Chapter 8
Cellobiose Dehydrogenase A
Hemoflavoenzyme
from
Phanerochaete chrysosporium
V . Renganathan a n d W e n j u n B a o D e p a r t m e n t o f C h e m i s t r y , Biochemistry, a n d M o l e c u l a r Biology, Oregon Graduate Institute of Science a n d Technology, 20000 Northwest W a l k e r R o a d , P . O . B o x 91000, P o r t l a n d , O R 97291-1000
Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by cellulose-degrading cultures of the lignocellulosedegrading basidiomycete Phanerochaete chrysosporium. C D H is monomelic and contains one heme b and one F A D on two distinct domains. In addition, C D H may also have a cellulose-binding domain. The heme iron of C D H is ferric, low spin, and is hexacoordinate with a histidine and a methionine as the fifth and sixth ligands. C D H oxidizes cellobiose to cellobionolactone in the presence of electron acceptors such as cytochrome c, dichlorophenol-indophenol, and ferricyanide. In the C D H reaction, flavin is the primary acceptor of electrons from cellobiose; the reduced flavin then transfers the electrons to the heme iron, one electron at a time. The reduced heme in turn transfers its electrons to ferricytochrome c. Whereas electron transfer to cytochrome c occurs only from the heme, electron transfer to other acceptors can occur directly from the reduced flavin. C D H enhances crystalline cellulose degradation by cellulases and this may be one of its physiological functions. This report reviews the progress made in understanding the structure, function, and mechanism of C D H , and its potential role in lignocellulose degradation.
M a n y cellulolytic fungi, including P. chrysosporium (1,2), Sporotrichum thermophile (3), Coniophora puteana (4), and Sclerotium rolfsii (5), produce extracellular cellobiose oxidizing enzymes, in addition to cellulases. A l l of these dehydrogenases appear to oxidize cellobiose to cellobionolactone. Cellobiose dehydrogenases ( C D H ) from P. chrysosporium (1,6,7), S. thermophile (3,8), and C. puteana (4,9), a brown-rot fungus, are characterized as hemoflavoenzymes containing one heme b and one F A D or F M N per monomer. In addition to C D H , P. chrysosporium produces another cellobiose-oxidizing flavoenzyme, cellobiose:quinone oxidoreductase (CBQase) (2).
0097-6156/94/0566-0179$08.00/0 © 1994 American Chemical Society
180
ENZYMATIC CONVERSION OF BIOMASS F O R FUELS PRODUCTION
The nature of cellobiose-oxidizing enzymes produced by S. rolfsii, Monilia sp., Chaetomium cellulolyticum, and Fomes annosus is not known (5,10-12). O n l y a few enzymes are known to contain a heme and a flavin on a monomelic subunit Apart from C D H , the following enzymes are characterized as hemoflavoenzymes: flavocytochrome b or lactate dehydrogenase from Saccharomyces cerevisiae (13), spermidine dehydrogenase from Serratia marcescens (14), rubredoxin oxidase from Desulfovibrio gigas (15), nitric oxide synthetase from murine macrophages (16), and a fatty acid monooxygenase from Bacillus megaterium (17). A m o n g these, only flavocytochrome b , which dehydrogenates lactate to pyruvate, is well characterized and this enzyme may serve as a model for C D H (13,18,19). Flavocytochrome b is a tetramer; each monomer contains one F M N and one heme b. The polypeptide chain is organized into two domains, a cytochrome domain to which the heme binds, and a flavodomain to which the F M N binds (13,18). The two domains are linked by a protease-sensitive segment (18). The heme iron is in the ferric oxidation state and is hexacoordinate. Histidine residues 43 and 66 of the apoprotein function as the fifth and the sixth ligands to the heme iron (13). The F M N group is the primary acceptor of electrons from lactate. The reduced flavin then transfers the electrons to the heme iron i n two single steps. The heme, i n turn, transfers the electrons to cytochrome oxidase v i a ferricytochrome c (13). C D H (also known as cellobiose oxidase) from P. chrysosporium was discovered by Eriksson and coworkers (1). Several methods for the purification of C D H from P. chrysosporium have been reported (1,6,7,1920); however, a procedure that we recently developed provides C D H in high yield and with high specific activity compared to other methods (6). C D H from P. chrysosporium is a monomer with a molecular mass of approximately 90,000. It contains one heme b and one F A D per monomer. It is a glycoprotein with a neutral carbohydrate content of 9.4% (6). C D H is very stable between p H 3 and 10.5, and below 50°C. However, below p H 2 or above 5 0 ° C , the activity is lost due to release of the flavin from the active site. The heme appears to be tightly bound to the apoprotein under these conditions (6). 2
2
2
Spectral Studies The heme iron of C D H exists i n the ferric state (1,6,7). The U V - v i s i b l e spectrum of oxidized C D H shows absorbances at 420, 529, and 570 nm; on the addition of cellobiose or dithionite, absorbances shift to 428, 534, and 564 nm (Figure 1) (1,6,7). These spectral characteristics of C D H are very similar to the those of the hemoflavoenzymes, flavocytochrome b and spermidine dehydrogenase (6,14,23). Our preliminary studies indicate that the resonance Raman spectral characteristics of C D H are also very similar to those of flavocytochrome b suggesting similar heme structures for both of these enzymes (23; Cohen, Bao, Renganathan, and Loehr, unpublished results). Ferric C D H does not bind azide or cyanide; this is indicative of a hexacoordinate heme iron as is found i n flavocytochrome b (6). T w o histidines serve as the fifth and sixth ligands to the heme iron of flavocytochrome b (13). However, based on nuclear magnetic resonance and infrared-magnetic circular dichroism studies, C o x et al. have concluded that C D H has a histidine and a methionine as the fifth and sixth coordinations to the heme iron (24). 2
2
2
2
F i g u r e 1. U V - v i s i b l e spectra of oxidized (a) and reduced (b) C D H . Reduced C D H was generated by the addition of cellobiose (50 p M ) to the oxidized form. (Reproduced with permission from ref. 6, copyright Academic Press, 1993.)
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ENZYMATIC CONVERSION OF BIOMASS FOR FUELS PRODUCTION
Structural Organization The heme and the F A D of C D H are bound noncovalently to different domains (2021)- O n treatment with a protease such as papain, C D H is hydrolyzed into two peptide fragments: a larger peptide ( M , - 55,000) containing the flavin, and a smaller peptide ( M , « 35,000) containing the heme (2021)- The flavin-containing peptide is catalytically active and oxidizes cellobiose to cellobionolactone in the presence of quinones or dichlorophenol-indophenol. However, unlike native C D H , the flavodomain cannot transfer its electrons to cytochrome c (2021)These characteristics of the flavodomain are very similar to those of CBQase, the second cellobiose-oxidizing extracellular enzyme identified from P. chrysosporium (2). It has been postulated that C B Q a s e is formed v i a the proteolytic hydrolysis of C D H (20 21). Our recent observations—that culture conditions favoring protease production increase the yield of C B Q a s e and decrease the yield of C D H - s u p p o r t this hypothesis (Bao, Lymar, and Renganathan, unpublished results). Our laboratory first reported (25) that C D H and C B Q a s e bind to microcrystalline cellulose, and we proposed that these enzymes may have a specific cellulose-binding domain similar to that of cellulases. Later, Henriksson et al. (79) demonstrated that the cellulose-binding domain of C D H is located on the flavodomain. y
Kinetic Studies Cellobiose, cellotriose, cellotetraose, cellopentaose, and lactose function as substrates for C D H (7). C D H oxidizes these sugars in the presence of electron acceptors such as cytochrome c, dichlorophenol-indophenol, quinones, ferricyanide, Mn(III)-malonate complex, and even ferric iron and its complexes (1,6,7202226). Our recent steadystate kinetic studies suggest that cellobiose, with a K,,, of 25 u M , is the preferred substrate, whereas lactose, with a K,,, of 630 p M , is the least preferred substrate. However, all the sugar substrates exhibited similar k ^ values (6). Our kinetic investigation also suggests that cytochrome c is the preferred electron acceptor for C D H (6). Cytochrome c is the physiological electron acceptor for the hemoflavoenzymes flavocytochrome b and spermidine dehydrogenase (13,14); however, unlike C D H , these enzymes are intracellular and membrane associated. A s in the flavocytochrome b reaction, the flavin moiety of C D H appears to receive the electrons from cellobiose first and then transfer them to the heme iron, one electron at a time. The reduced heme, in turn, transfers the electrons to ferricytochrome c (Scheme I). Jones and W i l s o n (27) observed that the rate constant for heme iron reduction in C D H increases with enzyme concentration and interpreted 2
2
Cellobiose
>^
FAD
0X
Cellobionolactone ^ - ^ ^ ^ F A D
Scheme I.
-^-^
red
- ^ ^ ^
Heme-Fe - * N . >^ 2+
Heme-Fe
3+
Catalytic cycle of C D H
Cytc-Fe
3+
Cyt c - F e
2+
8.
R E N G A N A T H A N AND BAO
Cellobiose Dehydrogenase
183
this as due to the intermolecular reduction of the heme center by flavin groups; whereas, i n the case of flavocytochrome b , electron transfer from flavin to heme is intramolecular (79). Samejima et al. (28) studied the effect of p H on the reduction of cytochrome c and dichlorophenol-indophenol by C D H . Although both of these electron acceptors are active at p H 4.2, only dichlorophenol-indophenol is active at p H 5.9. Furthermore, a stopped-flow kinetic study indicated that reduction of the flavin at p H 4.2 and 5.9 is fast, whereas the heme reduction is fast only at p H 4.2. These observations have led to the proposal that reduction of heme by the flavin decreases at a higher p H . Since the heme transfers electrons to cytochrome c, the reaction rate for cytochrome c reduction also has been suggested to decrease at a higher p H (28). 2
CDH
Reaction with Oxygen
C D H was first characterized as an oxidase because it consumes oxygen during its reaction (7). However, recent studies clearly indicate that oxygen is a very poor acceptor of electrons from C D H (622,29). Accordingly, we renamed the enzyme cellobiose dehydrogenase to correctly reflect its nature. In the absence of a suitable electron acceptor, 0 serves as the electron acceptor and is reduced to H 0 . W e have demonstrated the stoichiometric production of H 0 from cellobiose oxidation (6). Based on pre-steady-state kinetic studies, W i l s o n et al. identified the heme group as the site for reaction with oxygen (29). C D H can reduce 0 by one electron to produce a superoxide radical, or by two electrons to produce H 0 . However, superoxide radicals are unstable and readily disproportionate to H 0 and 0 . Thus, both one-electron and two-electron reduction of oxygen w i l l lead to H 0 generation. It has been hypothesized that a superoxide radical is formed i n the C D H reaction; however, formation of superoxide radical has not yet been established (26). 2
2
2
2
2
2
2
2
2
2
2
2
2
Interaction o f C D H w i t h L i g n i n Peroxidase The lignin degradative system of P. chrysosporium contains two important extracellular peroxidases: lignin peroxidase ( L i P ) and manganese peroxidase ( M n P ) (30,31). C D H , in the presence of cellobiose, inhibits a variety of reactions catalyzed by L i P , M n P , and horseradish peroxidase ( H R P ) (6,22). Oxidation of a phenol by the peroxidases proceeds through the corresponding phenoxy radical, and produces quinone and polymerized phenol as the final products. C D H inhibits phenol oxidation by reducing the phenoxy radicals (22). Phenoxy radicals are also produced during lignin depolymerization by phenol oxidases, and these radicals tend to condense with themselves and with the lignin substrate (32,33). It has been proposed that reduction of phenoxy radicals by cellobiose-oxidizing enzymes prevents these polymerization reactions and thus increases the rate of depolymerization (34). In addition to phenols, nonphenolic compounds such as 3,4-dimethoxybenzyl alcohol and methoxybenzenes serve as L i P substrates. L i P oxidation of these compounds proceeds through the corresponding aromatic cation radical intermediate (50,37). C D H is suggested to inhibit these L i P reactions by reducing the cation
184
ENZYMATIC CONVERSION OF BIOMASS FOR FUELS PRODUCTION
radical intermediates (22). In the L i P reaction, H 0 oxidizes the ferric native enzyme by two electrons to produce compound I. Suitable peroxidase substrates then reduce compound I by one electron to generate compound II. Donation of one more electron from the substrate to compound II completes the catalytic cycle and regenerates the ferric peroxidase (30,31,35). Our recent studies suggest electron transfer from reduced C D H to H 0 - o x i d i z e d forms of L i P and H R P (Bao and Renganathan, unpublished observations). A l s o , i n the presence of cellobiose and very low concentrations of veratryl alcohol, C D H protected L i P from H 0 -dependent inactivation (56,57,Bao and Renganathan, unpublished results). 2
2
2
2
2
2
R o l e o f C D H i n Cellulose Degradation Our laboratory examined the contribution of C D H to cellulose degradation by studying the effect of C D H addition on microcrystalline cellulose hydrolysis by Trichoderma viride cellulase (38). A t low concentrations (5-10 pg/mL), C D H enhances both glucose and cellobiose production (Figure 2). However, at higher levels, production of reducing sugar decreases and cellobionolactone increases. The decrease observed
Cellobiose
dehydrogenase
(jig/ml)
F i g u r e 2. Effect of P. chrysosporium C D H on T. viride cellulase-catalyzed conversion of microcrystalline cellulose to glucose ( • ) , cellobiose (±), and cellobionolactone (•). (Reproduced with permission from ref. 38, copyright Elsevier Science Publishers, 1992.)
8.
RENGANATHAN A N D BAO
185
Cellobiose Dehydrogenase
at higher C D H concentrations is attributed to the increased oxidation of cellobiose to cellobionolactone. Cellulose weight loss, in the presence of 10 pg mL" C D H , increases by 19% compared to the control cellulase incubations (Table I). However, at 60 pg mL" , C D H weight loss decreases by 24% compared to that of the control. In these reactions, oxygen functions as the electron acceptor for C D H leading to the production of H 0 . W e suspected that oxy-radicals generated from H 0 might be inactivating the cellulases, particularly at high C D H concentrations. In support of this hypothesis, we demonstrated that catalase addition increases the cellulose weight loss (Table I) and reducing sugar production. Hydrolysis of crystalline cellulose is recognized as the rate-limiting step in the saccharification of cellulose to glucose (39). Our findings suggest that C D H increases the velocity of this rate-limiting step. 1
1
2
2
2
2
Kremer and W o o d (26) proposed that hydroxyl radical is produced by the reaction of ferrous iron, generated through the one-electron reduction of ferric iron by C D H , and H 0 , generated from the C D H reaction with oxygen. H y d r o x y l radicals generated in this fashion are suggested to disrupt the crystalline structure of cellulose 2
2
T a b l e I. T. viride Cellulase Catalyzed Hydrolysis of Microcrystalline Cellulose. C D H and/or Catalase Addition on Cellulose Weight Loss.
Effect of
a
Enzymes
Weight loss (mg)
Percentage weight loss relative to control b
1
1. Cellulase (0.2 mg mL" ) (control) 2. Cellulase (0.2 mg mL" ) C D H (10 pg m L ) 3. Cellulase (0.2 mg mL" ) C D H (60 pg m L ) 4. Cellulase (0.2 mg m L ) C D H (10 pg m L ) + catalase (5 pg mL" ) 5. Cellulase (0.2 mg m L ) C D H (60 pg mL" ) + catalase (5 pg mL" )
91.5 ± 1.0
1
+
1
+
1
1
1
+
1
+
100
± 1.1
108.5 ± 0.6
118.6 ± 0 . 6
69.5 ± 0.1
76.0 ± 0 . 1
109.2 ± 2.3
119.3 ± 2 . 1
86.7 ± 0.2
94.8 ± 0.2
1
1
1
1
a
b
Enzymes were incubated with 600 mg microcrystalline cellulose in 50 m M acetate, p H 5, in a total volume of 30 m L . Weight loss was determined as described (36). Cellulose weight loss observed in the absence of C D H and catalase was considered to be 100%.
S O U R C E : Reproduced with permission from ref. 38, copyright Elsevier Science Publishers, 1992.
186
ENZYMATIC CONVERSION OF BIOMASS FOR FUELS PRODUCTION
(2(5). W e observed very similar enhancement of cellulose hydrolysis by C D H in the presence or absence of catalase. In the presence of catalase, H 0 is decomposed, severely inhibiting hydroxyl radical formation. Therefore, hydroxyl radicals are not responsible for the enhancement of cellulose hydrolysis observed in our study. A n alternate explanation for enhanced cellulose hydrolysis is that cellobiose is an inhibitor of cellulases (40), and by dehydrogenating cellobiose to cellobionolactone C D H is removing this inhibition. In addition, C D H appears to be capable of oxidizing cellulose directly (826); however, the nature of the degradation products and the possible significance of this reaction is not known. Further studies are necessary to fully understand the mechanism by which C D H enhances cellulose degradation. 2
2
Though C D H was discovered i n 1978, most information on this enzyme has accumulated over the past five years through the efforts of several laboratories. In the next few years, further research should provide a more thorough understanding of the structure, function, and mechanism of C D H , and the role of C D H in lignocellulose degradation. Acknowledgments Our research was supported by grants D E - F G 0 6 - 8 7 E R 13715 and D E - P R 0 6 - 9 2 E R 20065 from the U . S . Department of Energy, Office of Basic Energy Sciences. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Ayers, A.R.; Ayers, S.B.; Eriksson, K . - E . Eur. J. Biochem. 1978, 90, 171. Westermark, U . ; Eriksson, K . - E . Acta Chim. Scan. 1975, B29, 419. Coudray, M.-R.; Canevascini, G.; Meier, H . Biochem. J. 1982, 203, 277. Schmidhalter, D.R.; Canevascini, G. Appl. Microbiol. Biotechnol. 1992, 37, 431. Sadana, J.C.; Patil, R.V.J. Gen. Microbiol. 1985, 131, 1917. Bao, W.; Usha, S.N.; Renganathan, V . Arch. Biochem. Biophys. 1993, 300, 705. Morpeth, F.F. Biochem. J. 1985, 228, 557. Canevascini, G.; Borer, P.; Dreyer, J.-L. Eur. J. Biochem. 1991, 198, 43. Schmidhalter, D.; Canevascini, G. Arch. Biochem. Biophys. 1993, 300, 559. Dekker, R.F.H. J. Gen. Microbiol. 1980, 120, 309. Fahnrich, P.; Irrgang, K . Biotechnol. Lett. 1982, 4, 775. Hutterman, A . ; Noelle, A . Holzforschung 1982, 36, 283. X i a , Z.-X.; Shamala, N . ; Bethege, P.H.; Lim, L.W.; Bellamy, H.D.; Xuong, N . H . ; Lederer, F.; Mathews, F.S. Proc. Natl. Acad. Sci. USA 1987, 84, 2629. Tabor, C.W.; Kellogg, P.D. J. Biol. Chem. 1970, 245, 5424. Chen, L . ; Liu, M . - Y . ; LeGall, J.; Fareleira, P.; Santos, H . ; Xavier, A . V . Biochem. Biophys. Res. Commun. 1993, 193, 100. Marietta, M . A . J. Biol. Chem. 1993, 268, 12231. Narhi, L.O.; Fulco, A.J. J. Biol. Chem. 1986, 261, 7160. Celerier, J.; Risler, Y . ; Schwenke, J.; Janot, J.-M.; Gervais, M . Eur. J. Biochem. 1989, 182, 67.
8.
19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
32. 33. 34. 35. 36. 37. 38. 39. 40.
RENGANATHAN
A N D BAO
Cellobiose Dehydrogenase
187
Walker, M.C.; Tollin, G . Biochemistry 1992, 31, 2798. Henriksson, G.; Pettersson, G.; Johansson, G.; Ruiz, A.; Uzcategui, E. Eur. J. Biochem. 1991, 196, 101. Wood, J.D.; Wood, P.M. Biochim. Biophys. Acta 1992,119, 90. Samejima, M.; Eriksson, K.-E. Eur. J. Biochem. 1992, 207, 103. Desbois, A.; Tegoni, M . ; Gervais, M . ; Lutz, M . Biochemistry 1989, 28, 8011. Cox, M.C.; Rogers, M.S.; Cheesman, M.; Jones, J.D.; Thomson, A.J.; Wilson, M.T.; Moore, G.R. FEBS Lett. 1992, 307, 233. Renganathan, V . ; Usha, S.N.; Lindenburg, F. Appl. Microbiol. Biotechnol. 1990, 32, 609. Kremer, S.M.; Wood, P.M. Eur. J. Biochem. 1992, 208, 807. Jones, J.D.; Wilson, M . Biochem. J. 1988, 256, 713. Samejima, M.; Phillips, R.S.; Eriksson, K.-E. FEBS Lett. 1992, 306, 165. Wilson, M.T.; Hogg, N . ; Jones, G.D. Biochem. J. 1990, 270, 265. Kirk, T.K.; Farrell, R.F. Ann. Rev. Microbiol. 1987, 41, 465. Gold, M . H . ; Wariishi, H.; Valli, K . In Biocatalysis in Agricultural Biotechnology; Whitaker, J.R.; Sonnet, P.E., Eds.; American Chemical Society: Washington, DC, 1989; pp 127-140. Hammel, K.E.; Moen, M . A . Enzyme Microb. Technol. 1991, 13, 15. Wariishi, H.; Valli, K.; Gold, M . H . Biochem. Biophys. Res. Commun. 1991, 176, 269. Ander, P.; Mishra, C.; Farrell, R.L.; Eriksson, K.-E.L. J. Biotechnol. 1990, 13, 190. Renganathan, V.; Gold, M . H . Biochemistry 1986, 25, 1626. Wariishi, H.; Gold, M . H . J. Biol. Chem. 1990, 265, 2070. Cai, D.; Tien, M . J. Biol. Chem. 1992, 267, 11149. Bao, W.; Renganathan, V . FEBS Lett. 1992, 302, 77. Ohmine, K.; Ooshima, H.; Harano, Y . Biotechnol. Bioeng. 1983, 25, 2041. Gritzali, M . ; Brown, R.D. In Hydrolysis of Cellulose: Mechanisms of Enzymatic and Acidic Catalysis; Brown, R.D.; Jurasek, L . , Eds.; American Chemical Society: Washington, DC, 1979; pp 237-260.
R E C E I V E D January 19, 1994
