Biosci. Biotechnol. Biochem., 66 (4), 717–721, 2002

NADH Oxidation by Manganese with or without a-Hydroxy

Tetsuya DEGUCHI, Masaaki MATSUBARA,andTomoakiNISHIDA*

Chemical and Environmental Technology Laboratory, KOBE STEEL, LTD., Kobe 651-2271, Japan *Department of Forest Resources Science, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan

Received August 3, 2001; Accepted November 28, 2001

NADH oxidation by manganese peroxidase (MnP) organic chelate the generated Mn(III) and was done in a reaction mixture including either a- release the Mn(III) from the -manganese hydroxy acid or acetate. The oxidation in the former complex. The released Mn(III)-organic acid, in turn, reaction mixture was inhibited by a and was ac- oxidizes various substrates.7,8) This oxidizing system celerated by exogenous H2 O2, while the oxidation in the in which the low molecular weight compound, latter reaction mixture was inhibited by a superoxide Mn(III)-organic acid, acts as the direct oxidizing dismutase and was not accelerated by the exogenous agent is believe to be e‹cient for oxidation of poly-

H2O2. These results indicated that there are signiˆcant meric substrates such as because the low diŠerences between the two reaction systems, particu- molecular weight compound is mobile in polymeric larly, in the active oxygen species involved in the reac- substrates which may be inaccessible to polymeric en- tions. Additionally, the experiment of MnP reduction zymes. with Mn(II) suggests that MnP has a separate catalytic On the other hand, we have reported that the white activity other than an oxidation of Mn(II) to Mn(III) in rot fungi were able to oxidatively degrade nylon un- the reaction mixture including acetate. der ligninolytic conditions9) and that puriˆed MnP was able to degrade nylon without the exogenous 10,11) Key words: manganese peroxidase; NADH; nylon H2O2. In addition, surprisingly, the a-hydroxy degradation acid was a strong inhibitor of the nylon degradation. These results cannot be explained by the known fea- White rot fungi are the best-known and most eŠec- tures of the MnP catalytic cycle. tive lignin-degrading microorganisms. These fungi Recently we recognized that MnP could oxidize have recently received worldwide attention because NADH either with or without a-hydroxy acid. The of their detoxiˆcation of recalcitrant environmental NADH oxidation by MnP is very interesting and may pollutants such as dioxins,1–3) DDT,4) and PCB,5,6) un- be useful to increase the understanding of the MnP der ligninolytic conditions. The process of their lig- reaction without a-hydroxy acid. In this paper, we in- nin degradation is nonspeciˆc and nonstereoselec- vestigate the NADH oxidation by MnP and reveal tive, which explains why these fungi can degrade the diŠerences in the NADH oxidation between with various organic materials. and without a-hydroxy acid. Under ligninolytic conditions, many white rot fungi secrete extracellular . Manganese Materials and Methods peroxidase (MnP) is one of the most frequently en- countered peroxidases among white rot fungi. MnP Chemicals. NADH and superoxide dismutase has the same catalytic cycle as other peroxidases but (SOD) were purchased from Wako Pure Chemical MnP is unique in its ability to catalyze the oxidation Industries (Osaka, Japan). Catalase was purchased of Mn(II) to Mn(III). Resting MnP is oxidized by fromSigmaChemicalCo.(St.Levis,Mo.).

H2 O2 in a single two-electron step to form MnP com- pound I and the latter is reduced by Mn(II) back to MnP preparation. The white rot fungus strain the resting MnP. In this reduction step, a total of two IZU-154, which had been isolated in our laborato- equivalents of Mn(III) is formed. In this catalytic ry,12) was used for the production of MnP. MnP was cycle, a-hydroxy acids such as lactate, malate, and puriˆed from the extracellular ‰uid of the IZU-154 tartrate are known to have an important role. These cultures (6 days incubation) as described previous-

To whom correspondence should be addressed. Tetsuya DEGUCHI, Phone: +81-78-992-5618; Fax: +81-78-992-5604; E-mail: te-deguchi＠ rd.kcrl.kobelco.co.jp Abbreviations: MnP, manganese peroxidase; 2,6-DMP, 2,6-dimethoxyphenol; SOD, superoxide dismutase 718 T‚ DEGUCHI et al. ly.10)

NADH oxidation. The reaction mixture, contained

50 mM organic acid and 1 mM MnSO4,wasadjusted to pH 4.5 by sodium hydroxide. The organic acid was selected from lactate, tartrate, malate, and acetate. The MnP was added to the reaction mixture and reaction was initiated by the addition of NADH (ˆnal conc. of 0.17 mM). After mixing them, NADH oxida- tion was followed by monitoring the absorbance at 340 nm. In experiments investigating the eŠects on

H2 O2,SOD,andcatalase,theywereaddedtothe reaction mixture before the addition of NADH. In order to investigate whether the reaction involved the production of low molecular weight compounds which were able to oxidize NADH or not, an experiment using an ultra-ˆltration mem- brane, Ultrafree-PFL (10,000-molecular-weight Fig. 1. NADH Oxidations in Various Reaction Mixtures. Symbols: , acetate 50 mM; , lactate 50 mM; $, tartrate 50 cutoŠ; Millipore), was done. The ˆrst reaction mix- mM; ,malate50mM. ture and MnP were placed in the Ultrafree-PFL (total vol. of 0.5 ml). Then the ˆrst reaction was started by the addition of NADH (ˆnal conc. of 0.17 mM). After 5 minutes of reaction, the Ultrafree-PFL was pressurized and a solution containing low molecular weight compounds but no MnP was collected in a receiver. One-tenth of a ml of the solution was then added to the second reaction mixture (0.5 ml) con- taining 0.17 mM NADH and no MnP. The absor- bance at 340 nm of the second reaction mixture was monitored.

Reduction of MnP with manganese. In order to in- vestigate the MnP reduction rate with manganese, the absorbance at 406 nm, which diŠers in extinction coe‹cient between resting and oxidized MnP,8) was monitored. Puriˆed MnP in the reaction mixture in- cluding either malate or acetate was oxidized by H2O2 (ˆnal conc. of 0.1 mM) and then MnSO4 was added to the reaction mixture (ˆnal conc. of 1 mM). The absor- Fig. 2. NADH Oxidation Rates in Various Reaction Mixtures. bance at 406 nm of the reaction mixture was immedi- Symbols: , acetate 50 mM; , lactate 50 mM; $, tartrate 50 ately monitored after the addition of MnSO . mM; ,malate50mM. The decreasing rates of absorbance at 4 340 nm were calculated as the average for 0.1 min. Results trapolation to the intercept of Fig. 2, was NADH oxidation proˆle 0.18 Abs.Wmin. and maximum. The oxidation rate Figure 1 shows the courses of absorbance at gradually decreased. 340 nm, indicating a NADH concentration. In the Figure 3 shows the course of absorbance in the reaction mixture including a-hydroxy acid, i.e. lac- presence of both acetate and malate. The proˆle of tate, tartrate, or malate, the initial oxidation rate, the reaction course resembled that of malate very whichwasestimatedbyextrapolationtotheintercept closely, indicating that malate is predominant in of Fig. 2, was close to zero and the oxidation rate deciding the reaction type. increased. The oxidation rate reached maxima, 1.72, 0.35, and 0.50 Abs.Wmin., after acceleration periods EŠectsofH2O2 of 0.3, 2.7, and 2.1 minutes in the reaction mixture The eŠects of exogenous H2 O2 on NADH oxida- including lactate, tartrate, and malate, respectively. tion in the reaction mixture including malate or On the other hand, in the reaction mixture including acetate are shown in Figs. 4(a) and 4(b), respectively. acetate, this acceleration period was not observed. In the former reaction mixture, the additional H2O2 The initial oxidation rate, which was estimated by ex- increased the initial oxidation rate and reduced the NADH Oxidation by Manganese Peroxidase with or without a-Hydroxy Acid 719

Fig. 5. EŠects of SOD and Catalase on NADH Oxidation in the Reaction Mixture Including Malate (a) and Acetate (b). Symbols: , no addition; , 340 uWml of SOD (ˆnal concen- tration); #, 2600 uWml of catalase (ˆnal concentration).

Fig. 3. NADH Oxidation in the Reaction Mixture Including both Malate and Acetate. Symbols: , acetate 50 mM; #, malate 50 mM; ; acetate 50 mM+malate 50 mM.

Fig. 4. EŠects of Exogenous H2 O2 onNADHOxidationinthe Fig. 6. NADH Oxidation by Low Molecular Weight Compounds Reaction Mixture Including Malate (a) and Acetate (b). Obtained from the Reaction Mixture Including Malate or Symbols: , no addition; , 100 mM of H2 O2 (ˆnal concen- Acetate. tration); $,10mM; ,1mM. Symbols: , malate; , acetate; #, control. The control was done by low molecular weight compounds obtained from the reaction mixture including malate but not enzyme. acceleration period. Particularly the 100 mM H2O2 completely extinguished the acceleration period. These results suggest that the NADH oxidation in the lase, on the whole, slightly accelerated the NADH reaction mixture including malate requires H2O2, oxidation (Fig. 5(b)). These results indicate that the especially in the initiation, and the acceleration two reaction mixtures diŠer in the active oxygen spe- period is caused by an insu‹ciency of H2O2. cies involved in the reactions. On the other hand, in the latter reaction mixture, the acceleration resulting from the addition of H2O2 NADH oxidation with low molecular weight com- was not observed. Besides, the inhibition of the reac- pounds tion was observed by the 100 mM H2O2, perhaps due Figure 6 shows the courses of absorbance at to the oxidative stress of H2 O2. 340 nm of the second reaction mixtures after the addition of the low molecular weight compounds EŠects of SOD and catalase obtained in the ˆrst reaction mixtures. Only low Figure 5 shows the eŠects of SOD and catalase on molecular weight compounds, obtained from the NADH oxidation. In the reaction mixture including reaction mixture including malate, had NADH oxi- malate, the NADH oxidation was strongly inhibited dation activity. by catalase but not by SOD (Fig. 5(a)). On the other hand, in the reaction mixture including acetate, SOD Reduction of MnP with manganese strongly inhibited the NADH oxidation and catalase Figure 7 shows the courses of the absorbance at slowed down the initial oxidation rate although cata- 406 nm. Although the reduction of MnP in the reac- 720 T‚ DEGUCHI et al. including malate was accelerated by the exogenous

H2O2 but the reaction system including acetate is in- sensitive to the exogenous H2O2. These results sug- gest that the two reaction systems diŠer in the

amount of H2O2 required although both systems re- quire some H2O2. Another diŠerence is whether active low molecular weight compounds are involved in the reaction sys- tem or not. The experience using ultra-ˆltration showed that the reaction system including a-hydroxy acid produces active low molecular weight com- pounds for NADH oxidation (Fig. 6). It is well- known that MnP produces Mn(III)-a-hydroxy acid complex in the presence of three items, Mn(II), a-

hydroxy acid, and H2O2, in a peroxidase catalytic cycle. All items except the H2 O2 are apparently present in the reaction mixture including malate. Fig. 7. Reduction of Manganese Peroxidase with Manganese in Moreover, the H2 O2 can also come from, for exam- the Reaction Mixture Including Malate (a) or Acetate (b). ple, the NADH autooxidation as mentioned above. The reaction was started by the addition of manganese. Dashed lines are the predicted amounts. Considering this, the active low molecular weight compounds are probably a Mn(III)-a-hydroxy acid complex. This complex is known to have su‹cient tion mixture including malate momentarily occurred, oxidation potential to oxidize NADH.7) that in the reaction mixture including acetate was sig- On the other hand, the active low molecular weight niˆcantly slower. compounds were not found in the reaction system in- cluding acetate. This is a very interesting result be- Discussion cause MnP has been recognized to be an enzyme whichoxidizesMn(II)toMn(III),whichinturnoxi- The studies described here show that there are sig- dizes the . In addition, our result shows that niˆcant diŠerences between the two reaction systems, the reduction rate of MnP with Mn(II), i.e. the oxi- especially in active oxygen species involved in the dation rate of Mn(II) by MnP, is extremely slow in reactions. The experiments using SOD suggest that the reaction system including acetate. These results the superoxide anion essentially relates to progress of suggest that in the reaction system including acetate, the NADH oxidation in the reaction system including MnP has a separate catalytic activity other than acetate, but does not relate to the reaction system in- catalyzing the Mn(II) to Mn(III) reaction. In addi- cluding a-hydroxy acid such as malate. tion, the result that malate, which has the role of

The eŠect of H2O2 may be rather more complicat- releasing the manganese from the enzyme-manganese ed than that of the superoxide anion. In the reaction complex, is predominant in deciding the reaction system including a-hydroxy acid, catalase is a strong type (Fig. 3) suggests that that activity appears when inhibitor and exogenous H2O2 accelerates the reac- MnP is in the form of enzyme-manganese complex. tion, indicating that this reaction system apparently a-Hydroxy acid is an essential component of the requires H2O2 to progress the reaction. On the other well-known MnP reaction system. This reaction sys- hand, in the reaction system including acetate, tem is very eŠective in degrading polymeric sub- NADH oxidation is not inhibited by catalase and is strates such as lignin that are inaccessible to polymer- not accelerated by the exogenous H2O2 (Figs. 4 and ic , because this system produces the 5). In spite of these results, it seems that the reaction Mn(III)-a-hydroxy acid complex, as a direct oxidiz- system including acetate also requires H2O2, especial- ing agent, which is mobile in the polymeric substrate. ly in the initiation of the reaction, because an acceler- From this ability, MnP has been believed to be one of ation period was observed when catalase was added the main forces of lignin degradation by white-rot in the reaction mixture. fungi. On the other hand, we show in this paper that

One source of H2O2 may come from the autooxi- MnP has another catalytic activity in the absence of dation of NADH, reaction (1): a-hydroxy acid, and suggest that the activity is other than the catalyzing Mn(II) to Mn(III) reaction and is NADH+O +H+ªNAD++H O (1) 2 2 2 expressed when MnP is in the form of enzyme-man- This reaction occurs spontaneously but very ganese complex. These results show the further pos- 13) slowly and therefore the H2O2 produced is very sibility of a role for MnP in lignin degradation and slight. The experiments using exogenous H2O2 an extension of the substrates that MnP can oxidize. showed that the oxidation rate in the reaction system We are hopeful that continuing studies will yield fur- NADH Oxidation by Manganese Peroxidase with or without a-Hydroxy Acid 721 ther insight into the function and mechanism of this 3904–3909 (1995). activity. 7) Glenn, J. K., Akileswaran, L., and Gold, M. H., Mn(II) oxidation is the principal function of the References extracellular Mn-peroxidase from chrysosporium. Arch. Biochem. Biophys., 251, 1) Bumpus,J.A.,Tien,M.,Wright,D.,andAust,D., 688–696 (1986). Oxidation of present environmental pollutants by a 8) Wariishi, H., Dunford, H. B., MacDonald, I. D., white rot fungus. Science, 227, 1434–1436 (1985). and Gold, M. H., Manganese peroxidase from the 2) Valli, K., Wariishi, H., and Gold, M. H., Degrada- lignin-degrading basidiomycete Phanerochaete tion of 2,7-dichlorodibenzo-p-dioxin by the lignin- chrysosporium. J. Biol. Chem., 264, 3335–3340 degrading basidiomycete Phanerochaete chrysospori- (1989). um.J.Bacreriol., 62, 2131–2137 (1992). 9) Deguchi,T.,Kakezawa,M.,andNishida,T.,Nylon 3) Takeda, S., Nakamura, M., Matsuda, T., Kondo, R., degradation by lignin-degrading fungi. Appl. Envi- and Sakai, K., Degradation of polychlorinated diben- ron. Microbiol., 63, 329–331 (1996). zo-p-dioxins and polychlorinated dibenzofurans by 10) Deguchi, T., Kitaoka, K., Kakezawa, M., and the white rot fungus Phanerochaete sordida YK-624. Nishida, T., Puriˆcation and characterization of a Appl. Environ. Microbiol., 62, 4323–4328 (1996). nylon-degrading enzyme. Appl. Environ. Microbiol., 4) Bumpus,J.A.andAust,S.D.,Biodegradationof 64, 1366–1371 (1996). DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane] 11) Nomura, N., Deguchi, T., Shigeno-Akutsu, Y., by the white rot fungus Phanerochaete chrysospori- Nakajima-Kambe, T., and Nakahara, T., Gene struc- um. Appl. Environ. Microbiol., 53, 2001–2008 tures and catalytic mechanisms of microbial enzymes (1987). able to biodegrade the synthetic solid polymers nylon 5) Yadav, J. S., Wallace, R. E., and Reddy, C. A., and polyester polyurethane. Biotechnology and Mineralization of mono- and dichlorobenzenes and Genetic Engineering Reviews, 18, 125–147 (2001). simultaneous degradation of chloro- and methyl- 12) Nishida, T., Kashino, Y., Mimura, A., and substituted benzenes by the white rot fungus Takahara, Y., Lignin biodegradation by wood- Phanerochaete chrysosporium. Appl. Environ. rotting fungi I.-Screening of lignin-degrading fungi. Microbiol., 61, 677–680 (1995). Mokuzai Gakkaishi, 34, 530–536 (1988). 6) Dietrich,D.,Hickey,W.J.,andLamar,R.,Degra- 13) Yokota, K. and Yamazaki, I., Analysis and computer dation of 4,4?-dichlorobiphenyl, 3,3?,4,4?- simulation of aerobic oxidation of reduced tetrachlorobiphenyl, and 2,2?,4,4?,5,5?-hexachloro- nicotinamide adenine dinucleotide catalyzed by biphenyl by the white rot fungus Phanerochaete . Biochemistry, 16, 1913–1920 chrysosporium. Appl. Environ. Microbiol., 61, (1977).