Type III Cu mutants of Myrothecium verrucaria bilirubin oxidase

Type III Cu ligand, His456 and His458, of Myrothecium verrucaria (MT-1) bilirubin oxidases (BO) [EC 1.3.3.5] were doubly mutated as to Lys, Asp, and Val. In spite of perturbation of the type III Cu centers, these mutants were pale blue or colourless when isolated. However, they became intense blue on reaction with reducing agents such as dithionite, ascorbate, hexacyanoferrate(II), and octacyanotangstate(IV) under air, or with an oxidizing agent such as hexacyanoferrate(III), indicating that they are in mixed forms when expressed in Aspergillus oryzae. His456.458Lys and His456.458Asp mutated as to potential coordinating groups showed weak BO and ferroxidase activities, while His 456.458Val mutated as to non-coordinating groups showed no enzyme activity at all.     

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.     

胆红素氧化酶产酶菌株的分离及最佳产酶条件的研究

A bilirubin oxidase (EC 1.3.3.5) producing strain, Mv 2.1089, was isolated from several strains of Myrothecium verrucaria by dilution method. The optimum conditions of enzyme production were investigated and the results were as follows: the suitable medium was cultured at 25 degrees C on a rotating shaker glucose and peptone, at pH 6.0. The strain was cultured at 25 degrees C on a rotating shaker (150 r/min) for 96 h. Bilirubin oxidase with 0.5-1.5 u/ml was obtained in the culture medium.     

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.     

Characterization of alkaliphilic laccase activity in the culture supernatant of Myrothecium verrucaria 24G-4 in comparison with bilirubin oxidase

An enzyme showing alkaliphilic laccase activity was purified from the culture supernatant of Myrothecium verrucaria 24G-4. The enzyme was highly stable under alkaline conditions, showed an optimum reaction pH of 9.0 for 4-aminoantipyrine/phenol coupling, and decolorized synthetic dyes under alkaline conditions. It showed structural and catalytic similarities with bilirubin oxidase, but preferably oxidized phenolic compounds. The enzyme catalyzed veratryl alcohol oxidation at pH 9.0 with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as a mediator, suggesting that the laccase mediator system functioned well under alkaline conditions.     

Point mutations at the type I Cu ligands, Cys457 and Met467, and at the putative proton donor, Asp105, in Myrothecium verrucaria bilirubin oxidase and reactions with dioxygen

The type I Cu site in the Cys457Ser mutant of Myrothecium verrucaria bilirubin oxidase was vacant, but the trinuclear center composed of a type II Cu and a pair of type III Cu's was fully occupied by three Cu ions. Cys457Ser could react with dioxygen, affording reaction intermediate I with absorption maxima at 340, 470, and 675 nm. This intermediate corresponds to that obtained from laccase, whose type I Cu is cupric and type II and III Cu's are cuprous [Zoppellaro, G., Sakurai, T., and Huang, H. (2001) J. Biochem. 129, 949-953] or whose type I Cu is substituted with Hg [Palmer, A. E., Lee, S. K., and Solomon, E. I. (2001) J. Am. Chem. Soc. 123, 6591-6599]. Another type I Cu mutant, Met467Gln, with modified spectroscopic properties and redox potential, afforded reaction intermediate II with absorption maxima at 355 and 450 nm. This intermediate corresponds to that obtained during the reaction of laccase [Sundaram, U. M., Zhang, H. H., Hedman, B., Hodgson, K. O., and Solomon, E. I. (1997) J. Am. Chem. Soc. 119, 12525-12540; Huang, H., Zoppellaro, G., and Sakurai, T. (1999) J. Biol. Chem. 274, 32718-32724]. According to a three-dimensional model of bilirubin oxidase, Asp105 is positioned near the trinuclear center. Asp105Glu and Asp105Ala exhibited 46 and 7.5% bilirubin oxidase activity compared to the wild-type enzyme, respectively, indicating that Asp105 conserved in all multi-copper oxidases donates a proton to reaction intermediates I and II. In addition, this amino acid might be involved in the formation of the trinuclear center and in the binding of dioxygen based on the difficulties in incorporating four Cu ions in Asp105Ala and Asp105Asn and their reactions with dioxygen.     

Compensatory binding of an asparagine residue to the coordination-unsaturated type I Cu center in bilirubin oxidase mutants

Met467, the axial ligand to type I Cu in a multicopper oxidase, Myrothecium verrucaria bilirubin oxidase was substituted with a non-coordinating Phe and Leu to transform the spectral and magnetic properties and oxidase activities of the enzyme into those of fungal laccases, but the mutated type I Cu center showed properties characteristic of phytocyanins, blue copper proteins with an axial coordination of Gln, due to compensatory binding of the distal Asn459 as evidenced by a double mutation.     

Electrochemical consideration on the optimum pH of bilirubin oxidase

Steady-state current-potential curves were obtained for the direct electron transfer (DET) of bilirubin oxidase (BOD) at a highly oriented pyrolytic graphite electrode, and the theoretical analysis based on nonlinear regression enabled us to determine the formal redox potential (E degrees') of BOD in a wide pH range of 2.0 to 8.5. Cyclic voltammetric measurements were also performed for substrates, including p-phenylenediamine (PPD), o-aminophenol (OAP), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and their E degrees ' values or the anodic peak potentials (for OAP) were determined at various pH values. The difference in the redox potentials between BOD and substrates (DeltaE degrees') showed a maximum at pH 6.5 to 8.0, pH 6.5 to 8.0, and pH 3.5 to 4.5 for PPD, OAP, and ABTS, respectively. These pH ranges should be thermodynamically most favorable for the electron transfer between BOD and the respective substrates. In practice, the pH ranges showing a maximum DeltaE degrees' corresponded well with the optimum pH values for the O(2) reduction activity of BOD: pH 6.5 to 7.5, pH 8.0 to 8.5, and pH 4.0 for PPD, OAP, and ABTS, respectively. Thus, it was suggested that DeltaE degrees ' should be one of the primary factors determining the activity of BOD with the substrates.     

Cu(I)-dependent biogenesis of the galactose oxidase redox cofactor

Galactose oxidase is a copper metalloenzyme containing a novel protein-derived redox cofactor in its active site, formed by cross-linking two residues, Cys228 and Tyr272. Previous studies have shown that formation of the tyrosyl-cysteine (Tyr-Cys) cofactor is a self-processing step requiring only copper and dioxygen. We have investigated the biogenesis of cofactor-containing galactose oxidase from pregalactose oxidase lacking the Tyr-Cys cross-link but having a fully processed N-terminal sequence, using both Cu(I) and Cu(II). Mature galactose oxidase forms rapidly following exposure of a pregalactose oxidase-Cu(I) complex to dioxygen (t(1/2) = 3.9s at pH7). In contrast, when Cu(II) is used in place of Cu(I) the maturation process requires several hours (t(1/2) = 5.1 h). EDTA prevents reaction of pregalactose oxidase with Cu(II) but does not interfere with the Cu(I)-dependent biogenesis reaction. The yield of cross-link corresponds to the amount of copper added, although a fraction of the pregalactose oxidase protein is unable to undergo this cross-linking reaction. The latter component, which may have an altered conformation, does not interfere with analysis of cofactor biogenesis at low copper loading. The biogenesis product has been quantitatively characterized, and mechanistic studies have been developed for the Cu(I)-dependent reaction, which forms oxidized, mature galactose oxidase and requires two molecules of O2. Transient kinetics studies of the biogenesis reaction have revealed a pH sensitivity that appears to reflect ionization of a protein group (pKa = 7.3) at intermediate pH resulting in a rate acceleration and protonation of an early oxygenated intermediate at lower pH competing with commitment to cofactor formation. These spectroscopic, kinetic, and biochemical results lead to new insights into the biogenesis mechanism.     

Trehalose as an endogenous reserve in spores of the fungus Myrothecium verrucaria

Mandels, G. R. (U.S. Army Natick Laboratories, Natick, Mass.), Rasma Vitols, and Frederick W. Parrish. Trehalose as an endogenous reserve in spores of the fungus Myrothecium verrucaria. J. Bacteriol. 90:1589-1598. 1965.-Gross analysis of Myrothecium verrucaria spores showed approximately 3% fat, 33% carbohydrate, and 9.5% nitrogen. The water-soluble carbohydrates were trehalose, glucose, mannitol, and an unidentified phosphorylated compound. Water-soluble amino acids include leucine or norleucine (or both), valine, gamma-amino-n-butyric acid, beta-amino-n-butyric acid, ergothionine, glutamic acid, glutamine, glycine, aspartic acid, asparagine, cystine, and cystathionine. Ergosterol was also present. alphaalpha-Trehalose is the major reserve (20% of the dry weight), although approximately 30% of it appeared to be at the spore surface and was released by nonlethal treatment with 0.1 n HCl. Treatment with toluene or exposure to heat sufficient to kill the spores (20 min at 60 C) caused rapid liberation of all of the trehalose. Although spores could utilize exogenous trehalose with no appreciable lag, some stimulus, such as exposure to heat (10 min at 55 C), incubation with azide, or germination on exogenous substrates, was necessary to effect utilization of trehalose reserves. Spores have trehalase, but it is apparently at the spore surface, since it is inactivated by acid treatment which does not kill the spores. The metabolic pathway for utilization of trehalose is not known, but presumably it is not mediated by trehalase. The involvement of mannitol is indicated, since it tends to increase as trehalose decreases, although the changes are not quantitatively equivalent.     

Constitutive and Induced Trehalose Transport Mechanisms in Spores of the Fungus Myrothecium verrucaria

Trehalose is absorbed by two distinct systems-one constitutive, the other induced by turanose and to a lesser extent by nigerose but not by trehalose. The constitutive system is apparently mediated by a surface trehalase; the induced system has the characteristics of a permease. The specificity of the induced system is apparently limited to the alpha glucosyl-glucose or glucosyl-fructose linkage, because absorption of kojibiose, nigerose, maltose, isomaltose, turanose, sucrose, and melezitose, in addition to that of trehalose, was increased. Absorption of beta-linked or of galactose-containing disaccharides was not increased. The constitutive and induced trehalose-absorbing systems differ in their activity, specificity, lability to acid treatment, effects of substrate concentration, and pH optima. Both systems require oxygen, and no marked differential effects of inhibitors were observed. The activity of the induced system is proportional to log turanose concentration (from about 1 to 300 mug/ml), and is an approximate linear function of time of exposure (from about 1 to 50 min). Accumulation of trehalose occurred against a concentration gradient in both systems but particularly in the induced. No leakage was observed. The activity of the induced system declined slowly upon removal of the inducer. Accumulated trehalose is metabolized after activation by azide as are the endogenous trehalose reserves. The accumulated trehalose appears to enter the endogenous trehalose pool found in these spores, although some data suggest it may be more accessible. Respiratory data indicate that absorbed trehalose is available for metabolism while in transit from the external membrane to the internal pool.     

The effects of reversible freezing inactivation and inhibitor binding on redox properties of l-amino-acid oxidase

We measured the redox potentials of frozen inactivated l-amino-acid oxidase (l-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.2) and inhibitor-bound (anthranilic acid) enzyme, and compared these redox properties to those of active l-amino-acid oxidase and benzoate-bound d-amino-acid oxidase (EC 1.4.3.3), respectively. The redox properties of the inactive enzyme are similar to the properties of free flavin; the potential is within 0.015 V of free flavin and no radical stabilization is seen. This corresponds to the loss of most interactions between apoprotein and flavin. In contrast, the anthranilic acid lowers the amount of radical stabilized from 85% to 35%. The potentials are still 0.150 V positive of free flavin, indicating that in the presence of inhibitor, many flavin-protein interactions remain intact. The difference between this behavior and that of d-amino-acid oxidase bound to benzoate, where the amount of radical declined from 95% to 5%, is explained on the basis of the relative tightness of binding of apoprotein to FAD. d-Amino-acid oxidase apoprotein has a relatively low K_a (10⁶) for FAD, and benzoate has a relatively high K_a (10⁵) for the enzyme. Therefore, the binding of benzoate increases the tightness of FAD binding to apo-d-amino-acid oxidase (10¹¹), indicating significant changes in flavin-protein interactions. In contrast, apo-l-amino-acid oxidase binds flavin tightly (the K_a is greater than 10⁷) and the enzyme binds to anthranilate much less tightly, with a K_a of 10³. The l-amino-acid oxidase apoprotein binding to FAD is tight initially, and the binding of anthranilate changes it only slightly. Therefore, redox studies indicate that the ability of a flavoprotein to be regulated may be influenced by the strength of the interaction of flavin with the apoprotein, as well as the strength of interaction of the substrate or activator.