We previously compared the kinetic characteristics of classes I and II CDHs from P. 10) 26) 27) However, the pH dependence of ascomycetes class II CDH is species-dependent, and no clear pattern has been found in this class. 24) 25) The basiomycetes CDH commonly exhibits acidic pH-dependence of cytochrome c reduction, whereas reduction of quinone-like electron acceptors is flavin-dependent and shows a rather broad pH dependence. 23) A recent phylogenetic analysis of the amino acid sequence of the flavin domain indicated that CDHs can be divided into class I (from basidiomycetes) and class II (from ascomycetes), together with class III, which contains putative ascomycetes gene products. 21) 22) Some CDHs from ascomycetes carry a family 1 carbohydrate-binding module in addition to the flavin and cytochrome domains. 10) 16) 18) The flavin domain belongs to the glucose-methanol-choline (GMC) oxidoreductase family, 19) 20) although the heme domain is unique to CDH. 4) 16) 17) The flavin chromophore in CDH is FAD, which is responsible for the oxidation of cellobiose. 11) 12) 13) Therefore, CBO and CBQ are now both categorized as CDH, and the physiological function of the enzyme has been characterized primarily as cellulose degradation in recentĬDH consists of two major domains, i.e., the flavin- and heme-containing domains. However, later reports established that CBQ is a proteolytic product of CBO, 7) CBO utilizes oxygen more slowly than other redox compounds, 8) 9) 10) and CDH is present in many other non-lignolytic cellulolytic fungi. 5) 6) These enzymes were thought to be interface enzymes between cellulose and lignin degradation because of their substrate specificity for cellulolytic and lignolytic products as the electron donor and acceptor, respectively. 1) 2) 3) This enzyme was initially isolated from the wood-rotting basidiomycete Phanerochaete chrysosporium as two different proteins, i.e., cellobiose oxidase (CBO, EC 1.1.3.25), which is a flavocytochrome oxidizing cellobiose with molecular oxygen as an electron acceptor, 4) and cellobiose:quinone oxidoreductase (CBQ, EC 1.1.5.1), which is a flavo-protein utilizing quinones as electron acceptors. It catalyzes oxidation of the reducing-end of cellobiose and cellooligosaccharides (hydrolytic products of cellulose) and produces the corresponding δ-lactones, using various quinones and ferric compounds as electron acceptors. These differences can be explained in terms of the effect of the side chain of the amino acid residue at position 734 on the reactivity of the flavin cofactor.Ĭellobiose dehydrogenase (CDH, EC 1.1.99.18) is an extracellular flavocytochrome produced by cellulolytic fungi. As for the pH-dependence of the specific activity, Q734S did not have an apparent optimum pH in the pH range tested, whereas Q734T showed an acidic optimum pH profile compared with WT. However, k cat/ K m for Q734S was similar at both pHs, whereas k cat/ K m for Q734T was 3.5 times higher at pH 4.0 than that at pH 7.0. When the steady-state kinetic parameters were compared at pH 4.0 and 7.0, WT showed the highest activity at both pH values. The two mutant enzymes showed almost identical absorption spectra, although that of WT was slightly different. Here, we mutated glutamine 734 (Q734) in the flavin domain of class I CDH from the basidiomycete Phanerochaete chrysosporium to the corresponding amino acid in class II CDHs (serine or threonine), and compared the kinetics of the mutant enzymes (Q734S and Q734T) and wild-type enzyme (WT). Fungal cellobiose dehydrogenases (CDHs) are divided on the basis of amino acid sequence into class I (from basidiomycetes) and class II (from ascomycetes), which show quite different pH-dependence of the activity.