CLONING, SEQUENCING, AND HETEROLOGOUS EXPRESSION OF A GENE CODING FORARTHROMYCES-RAMOSUS PEROXIDASE

To understand the relationship between the structure and functions of the peroxidase of Arthromyces ramosus, a novel taxon of hyphomycete, a nd the evolutionary relationship of the A. ramosus peroxidase (ARP) wi th the other peroxidases, we isolated complementary and genomic DNA cl ones encoding ARP and characterized them, The sequence analyses of the ARP and cDNA coding for ARP showed that a mature ARP consists of 344 amino acids with a N-terminal pyroglutamic acid preceded by a signal p eptide of 20 amino acid residues. The amino acid sequence of ARP was 9 9% identical to that of the peroxidase of Coprinus cinereus, a basidio mycete, and also had very high similarities (41-43% identity) to those of basidiomycetous lignin peroxidases, although we could find no lign in peroxidase activities for ARP when assayed with lignin model compou nds, We could identified His184 and His56 as proximal and distal ligan ds to heme, respectively, and Arg52 as an essential Arg. Comparison of the sequences of complementary and genomic DNAs found that protein-en coding DNA is interrupted by 14 intervening sequences. The ARP cDNA wa s expressed in the yeast Saccharomyces cerevisiae under the promoter o f the glyceraldehyde 3-phosphate dehydrogenase gene, yielding 0.02 uni ts/ml of a secreted active peroxidase.