruit and vegetables.

In this paper, we compare and evaluate the applicability of three UV-VIS absorbance based assays for high-throughput quantification of ascorbic acid in horticultural products. All the methods involve the use of a common enzyme (ascorbate oxidase) in combination with a different indicator molecule. The three methods were retrieved from literature: a direct oxidase-method, an OPDA coupled oxidase-method and a PMS-method, which is commercially available. The analysis in high-throughput context involved the analysis in microplates in combination with the use of an automated liquid handling system. We checked (i) the performance factors of the selected methods on standard solutions, (ii) the applicability of the defined methods in high-throughput context, and, (iii) the accuracy of the methods on real samples using HPLC as a reference technique. The OPDA-method was found to be the most appropriate method for the quantification of ascorbic acid in high-throughput context with a linear range between 7.0 and 950 mgL(-1) and excellent correlation parameters (slopes close to 1, intercepts close to 0, R(2)>0.91) with the reference technique when real samples were analyzed. Finally, this method was optimized for assay cost and assay time. Hereto the enzymatic reaction was mathematically described using a model for enzyme kinetics, which was then used to calculate the optimal concentrations of ascorbate oxidase and OPDA. As a result of the modeling the amount of enzyme in the assay could be reduced with a factor 2.5 without affecting significantly the reaction time. In a last step the optimal concentrations were used for a successful validation with the HPLC-reference technique.

Another multiheme protein, hydroxylamine oxidoreductase, abundantly produced in an anammox bacterium besides the hydrazine-oxidizing enzyme.

A hydroxylamine oxidoreductase (HAO) was purified from anammox sludge in which an anammox bacterium, strain KSU-1, was dominant. The enzyme was a 118-kDa homodimer composed of a 53-kDa subunit. With phenazine methosulfate and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide as electron acceptors, the V(max) and K(m) for hydroxylamine were determined as 9.6+/-0.2 micromol/min x mg and 33+/-2 microM, while those for hydrazine were 0.54+/-0.0 micromol/min x mg and 25+/-2 microM, respectively. The HAO had a P468 chromophore. These enzymatic properties were different from those of the hydrazine-oxidizing enzyme (HZO), a multiheme protein abundantly produced by the KSU-1 strain, but were similar to those of the HAO purified from Candidatus Brocadia anammoxidans. The hao gene exists upstream of the hzoB gene, which codes for the HZO. The sequence deduced from the hao gene indicated eight c-type heme binding motifs and showed 87% identity with a polypeptide encoded by an open reading frame (kustc1061) in the genome of an anammox bacterium Candidatus Kuenenia stuttgartiensis. These suggested that the HAO is an indispensable enzyme and well conserved in anammox bacteria, similar to the HZO. This enzyme might therefore be a specific hydroxylamine oxidoreductase for anammox bacteria.

Chemiluminescence assay for tetrahydrobiopterin based on the generation of hydrogen peroxide using isoluminol-microperoxidase in the presence of 1-methoxy PMS.

We developed a novel highly sensitive chemiluminescence (CL) method for BH(4). The principle of the proposed method is based on active oxygen formation induced by 1-methoxy-5-methyl phenazinium methyl sulphate (1-methoxy PMS) in the presence of dissolved oxygen. Furthermore, active oxygen is determined by a CL assay involving the luminol reaction with microperoxidase. In this report, we examined the mechanism of formation and identified the reactive oxygen species derived from BH(4) employing 1-methoxy PMS. Additionally, optimum conditions for the CL assay of BH(4) were established.