High-level expression of Myrothecium verrucaria bilirubin oxidase in Pichia pastoris, and its facile purification and characterization

Bilirubin oxidase (BO) from Myrothecium verrucaria (authentic BO) catalyzing the oxidation of bilirubin to biliverdine was overexpressed in the methylotrophic yeast, Pichia pastoris. The cDNA encoding BO was cloned into the P. pastoris expression vector pPIC9K under the control of the alcohol oxidase 1 promoter and its protein product was secreted using the Saccharomyces cerevisiae alpha-mating factor signal sequence. The productivity of recombinant BO (rBO) in P. pastoris was approximately 5000 U/L of culture broth, being about 2.5- and 250-fold higher than rBO expressed in Aspergillus oryzae and S. cerevisiae, respectively. The calculated molecular mass of rBO consisting of 538 amino acids was 60,493 kDa, however, that of SDS-PAGE was 66 kDa because of non-native type N-linked sugar chains. The spectroscopic properties of rBO were typical of multicopper oxidase containing four Cu ions per protein molecule. The specific activity to oxidize bilirubin was 57 U/mg, having a value about twice that of authentic BO and rBO expressed in A. oryzae. Moreover, the thermostability of rBO expressed in P. pastoris was significantly high compared to the authentic BO previously reported. Accordingly, a heterologous expression system of rBO to meet clinical and industrial needs was constructed.     

The interactions of cytochrome c and porphyrin cytochrome c with cytochrome c oxidase. The resting, reduced and pulsed enzymes

Cytochrome c oxidase forms tight binding complexes with the cytochrome c analog, porphyrin cytochrome c. The behaviour of the reduced and pulsed forms of the oxidase with porphyrin cytochrome c have been followed as functions of ionic strength; this behaviour has been compared with that of the resting oxidase [Kornblatt, Hui Bon Hoa and English (1984) Biochemistry 23, 5906-5911]. All forms of the cytochrome oxidase studied bind one porphyrin cytochrome c per 'functional' cytochrome oxidase (two heme a); it appears as though porphyrin cytochrome c and cytochrome c compete for the same site on the oxidase. The resting enzyme binds cytochrome c 8 times more strongly than porphyrin cytochrome c; the reduced enzyme, in contrast, binds the two with almost equal affinity. In all three cases, resting, pulsed and reduced, the heme-to-porphyrin distance is estimated to be about 3 nm. The tight-binding complexes formed between cytochrome oxidase and porphyrin cytochrome c can be dissociated by salt. Debye-Hückel analysis of salt titrations indicate that the resting enzyme and the reduced enzyme are similar in that the product of the interaction charges on the two proteins is about -14. The product of the charges for the pulsed enzyme is -25, indicating that on average another positive and negative charge take part in the interaction of the two proteins. While there is one tight binding site for cytochrome c per two heme a, cytochrome c is able to 'communicate' with four heme a. In the absence of cytochrome c, electron transfer from tetramethylphenylenediamine to the oxidase to oxygen results in the conversion of the resting form to the 'oxygenated'; in the presence of cytochrome c, the same electron transfer results in the appearance of the 'pulsed' form. Cytochrome c titrations of the enzyme show that a ratio of only one cytochrome c to four heme a is sufficient to convert all the oxidase to the 'pulsed' form. Porphyrin cytochrome c, like cytochrome c, catalyzes the same conversion with the same stoichiometry. The binding data and salt effects indicate that major structural alterations occur in the oxidase as it is converted from the resting to the partially reduced and subsequently to the pulsed form.     

A novel terminal oxidase, cytochrome baa3 purified from aerobically grown Pseudomonas aeruginosa: it shows a clear difference between resting state and pulsed state.

A novel type of cytochrome c oxidase was purified to homogeneity from Pseudomonas aeruginosa which was grown aerobically. The purified oxidase contained two molecules of heme a, two atoms of copper, and one molecule of protoheme per molecule. One of the two heme a molecules in the oxidase reacted with carbon monoxide, so that the enzyme was of baa3-type. The oxidase molecule was composed of three subunits with molecular weights of 38,000, 57,000, and 82,000. Although the oxidase oxidized ferrocytochrome c-550 obtained from the bacterial cells grown aerobically, the oxidizing activity was not high. The "resting form" and the "pulsed form" of the oxidase were observed clearly with this enzyme, and the transition from the resting form to the pulsed form was accompanied by a distinct change of the enzymatic activity. The difference in the kinetics of the catalytic reactions between the two forms is discussed.     

Myrothecium verrucaria bilirubin oxidase and its mutants for potential copper ligands

Bilirubin oxidase (EC:1.3.3.5) purified from a culture medium of Myrothecium verrucaria MT-1 (authentic enzyme) catalyzes the oxidation of bilirubin to biliverdin in vitro and recombinant enzyme (wild type) was obtained by using an overexpression system of the bilirubin oxidase gene with Aspergillus oryzae harboring an expression vector. The absorption and ESR spectra showed that both bilirubin oxidases are multicopper oxidases containing type 1, type 2, and type 3 coppers similar to laccase, ascorbate oxidase, and ceruloplasmin. Site-directed mutagenesis has been performed for the possible ligands of each type of copper. In some mutants, Cys457 --> Val, Ala, His94 --> Val, and His134.136 --> Val, type 1 and type 2 copper centers were perturbed completely and the enzyme activity was completely lost. Differing from the holoenzyme, these mutants showed type 3 copper signals. However, the optical and magnetic properties characteristic of type 1 copper were retained even by mutating one of the type 1 copper ligands, i.e., a mutant, Met467 --> Gly, showed a weak but apparent enzyme activity. A double mutant His456.458 --> Val had only type 1 Cu, showing a blue band at 600 nm (epsilon = 1.6 x 10(3)) and an ESR signal with very narrow hyperfine splitting (A parallel = 7.2 x 10(-)3 cm-1). Since the type 2 and type 3 coppers are not present, the mutant did not show enzyme activity. These results strongly imply that the peculiar sequence in bilirubin oxidase, His456-Cys457-His458, forms an intramolecular electron-transfer pathway between the type 1 copper site and the trinuclear center composed of the type 2 and type 3 copper sites.     

Bilirubin oxidase activity of Bacillus subtilis CotA

The spore coat protein CotA from Bacillus subtilis was previously identified as a laccase. We have now found that CotA also shows strong bilirubin oxidase activity and markedly higher affinity for bilirubin than conventional bilirubin oxidase. This is the first characterization of bilirubin oxidase activity in a bacterial protein.     

Bilirubin Oxidase Activity of Bacillus subtilis CotA

The spore coat protein CotA from Bacillus subtilis was previously identified as a laccase. We have now found that CotA also shows strong bilirubin oxidase activity and markedly higher affinity for bilirubin than conventional bilirubin oxidase. This is the first characterization of bilirubin oxidase activity in a bacterial protein.     

Cancer isoform of a tumor-associated cell surface NADH oxidase (tNOX) has properties of a prion.

We have described a drug-responsive form of a cell surface NADH oxidase (hydroquinone oxidase) of cancer cells (tNOX) that exhibits unusual characteristics including resistance to proteases, resistance to cyanogen bromide digestion, and an ability to form amyloid filaments closely resembling those of spongiform encephalopathies and all of which are characteristics of PrP(sc) (PrP(res)), the presumed infective and proteinase K resistant particle of the scrapie prion. The tNOX protein from the HeLa cell surface copurified with authentic glyceraldehyde-3-phosphate dehydrogenase (muscle form) (GAPDH). Surprisingly, the tNOX-associated muscle GAPDH also was proteinase K resistant. In this paper, we show that combination of authentic rabbit muscle GAPDH with tNOX renders the GAPDH resistant to proteinase K digestion. This property, that of converting the normal form of a protein into a likeness of itself, is one of the defining characteristics of the group of proteins designated as prions.     

Purification, characterization and decolorization of bilirubin oxidase from Myrothecium verrucaria 3.2190

Myrothecium verrucaria 3.2190 is a nonligninolytic fungus that produces bilirubin oxidase. Both M. verrucaria and the extracellular bilirubin oxidase were tested for their ability to decolorize indigo carmine. The biosorption and biodegradation of the dye were detected during the process of decolorization; more than 98% decolorization efficiency was achieved after 7 days at 26°C. Additionally, the crude bilirubin oxidase can efficiently decolorize indigo carmine at 30°C~50°C, pH 5.5~9.5 with dye concentrations of 50 mg l(-1)~200 mg l(-1). Bilirubin oxidase was purified and visualized as a single band on native polyacrylamide gel electrophoresis (PAGE). Several enzymatic properties of the purified enzyme were investigated. Moreover, the identity of the purified bilirubin oxidase (BOD) was confirmed by matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). These results demonstrate that the purified bilirubin oxidase in M. verrucaria strain has potential application in dye effluent decolorization.     

An in vitro model of aneurysmal subarachnoid hemorrhage: oxidation of unconjugated bilirubin by cytochrome oxidase

Aneurysmal subarachnoid hemorrhage is a stroke subtype with high rates of mortality and morbidity. Cerebral vasospasm can lead to ischemic injury or death and is a common complication of aneurysmal subarachnoid hemorrhage, usually occurring 3-9 days afterwards. The cause of vasospasm is not known. Recently, there has been strong evidence that vasoactive oxidation products of bilirubin may be involved. Currently, the factors that lead to bilirubin oxidation are poorly characterized. In this study, we have designed an in vitro model of hemorrhagic stroke in order to investigate conditions that promote the oxidation of bilirubin to form vasoactive compounds. Using our model, we created a basic hematoma system of blood, CSF, and hemeoxygenase-1. We manipulated this system in various ways, incubated it and determined the concentration of vasoactive bilirubin oxidation products that resulted. Conditions where cytochrome oxidase was stimulated caused an increase bilirubin oxidation products (292.6 +/- 39.9 micromol/L respectively, vs. 79.3 +/- 1.3 micromol/L for the basic reaction, p < 0.05), which was attenuated by cyanide. Our data suggest that bilirubin oxidation products may be produced by oxidation(s) requiring an oxygen-utilizing enzyme like cytochrome oxidase.     

Identification of the NADPH-binding subunit of the respiratory burst oxidase.

The respiratory burst oxidase is a multicomponent membrane-bound enzyme that uses NADPH to reduce oxygen to O2-. When oxidase-containing membranes from activated neutrophils are treated with 0.3 M KCl, the NADPH-binding component of the oxidase elutes from the membranes in an active form. Treatment of this eluate with [32P]NADPH dialdehyde labels an approximately 32-kDa protein that is absent from eluates obtained from normal resting membranes or from resting or activated membranes from patients with one form of chronic granulomatous disease. We propose that this approximately 32-kDa protein is the NADPH-binding component of the oxidase.     

The reactivity of pulsed cytochrome c oxidase toward carbon monoxide

When pulsed cytochrome c oxidase is exposed to carbon monoxide in the absence of oxygen the enzyme is converted quickly to its CO-associated mixed valence state. The half-time for this reaction at 0 degree C is about 4 min. This is about 100 times faster than a similar reaction which begins with the resting form of the enzyme. The possible significance of this reaction in understanding the pulsed/resting phenomenon and the carbon monoxide oxygenase reactions of cytochrome oxidase is discussed.     

An immobilized enzyme reactor for the detoxification of bilirubin

An immobilized enzyme reactor has been developed for the degradation of bilirubin as a potential treatment for neonatal jaundice. It utilizes the enzyme bilirubin oxidase from Myrothecium verrucaria, which in the presence of molecular oxygen converts bilirubin to biliverdin and other products that are much less toxic than bilirubin. Bilirubin oxidase was covalently attached to agarose beads using cyano transfer activation. Forty percent of the specific activity of bilirubin oxidase was retained after immmobilization, and preparations with 20 units of enzymatic activity per gram of drained wet weight of gel were obtained. The stability of bilirubin oxidase at pH 7.4 and 37 degrees C was improved fivefold by immobilization. A 15-mL column containing immobilized bilirubin oxidase, through which a 37 degrees C solution of 332muM bilirubin and 450muM human serum albumin in 0.05M phosphate buffer (pH 7.4) was passed at 1 mL/min, converted more than 60 percent of the bilirubin per pass. The substrate specificity of the enzyme and the small volume of the reactor are important characteristics for this clinical application where it is desirable to remove only one compound from the blood and to minimize the volume of blood in the extracorporeal circuit. This reactor, by detoxifying the jaundiced infant's blood of bilirubin, would eliminate the risks associated with the use of donor blood as is done currently in treating severe neonatal jaundice.     

Enzymatic removal of bilirubin toxicity by bilirubin oxidase in vitro and excretion of degradation products in vivo

The toxic effects of the degradation products of bilirubin that were formed by reaction with bilirubin oxidase were investigated with the C 1300 mouse neuroblastoma cell line by examining the following parameters: growth inhibition, morphologic characteristics, membrane transport, DNA synthesis, and protein synthesis. The addition of bilirubin to the cells resulted in definite cytotoxic effects on all of these parameters in a dose-dependent fashion; the addition of bilirubin oxidase reversed the toxic effects on the C 1300 cells in vitro. Furthermore, we found that most of these enzymatic degradation products of bilirubin were excreted by the kidney into the urine in a few hours after intravenous injection of the degradation products; in contrast, no intact bilirubin was excreted. Thus, these findings suggest that hyperbilirubinemia in newborn infants (kernicterus) may be prevented by administering polyethylene glycol-conjugated bilirubin oxidase, with a longer plasma half-life which has been reported previously to oxidize bilirubin to its nontoxic components in the bloodstream.     

耐高温重组CotA蛋白的胆红素氧化酶反应动力学及活性鉴定

已知源于枯草芽孢杆菌内生孢子的CotA蛋白具有漆酶和胆红素氧化酶活性。然而,其分离纯化极为困难。本研究对表达与纯化的重组CotA蛋白的胆红素氧化酶特性及氧化还原功能进行鉴定。基因转染及筛选获得了表达CotA的P. pastoris菌株|继而,表达的重组CotA蛋白经DEAE-Sepharose FF 及Sephadex G-75层析分离与纯化,产物得率为25%,纯化产物的酶比活性为 4 U/mg。经SDS-PAGE 和 MALDI-TOF MS 分析显示,其分子质量为65 kD。纯化的CotA蛋白能够催化胆红素氧化,生成胆绿素,且催化反应速率受反应溶液中溶解氧含量的影响,提示纯化的重组CotA具有胆红素氧化酶活性。酶反应进一步证明,CotA的胆红素氧化酶反应最适pH值为pH 8.0,最适温度为60℃。该酶在90℃条件下的半衰期为7 h,提示CotA胆红素氧化酶具有高度的热稳定性。CotA修饰的摄谱仪石墨电极可直接电催化分子氧（O2）还原,具有很好的电流响应。我们的结果表明,重组的CotA蛋白具有耐高温胆红素氧化酶活性。更重要的是,我们的结果还提示重组的CotA蛋白在酶生物燃料电池阴极的制备上具有较好的应用潜能。     

Activation of the respiratory burst oxidase in a fully soluble system from human neutrophils

The O2(-)-forming respiratory burst oxidase is present in a dormant state in a fully soluble system containing both cytosol and a deoxycholate extract of membranes from resting human neutrophils. Sodium dodecyl sulfate at low concentrations converts this soluble dormant oxidase into its catalytically active form. The Vmax for the activated oxidase was 2.1 mumol of O2-/min/mg of membrane protein. Michaelis constants for NADPH and NADH (38 microM and 1.7 mM, respectively) were similar to those measured previously in other systems. Oxidase activity was not detected after sodium dodecyl sulfate treatment of systems containing solubilized neutrophil membranes obtained from patients with X-linked chronic granulomatous disease. These results suggest that the deoxycholate extract contains both the resting oxidase and those membrane-associated components needed for its activation, all in functioning states.     

Enhanced oxidation of bilirubin by an immobilized tri-enzyme system of glucose oxidase, bilirubin oxidase and horseradish peroxidase

Bilirubin oxidase was immobilized to nylon fibres. A tri-enzyme system composed of glucose oxidase, bilirubin oxidase and horseradish peroxidase was also immobilized to the fibres. Both immobilized systems were tested and it was found that the latter gave enhanced oxidation rates for bilirubin.     

Enzymatic oxidation of bilirubin by intestinal mucosa

Bilirubin oxidase, an aerobic enzyme which degrades bilirubin 'in vitro' to colourless diazo-negative compounds, including propentdyopents and trace amounts of biliverdin, has been demonstrated in homogenates of rat intestine, kidney and liver. The enzyme in the intestinal mucosa has been partially characterised and appears to be mitochondrial in origin; maximal activity was detected in the jejunum. Intestinal bilirubin oxidase has a mean activity of 0.51 +/- 0.03 (S.D.) nmol bilirubin degraded/min per mg protein. Similar bilirubin oxidase activities were found in the tissue of Sprague-Dawley and Gunn rats. The role of the enzyme 'in vivo' remains to be determined.     

疣孢漆斑菌(Myrothecium Verrucaria)J-1胆红素氧化酶的纯化及性质初步研究

从中国土样中筛选到一株能产生胆红素氧化酶的微生物(Myrothecium Verrucaria)J-1,培养后,分离纯化,最后经QAE—Sephadex A50柱层析,得到胆红素氧化酶比活为207.65 U/A 280nm,总产率为22.3％。纯酶紫外吸收峰为278 nm,凝胶电泳为单一色带。分子量估计为52000。它能迅速、特异地氧化胆红素为胆绿素,并进一步氧化成目前还不清楚的紫色化合物。最佳作用pH为7.0,最佳作用温度为40℃。     

Enzymic oxidation of unconjugated bilirubin by rat liver.

The presence of the enzyme bilirubin oxidase, which degrades bilirubin in vitro, was demonstrated in the liver. Subcellular-fractionation experiments indicate that bilirubin oxidase is located in both the inner and outer membranes of the mitochondria. The mean rate of the reaction is 1.57 +/- 0.38 (S.D.) nmol of bilirubin degraded/min per mg of mitochondrial protein (munits/mg of protein). With respect to the overall breakdown of bilirubin, the enzyme has a Km' of 136 microM-bilirubin and a Vmax.' of 9.13 munits/mg of protein. Its activity is influenced by the ionic strength of the media and is inhibited by KCN, thiol reagents, NADH and albumin. The enzyme is aerobic, and between 1 and 1.5 mol of O2 are consumed per mol of bilirubin degraded. The products of the reaction include propentdyopents. The hepatic bilirubin oxidase activity of the jaundiced Gunn-rat liver is not significantly different from that of the Sprague-Dawley rat, and it is not induced by beta-naphthoflavone.     

Peroxide interaction with pulsed cytochrome oxidase. Optical and EPR studies

EPR and optical analysis of the 420 nm form of cytochrome oxidase (Kumar, C., Naqui, A., and Chance, B. (1984) J. Biol. Chem. 259, 2073-2076) shows that 1) the 420 nm form possesses a 605 nm band, g = 5 EPR signals, and a slightly blue shifted 655 nm band; 2) the reaction of H2O2 with the 420 nm form generates the peroxide complex (Soret band at 427 nm) with the formation of a 580 nm band and abolition of both the 655 nm band and the g = 5 EPR signal. Comparison of our results with past data shows that various forms of oxidase formed from the resting oxidase through different protocols may be identified to be either the 420 nm or the 427 nm form and leads to identification of a peroxy intermediate during oxidase turnover.