Substrate binding modulates the activity of Mycobacterium smegmatis G, a flavin-dependent monooxygenase involved in the biosynthesis of hydroxamate-containing siderophores

Mycobacterium smegmatis G (MbsG) is a flavin-dependent monooxygenase that catalyzes the NAD(P)H- and oxygen-dependent hydroxylation of the terminal amino group on the side chain of l-lysine in the biosynthetic pathway of the siderophore mycobactin. Mycobactins are essential for mycobacterium growth under iron-limiting conditions encountered during infection in mammals. Thus, enzymes involved in the biosynthesis of mycobactin represent potential drug targets. MbsG was expressed in Escherichia coli and purified using metal affinity and ionic exchange chromatographies. Recombinant MbsG represents the first member of this class of enzymes isolated in the active form, with a tightly bound FAD cofactor. The k(cat) value for formation of hydroxylated l-lysine under steady-state conditions was 5.0 min(-1), and K(m) values of 0.21 mM for l-lysine, 1.1 mM for NADH, and 2.4 mM for NADPH were calculated. The enzyme functioned as an oxidase when the activity of MbsG was measured by monitoring oxygen consumption in the absence of l-lysine, oxidizing NADH and NADPH with k(cat) values of 59 and 49 min(-1), respectively. Under these conditions, MbsG produced both hydrogen peroxide and superoxide. In contrast, when l-lysine was present, the reaction became more coupled, producing hydroxylated l-lysine and decreasing the oxidase activity. These results suggest that substrate binding modulates the function of MbsG from an oxidase to a monooxygenase.     

Simultaneous demonstration of phagocytosis-connected oxygen consumption and corresponding NAD(P)H oxidase activity: Direct evidence for NADPH as the predominant electron donor to oxygen in phagocytizing human neutrophils

Phagocytosis-connected oxygen consumption by human neutrophils and corresponding NAD(P)H oxidase were measured by an oxygen electrode with sequential additions of opsonized zymosan, Renex 30 (0.067%), and NAD(P)H. At a concentration of 0.15 mM substrate, NADPH oxidase activity of stimulated neutrophils was twice that required to account for accompanying oxygen consumption, and was about 20 times higher than that activity obtained from resting cells. NADH oxidase activity of phagocytizing cells, however, was negligible at the same concentration of substrate. With high recovery of oxidase activity, these results strongly suggest that NADPH is the dominant electron donor to oxygen in phagocytizing human neutrophils.     

Butylated hydroxyanisole-stimulated NADPH oxidase activity in rat liver microsomal fractions

NADPH-dependent oxygen utilization by liver microsomal fractions was stimulated by the addition of increasing concentrations of butylated hydroxyanisole concomitant with the inhibition of benzphetamine N-demethylase activity. The apparent conversion of monooxygenase activity to an oxidase-like activity in the presence of the antioxidant was correlated with the partial recovery of the reducing equivalents from NADPH in the form of increased hydrogen peroxide production. The progress curve of liver microsomal NADPH oxidase activity in the presence of butylated hydroxyanisole displayed a lag phase indicative of the formation of a metabolite capable of uncoupling the monooxygenase activity. Ethyl acetate extracts of microsomal reaction mixtures obtained in the presence of butylated hydroxyanisole, oxygen, and NADPH stimulated the NADPH oxidase activity of either liver microsomes or purified NADPH-cytochrome c (P-450) reductase. Using high performance liquid chromatography, gas chromatography, and mass spectrometry techniques, two metabolites of butylated hydroxyanisole, namely t-butylhydroquinone and t-butylquinone, were identified. The quinone metabolite and/or its 1-electron reduction product interact with the flavoprotein reductase to directly link the enzyme to the reduction of oxygen which results in an inhibition of the catalytic activity of the cytochrome P-450-dependent monooxygenase.     

Polyvinylferrocenium modified Pt electrode for anaerobic glucose monitoring

An amperometric enzyme electrode for the determination of glucose under anaerobic solution conditions was developed by immobilizing glucose oxidase and then by adsorbing ferrocene in polyvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of ferrocene that the reduced flavin adenine dinucleotide centers of glucose oxidase was measured at a constant potential. The response characteristics of the enzyme electrode were investigated. The effects of the thickness of the polymeric film, the amount of the enzyme immobilized, the amount of the mediator, the glucose concentration, the applied potential, operating pH and temperature on the response of the enzyme electrode were studied. The response time and the optimum pH were found to be 30-40 s and pH 7.4 at 25 degrees C, respectively. The linear response was observed up to 5.0 mM glucose concentration that the produced detectable current was 0.0075 mM glucose concentration. The activation energy (E(a)) of immobilized enzyme reaction was calculated to be 41.3 kJ mol(-1) from the Arrhenius plot. The apparent Michaelis-Menten constant (K(Mapp)) was found to be 6.05 mM glucose according to the Lineweaver-Burk graph of the Michaelis-Menten equation under the optimum conditions. The interference signal due to the most common electrochemical interfering species was also evaluated.     

Regulation of the successive reaction catalyzed by rat neuronal nitric oxide synthase

The rat neuronal nitric oxide synthase (nNOS) catalyzes two monooxygenase reactions successively from L-arginine (L-Arg) to L-citrulline (L-Cit) via N(omega)-hydroxy-L-arginine (OH-Arg) without most of OH-Arg leaving the substrate-binding site. In the steady-state reaction conditions, the amount of OH-Arg produced is about 1/30-1/50 that of L-Cit. We found in this study using nNOS purified from an Escherichia coli expression system that the ratio of the amount of OH-Arg to L-Cit (OH-Arg/L-Cit) increased to about 1 at low concentration of NADPH. In one cycle of the nNOS reaction, the decrease in NADPH concentration was found to reduce the rates of two monooxygenase reactions but had little effect on the rate constant of OH-Arg dissociation from the enzyme. The addition of NADP(+), the competitive inhibitor for NADPH, caused the decrease in the rates of monooxygenase reactions in a single cycle of the reaction and the increase in the ratio of OH-Arg/L-Cit in the steady state. At low CaM concentrations, the ratio of OH-Arg/L-Cit was about the same as that at high CaM. In a single cycle of the nNOS reaction, the rate of monooxygenation was not altered by the CaM concentration but the amount of metabolized L-Arg decreased with the decrease in CaM concentration, showing that the amount of active nNOS was regulated by complex formation between nNOS and CaM. It becomes clear that there are two regulatory mechanisms for the successive reaction of nNOS. One controls the rates of monooxygenations and the other controls the amount of active species of nNOS.     

Application of the theory of oxygen transfer: determination of the Michaelis constant of glucose oxidase with respect to oxygen]

The oxidation of beta-D-glucose with glucose oxidase generally requires oxygen, which, under normal conditions is present at low concentrations in the reaction medium. Experiments show that glucose oxidase is no longer saturated by oxygen at enzyme concentrations greater than 0.4 mg.ml1. This is due to the decrease in the oxygen concentration of the solution. The value of the oxygen mass transfer coefficients and dissolved oxygen concentrations are determined. These dissolved oxygen concentrations are found to correlate with direct measurements with an oxygen electrode. From this, the Michaelis constant of glucose oxidase for oxygen is calculated. These experiments also show that oxygen is a limiting factor for this reaction.     

Making glucose oxidase fit for biofuel cell applications by directed protein evolution

Progress in miniature chip-design raises demands for implantable power sources in health care applications such as continuous glucose monitoring of diabetic patients. Pioneered by Adam Heller, miniaturized enzymatic biofuel cells (mBCs) convert blood sugars into electrical energy by employing for example glucose oxidase (GOx) on the anode and bilirubin oxidase on the cathode. To match application demands it is crucial to increase lifetime and power output of mBCs. The power output has been limited by the performance of GOx on the anode. We developed a glucose oxidase detection assay (GODA) as medium-throughput screening system for improving GOx properties by directed protein evolution. GODA is a reaction product detection assay based on coupled enzymatic reactions leading to NADPH formation which is recorded at 340 nm. The main advantage of the assay is that it detects the production of d-gluconolactone instead of the side-product hydrogen peroxide and enables to improve bioelectrochemical properties of GOx. For validating the screening system, a mutagenic library of GOx from Aspergillus niger (EC 1.1.3.4) was generated and screened for improved activity using Saccharomyces cerevisiae as host. Directed evolution resulted in a GOx mutant I115V with 1.4-1.5-fold improved activity for beta-d-glucose (Vmax from 7.94 to 10.81 micromol min(-1) mg(-1); Km approximately 19-21 mM) and oxygen consumption kinetics correlate well [Vmax (O2) from 5.94 to 8.34 micromol min(-1) mg(-1); Km (O2) from 700 to 474 microM]. The developed mutagenic protocol and GODA represent a proof-of-principle that GOx can be evolved by directed evolution in S. cerevisiae for putative use in biofuel cells.     

Determination of glucose oxidase immobilised as monolayer onto a flat surface

An assay to estimate the amount of glucose oxidase immobilised as a monolayer onto a flat surface is reported. This method is based on the electrochemical detection of the flavin adenine dinucleotide (FAD) cofactor released by the immobilised enzyme in acid solutions. FAD concentration in the acid solution was measured by amperometry, using a flow injection analysis (FIA) system equipped with a wall-jet electrode, and with a sensitivity of (9.2+/-2.0)x10(-2) nA/nM. By this method, the amount of glucose oxidase molecules present in a monolayer deposited on a silanised glass slide was easily detected, in which the detection limit is more than one order of magnitude lower than the maximum loading of the surface with an ordered monolayer of glucose oxidase.     

Effect of enzyme-matrix composition on potentiometric response to glucose using glucose oxidase immobilized on platinum

Glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) was immobilized in a crosslinked matrix of bovine serum albumin, catalase, glucose oxidase and glutaraldehyde on platinum foil. When placed in glucose solution, this enzyme-electrode elicited a potentiometric response that varied with the changes in glucose concentration. The immobilized glucose oxidase was present at 7.4-10.1 micrograms enzyme protein/ml of matrix, as determined with 125I-labelled enzyme. The coupled enzyme activity was stable over 120 h; however, the apparent activity of the immobilized glucose oxidase was markedly less than that for the same amount of enzyme free in solution. This indicated a significant level of diffusional resistance within the enzyme-matrix. The potentiometric response to glucose increased significantly as either the thickness of the enzyme-matrix or the glutaraldehyde content was reduced; this also was attributed to diffusional effects. Several enzyme-electrodes, constructed without exogenous catalase and with different amounts of glucose oxidase, showed greater sensitivity in potentiometric response at low glucose oxidase loadings. These results are consistent with the hypothesis that the potentiometric response arises from an interfacial reaction involving a hydrogen peroxide redox couple at a platinum surface. The data also suggest that an optimum range of hydrogen peroxide concentration exists for maximum electrode sensitivity.     

A spectrophotometric asay for cellobiase

Chemical methods for measuring cellobiase activity are based on increased reducing capacity, following conversion of cellobiose into glucose (1–3). The chemical methods are time consuming, of low sensitivity, and nonspecific. Enzymic methods use glucose oxidase to estimate the amount of glucose liberated (4,5). Although more specific and sensitive, the enzymic methods are also time consuming. The two-step method requires two successive incubations, which take several hours (4). Although very sensitive, the one-step method requires 75 min for a preincubation, a highly purified glucose oxidase with negligible disaccharidase activity, and an absolutely glucose-free substrate (5). In the method for assaying cellobiase described in this paper, glucose is measured enzymically by formation of NADPH with a coupling system using yeast hexokinase, glucose-6-P dehydrogenase, and NADP, similar to previous methods involving TPN (6). The highly reproducible assay evaluated here requires only 15 min and all reagents and auxiliary enzymes of adequate quality are available commercially.     

Activation of NADPH oxidase in human neutrophils permeabilized with Staphylococcus aureus alpha-toxin. A lower Km when the enzyme is activated in situ.

The NADPH oxidase is a multicomponent enzyme system that produces the reduced oxygen species essential for bacterial killing by polymorphonuclear leukocytes (PMN). Study of the oxidase has typically been carried out in cell-free systems in which Km values of 20-150 microM NADPH have been reported. However, when compared with affinities reported for other flavoprotein dehydrogenases and when considering the cellular concentration of NADPH/NADP+ of approximately 35 microM, the reported affinity of the oxidase for NADPH appears low. To investigate this apparent discrepancy we have studied the kinetics of NADPH oxidase activation in situ in human PMN permeabilized with Staphylococcus aureus alpha-toxin. alpha-Toxin permeabilization of human PMN did not initiate NADPH oxidase activation at physiologic concentrations of NADPH. If permeabilized cells were stimulated with 1 microM formyl-methionyl-leucyl-phenylalanine, 10 microM guanosine 5'-O-(3-thiotriphosphate), 0.5 mM Ca2+, 5 micrograms/ml cytochalasin B in the presence of varying concentrations of NADPH, we were able to demonstrate activation of the oxidase complex as shown by superoxide dismutase-inhibitable reduction of cytochrome c. In this system we determined that the Km for oxidase activation was 4-7 microM NADPH, a 4-10-fold decrease from reported values. The oxidase was the enzyme being studied as shown by the absence of enzymatic activity in patients with chronic granulomatous disease. In addition, if the enzyme was initially activated in permeabilized cells, the cells homogenized, and the Km for the oxidase determined in a cell-free system, the observed Km reverted to previously reported values (36 microM). These results indicate that NADPH oxidase, studied in situ, has a significantly higher substrate affinity than that observed in isolated membranes and, moreover, indicate that substrate affinity is optimal for catalysis at reported concentrations of cytosolic NADPH.     

Studies on the NADPH oxidation by subcellular particles from phagocytosing polymorphonuclear leucocytes: evidence for the involvement of three mechanisms

1. The NADPH-oxidizing activity of a 100 000 X g particulate fraction of the postnuclear supernatant obtained frm guinea-pig phagocytosing poymorphonuclear leucocytes has been assayed by simultaneous determination of oxygen consumption, NADPH oxidation and O2- generation at pH 5.5 and 7.0 and with 0.15 mM and 1 mM NADPH. 2. The measurements of oxygen consumption and NADPH oxidation gave comparable results. The stoichiometry between the oxygen consumed and the NADPH oxidized was 1:1. 3. A markedly lower enzymatic activity was observed, under all the experimental conditions used, when the O2- generation assay was employed as compared to the assays of oxygen uptake and NADPH oxidation. 4. The explanation of this difference came from the analysis of the effect of superoxide dismutase and of cytochrome c which removes O2- formed during the oxidation of NADPH. 5. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reactin at pH 5.5. The inhibition was higher with 1 mM NADPH than with 0.15 mM NADPH. 6. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 7.0 with 1 mM NADPH but less than at pH 5.5 with 1 mM NADPH. 7. The effect of superoxide dismutase at pH 7.0 with 0.15 mM NADPH was negligible. 8. In all instances the inhibitory effect of cytochrome c was greater than that of superoxide dismutase. 9. It was concluded that the NADPH-oxidizing reaction studied here is made up of three components: an enzymatic univalent reduction of O2; an enzymatic, apparently non-univalent, O2 reduction and a non-enzymatic chain reaction. 10. These three components are variably and independently affected by the experimental conditions used. For example, the chain reaction is freely operative at pH 5.5 with 1 mM NADPH but is almost absent at pH 7.0 with 0.15 mM NADPH, whereas the univalent reduction of O2 is optimal at pH 7.0 with 1 mM NADPH.     

Variations in tumor cell growth rates and metabolism with oxygen concentration, glucose concentration, and extracellular pH.

Tumors and multicellular tumor spheroids can develop gradients in oxygen concentration, glucose concentration, and extracellular pH as they grow. In order to calculate these gradients and assess their impact on tumor growth, it is necessary to quantify the effect of these variables on tumor cell metabolism and growth. In this work, the oxygen consumption rates, glucose consumption rates, and growth rates of EMT6/Ro mouse mammary tumor cells were measured at a variety of oxygen concentrations, glucose concentrations, and extracellular pH levels. At an extracellular pH of 7.25, the oxygen consumption rate of EMT6/Ro cells increased by nearly a factor of 2 as the glucose concentration was decreased from 5.5 mM to 0.4 mM. This effect of glucose concentration on oxygen consumption rate, however, was slight at an extracellular pH of 6.95 and disappeared completely at an extracellular pH of 6.60. The glucose consumption rate of EMT6/Ro cells increased by roughly 40% when the oxygen concentration was reduced from 0.21 mM to 0.023 mM and decreased by roughly 60% when the extracellular pH was decreased from 7.25 to 6.95. The growth rate of EMT6/Ro cells decreased with decreasing oxygen concentration and extracellular pH; however, severe conditions were required to stop cell growth (0.0082 mM oxygen and an extracellular pH of 6.60). Empirical correlations were developed from these data to express EMT6/Ro cell growth rates, oxygen consumption rates, and glucose consumption rates, as functions of oxygen concentration, glucose concentration, and extracellular pH. These empirical correlations make it possible to mathematically model the gradients in oxygen concentration, glucose concentration, and extracellular pH in EMT6/Ro multicellular spheroids by solution of the diffusion/reaction equations. Computations such as these, along with oxygen and pH microelectrode measurements in EMT6/Ro multicellular spheroids, indicated that nutrient concentration and pH levels in the inner regions of spheroids were low enough to cause significant changes in nutrient consumption rates and cell growth rates. However, pH and oxygen concentrations measured or calculated in EMT6/Ro spheroids where quiescent cells have been observed were not low enough to cause the cessation of cell growth, indicating that the observed quiescence must have been due to factors other than acidic pH, oxygen depletion, or glucose depletion.     

Studies on steroid monooxygenase from Cylindrocarpon radicicola ATCC 11011. Oxygenative lactonization of androstenedione to testololactone

A steroid monooxygenase of Cylindrocarpon radicicola was found to catalyze oxygenative lactonization of 17-ketosteroid, androstenedione, to yield D-homo-17 alpha-oxasteroid, testololactone, i.e., the androstenedione monooxygenase reaction, in addition to catalyzing the progesterone monooxygenase reaction. The reaction product was identified by TLC, GLC, and mass spectrometry. The oxygenation proceeded with unitary stoichiometry for 17-ketosteroid, NADPH, and molecular oxygen, indicating that it is a typical monooxygenase reaction of the external electron donor type. The enzyme catalyzed successively the side chain cleavage reaction of 17 alpha-hydroxy-20-ketosteroid to produce its 17-keto derivative and the lactonization of the product. The effects of pH and of the concentration of substrate steroids on the androstenedione monooxygenase reaction were different from those on the progesterone monooxygenase reaction. Progesterone is a strong and competitive inhibitor of the lactonization of 17-ketosteroids. The steroid monooxygenase is concluded to have the activities of both oxygenative esterification of 20-ketosteroids and oxygenative lactonization of 17-ketosteroids.     

Stoichiometry of microsomal oxidation reactions. Distribution of redox-equivalents in monooxygenase and oxidase reactions catalyzed by cytochrome P-450

The stoichiometry of NADPH oxidation in rabbit liver microsomes was studied. It was shown that in uncoupled reactions cytochrome P-450, besides O2- generation catalyzes direct two- and four-electron reduction of O2 to produce H2O2 and water, respectively. With an increase in pH and ionic strength, the amount of O2 reduced via an one-electron route increases at the expense of the two-electron reaction. In parallel, with a rise in pH the steady-state concentration of the oxy-complex of cytochrome P-450 increases, while the synergism of NADPH and NADH action in the H2O2 formation reaction is replaced by competition. The four-electron reduction is markedly accelerated and becomes the main pathway of O2 reduction in the presence of a pseudo-substrate--perfluorohexane. Treatment of rabbit with phenobarbital, which induces the cytochrome P-450 isozyme specific to benzphetamine results in a 2-fold increase in the degree of coupling of NADPH and benzphetamine oxidation. The experimental results suggest that the ratio of reactions of one- and two-electron reduction of O2 is controlled by the ratio of rates of one- and two-electron reduction of cytochrome P-450. In the presence of pseudo-substrates cytochrome P-450 acts predominantly as a four-electron oxidase; one of possible reasons for the uncoupling of microsomal monooxygenase reactions is the multiplicity of cytochrome P-450 isozymes.     

Oxidative stress and increased eNOS and NADPH oxidase expression in mouse microvessel endothelial cells

Elevated oxidative stress plays a key role in diabetes-associated vascular disease. In this study, we tested the hypothesis that high glucose-induced oxidative stress was associated with changes in the expression of NADPH oxidase, superoxide dismutase (SOD) and endothelial nitric oxide synthase (eNOS). Oxidative stress was assessed in cell cultures of mouse microvessel endothelial cells (MMECs) by fluorescence labelling with dihydroethidium, lucigenin-enhanced chemiluminescence and determining NADPH oxidase subunit and eNOS expression with real-time polymerase chain reaction protocol and Western blotting. Oxidative stress and expression of the NADPH oxidase subunit, p22phox, were both increased, SOD1 and 3 expression lowered and eNOS significantly elevated in MMECs treated with 40 mM glucose for 72 h compared to low glucose medium. Oxidative stress, p22phox mRNA, eNOS mRNA, and protein were lowered by concurrent incubation with sepiapterin. When eNOS protein expression in endothelial cells was significantly decreased by eNOS siRNA treatment, superoxide generation was significantly higher in the MMECs grown in low glucose, but reduced in those grown in high glucose for 72 h. Thus, exposure of MMECs to high glucose results in increased oxidative stress that is associated with increased eNOS and NADPH oxidase subunit expression, notably p22phox, and decreased expression of SOD1 and 3.     

Development of a D-alanine sensor for the monitoring of a fermentation using the improved selectivity by the combination of D-amino acid oxidase and pyruvate oxidase

A D-alanine (D-Ala) sensor for the monitoring of a fermentation process was developed using flow injection analysis (FIA). The FIA system consisted of a D-amino acid oxidase (D-AAOx) reactor, a Pyruvate oxidase (PyOx) electrode and a contrast electrode in the flow cell, and through the oxidation of D-amino acids in the D-AAOx reactor, pyruvic acid was formed only from D-Ala. The pyruvic acid was further oxidized with PyOx via the D-AAOx reaction. The amount of oxygen consumed in the PyOx reaction was proportional to the amount of D-Ala. It was possible to continuously repeat the assay up to 60 times at pH 6.8 and a flow rate of 0.18-ml min(-1). A linear relationship was obtained in the range of 0.1-1 mM D-Ala with a correlation coefficient of 0.987 and the detection limit was 0.05 mM. The relative standard deviation (R.S.D.) was 4.9% (n=5) for 0.5 mM D-Ala. The D-Ala content in some fish sauces was also determined using the proposed sensor system. The results obtained indicated a linear relationship between the amounts of D-Ala determined by the proposed sensor system and the conventional method. From the results, even if the substrate specificity of the enzyme (D-AAOx) was low, it was evident that the concentration of the original material (D-Ala) could be determined specifically when the first reaction product was changed by the second reaction (PyOx).