Cloning and characterization of a thermostable cellobiose dehydrogenase from Sporotrichum thermophile

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi, CDH contains one heme b and one FAD per m olecule and oxidizes cellobiose to cello-bionolactone in the presence of cy tochrome c. In this report, a thermostable CDH from the thermophilic ascomy cete Sporotrichum thermophile has been purified, cloned, and characterized. The temperature optimum for this CDH reaction was 60 degrees C, and the ac tivation energy for the reaction was 26.3 kJ/mol. The K-m and K-cat were te mperature-dependent and increased as reaction temperature increased. These kinetic properties prove that this CDH is truly thermophilic. A 2.8-kb cDNA was isolated by screening an expression library of S. thermophile with a p olyclonal antisera raised against Phanerochaete chrysosporium CDH, The cDNA encoded an 807-amino-acid protein with a predicted mass of 86,332 Ha. S. t hermophile CDH is organized into three domains, an N-terminal flavin domain , a middle heme domain, and a C-terminal cellulose-binding domain, which sh ows sequence similarity with the cellulose-binding domains of endoglucanase s and cellobiohydrolases from Trichoderma reesei. Comparison with the CDH s equences of P. chrysosporium and Trametes versicolor identified Met 95 and His 143 as potential heme coordinations. EFIG, LGGPM, and VNSTH motifs in t he heme domain and the XRXPXTDXPSXDGXRY motif in the flavin domain were ide ntified as CDH-specific motifs, With regard to the amino acid composition, S. thermophile CDH has more disulfide linkages and acidic and basic amino a cids compared to CDHs from P. chrysosporium and T. versicolor. (C) 1999 Aca demic Press.