CLONING AND SEQUENCING OF A GENE ENCODING CELLOBIOSE DEHYDROGENASE FROM TRAMETES-VERSICOLOR

Cellobiose dehydrogenase (CDH) is an enzyme produced under lignocellul ose-degrading conditions by Trametes versicolor strain 52J (Tv) and se veral other wood-degrading fungi, including Phanerochaete chrysosporiu m (Pc). In order to understand better the nature and properties of thi s enzyme, we isolated a genomic clone of Tv cdh using heterologous pro bes derived from the sequence of Pc cdh. DNA sequence analysis reveale d that Tv cdh consists of 3091 bp of coding sequence interrupted by 14 introns. Southern blotting showed that the gene was present in a sing le copy in the strain of Tv analyzed. Te cdh was shown by Northern blo t analysis to be expressed as a single transcript under cellulolytic c onditions. RT-PCR of poly(A)(+) RNA isolated under cellulolytic condit ions was used to generate a near full-length cDNA copy of the cdh mRNA . The deduced protein encoded by Tv cdh consists of 768 amino acids (a a), including a predicted 19 aa signal peptide. The protein had 73% id entity to the corresponding protein from Pc, which is the only other C DH-encoding gene that has been cloned. Based upon its deduced primary structure and alignment to similar sequences, Tv CDH shares a general structural organization with Pc CDH and other hemoflavoenzymes. Amino acid residues H-109 and M-61 in the N-terminal heme domain are hypothe sized to function in heme binding; the C-terminal flavin domain contai ned a consensus sequence for flavin binding between residues 217-222. Although the protein is known to bind to cellulose, no obvious homolog y to bacterial or fungal cellulose binding domains was observed. Howev er, a strong homology was observed to a region of Pc CDH that is hypot hesized to be involved in cellulose binding. (C) 1998 Elsevier Science B.V.