Chapter 16
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Cellobiose Dehydrogenase: An Extracellular Flavocytochrome from the Phytopathogenic Basidiomycete Sclerotium (Athelia) rolfsii 1
1
1,2
Dietmar Haltrich , Roland Ludwig , and Marcel Zámocky 1
Division of Biochemical Engineering, Institute of Food Technology, B O K U - University of Natural Resources and Applied Life Sciences at Vienna (Universität für Bodenkultur Wien), Muthgasse 18, A-1190 Wien, Austria Institute of Molecular Biology, Slovak Academy of Sciences, 845 51 Bratislava, Slovak Republic 2
Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome that is produced by wood-degrading and plant pathogenic fungi, both basidiomycetes and ascomycetes. Typically, CDH is a monomeric protein with a bipartite domain organization, consisting of an N-terminal heme domain containing a cytochrome b type heme, and a Cterminal flavin domain with a noncovalently bound FAD. These are linked by a protease-sensitive linker region. Based on the currently known sequences CDH can be divided in two distinct classes, class-1 CDH comprising five known sequences from basidiomycetes, and class-2 CDH represented by three sequences from ascomycetes. C D H oxidizes cellobiose and higher cellodextrins efficiently to their corresponding lactones. Concurrently a wide spectrum of different electron acceptors including various quinones, organic radical species and metal ion complexes are reduced. This report reviews recent progress made in understanding the physiology, structure and function of CDH.
© 2004 American Chemical Society
In Lignocellulose Biodegradation; Saha, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
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272 Sclerotium rolfsii (teleomorph: Athelia rolfsii) was first described by Peter Henry Rolfs more than 100 years ago as a plant pathogen causing tomato blight. S. rolfsii causes disease (Southern blight or Rolfs Disease) in over 500 plant species, many of which are important crop plants such as tomatoes, peppers, sunflowers, peanuts, etc., and causes enormous economic damage. During its attack on plant material S. rolfsii produces large amounts of different enzymes which rapidly destroy host tissue and cell walls, thus enabling it to enter the host organism. Additionally, it secretes copious amounts of oxalic acid which binds calcium ions from calcium pectate of the host cell wall which enables together with acidification the polygalacturonase to hydrolyze pectates of the middle lamella. 5. rolfsii is known to produce cellulolytic and hemicellulolytic enzymes in high amounts (1-3), as a matter of fact it has been recognized as one of the few producers of these hydrolases that are of industrial interest (4). S. rolfsii secretes a complete cellulose degrading enzyme system consisting of endoglucanases (5, 6), cellobiohydrolases (7), and β-D-glucosidases (8) which have been isolated and characterized extensively in the past. In addition, cellobiose dehydrogenase was reported as part of the cellulolytic enzyme system of 5. rolfsii (9, 10). Cellobiose-oxidizing enzymes were first described 30 years ago in cellulolytic cultures of the white rot fungi Phanerochaete chrysosporiam and Trametes versicolor (11). In the scientific literature two types of these enzymes are described: the hemoflavoprotein cellobiose dehydrogenase (CDH, EC 1.1.99.18; formerly cellobiose oxidase) and the flavoprotein cellobiose:quinone oxidoreductase (CBQ, EC 1.1.5.1), which for a long time was believed to be a separate enzyme. However, it was shown that the intact hemoflavoprotein can be cleaved by proteases, either by endogenous ones formed by the organism itself (12, 13) or by proteases such as papain (14, 15), into a catalytically active flavin domain, which is similar to CBQ, and an inactive heme domain. Recently, it was shown that the flavin fragment originates from the cdh gene and that the primary RNA transcript is not differently spliced. This implies that this hemedevoid fragment is formed at the protein level by limited proteolysis (16).
Characterization of Cellobiose Dehydrogenase Cellobiose oxidoreductases, both intact CDH and/or the flavin-only fragment CBQ, were purified and characterized from a number of fungi, including P. chrysosporium (Sporotrichum pulveritlentum) (17, 18), Chrysonilia (Monilia) sitophila (19), Coniophora puteana (20), Heterobasidion annosum (Fomes annosus) (21), Humicola insolens (22), Irpex lacteus (23), Pycnoporus cinnabarinus (24, 25), Schizophyllum commune (26), S. rolfsii (10), Thielavia heterothallica (Sporotrichum thermophile) (27, 28), T. versicolor (29), Trametes pubescens and Trametes villosa (30). Typically, CDH is a monomeric enzyme consisting of the N-terminal heme domain with one heme b, which is hexacoordinate (31), and the flavin domain containing one noncovalently bound
In Lignocellulose Biodegradation; Saha, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
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273 FAD (or alternatively one 6-hydroxy-FAD (32)). These two domains are connected by a protease-sensitive linker region that is rich in serine and threonine. In accordance with reports on other CDH enzymes, intact CDH from S. rolfsii contains both the heme and the flavin as prosthetic groups. The heme cofactor was identified as protoheme IX (heme b) and estimated as one heme per CDH molecule. Similarly, FAD was quantified as one nucleotide per molecule by spectrophotometric analysis. Both the oxidized and the reduced spectrum of CDH are shown in Figure 1. The major peak of the oxidized spectrum at 421 nm can be attributed to the heme cofactor, whereas the broad absorbance shoulder between 450 and 500 nm can be mainly attributed to the FAD group. The extinction coefficients for the oxidized state of the enzyme at 421, 460, 531 and 563 nm were 105,24, 12 and 9 mM^cm* , respectively. Upon reduction of CDH at pH 4 by its substrate cellobiose or reducing agents such as sodium dithionite, strong peaks appeared at 429, 533 and 564 nm, representing the Soret, β and α peaks of a typical heme protein. Absorption between 450 and 500 nm decreased drastically, presumably representing the reduced form of FAD. The extinction coefficients for 429, 460, 533 and 564 nm were determined to be 140, 10, 17 and 28 mM'^cm" , respectively, for the reduced enzyme. 1
1
Figure I. Absorption spectra of CDHfrom S. rolfsii in the oxidized and reduced state. The reduction was performed with cellobiose. Table I lists some properties of CDH enzymes isolated from different organisms. Typically, CDH is a monomeric glycoprotein with a molecular mass of approximately 90-100 kDa and an acidic pi-value. Cellobiose oxidoreductases oxidize various sugars at their anomeric carbon atoms to the
In Lignocellulose Biodegradation; Saha, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
In Lignocellulose Biodegradation; Saha, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.
11 34 3.4 25 110 30 210 120 210
6.5-7.0 3.0-5.0* 4.5-5.0 4.5 4.5 4.5 4.5-5.0 4.5-5.0 4.5-5.0
nr
9.9
7.8
4.9
10
23
3.6
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26
1.8
7.8
0.69
2.8
31
1.2
4.0 0.67
93
d
pH K value (μΜ) cytc optimum" cellobiose DClh+ 120 0.30 3.2-4.8 15 4.0 84 14 nr
400
51
380
430
630
960
430 1,80
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c
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1,30
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6,301
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620
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