Cloning and biochemical characterization of a thermostable cellobiose dehydrogenase from Sporotrichum thermophile and its role in cellulose degradation (Phanerochaete chrysosporium)

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by cellulose-degrading fungi. CDH oxidizes cellobiose to cellobionolactone in the presence of electron acceptors such as cytochrome c, quinones, Fe(III) and Mn(III). In the absence of these electron acceptors oxygen functions as an alternate electron acceptor. Addition of CDH to a cellulolytic system enhances the degradation of cellulose. However, the mechanism by which CDH enhances cellulose degradation is not understood. In this work, a thermostable CDH from Sporotrichum thermophile (CDH_st) has been purified, cloned and characterized. In addition, the aerobic reaction of CDH and the mechanism of CDH-dependent enhancement of cellulose hydrolysis have been examined.; Purified CDH_st has a maximum activity of 13.4 U/mg. Its kinetics for cellobiose oxidation was temperature dependent; thus the K_m and k_cat increased with temperature. Analysis of the cdh CDNA sequence suggested that CDH_st is organized into three domains; (1) an N-terminal heme domain to which one heme b binds; (2) a middle flavin domain to which an FAD binds; and (3) C-terminal cellulose-binding domain which presumably enables this CDH to bind to cellulose. Comparison of amino acid sequences of the CDHs from S. thermophile and P. chrysosporium identified Met 95 and His 143 as the axial coordinations to the heme iron. Multiple alignment analysis of the flavin domain sequences of CDHs and the glucose-methanol-choline oxidoreductase family suggested a unique CDH motif within the flavin domain.; When CDH from P. chrysosporium oxidized cellobiose in the presence of oxygen, hydrogen peroxide and hydroxyl radicals were generated. Inclusion of iron-chelators (ferrozine, apotransferrin) abolished hydroxyl radical generation and enhanced H₂O₂ generation; in addition, chelators also strongly inhibited O₂ uptake. These data indicate the involvement of an enzyme-bound iron as the site of oxygen reaction.; The ability of CDH from P. chrysosporium to bind to and to enhance the hydrolysis of different types of celluloses by Trichoderma viride cellulase was examined. CDH enhanced the hydrolysis of only those celluloses to which it could bind. A model for cellulose hydrolysis enhancement is proposed.