Cloning, sequencing, and heterologous expression of a gene coding for Arthromyces ramosus peroxidase.
To understand the relationship between the structure and functions of the peroxidase of Arthromyces ramosus, a novel taxon of hyphomycete, and the evolutionary relationship of the A.ramosus peroxidase (ARP) with the other peroxidases, we isolated complementary and genomic DNA clones 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 peptide of 20 amino acid residues. The amino acid sequence of ARP was 99% identical to that of the peroxidase of Coprinus cinereus, a basidiomycete, and also had very high similarities (41-43% identity) to those of basidiomycetous lignin peroxidases, although we could find no lignin peroxidase activities for ARP when assayed with lignin model compounds. We could identified His184 and His56 as proximal and distal ligands to heme, respectively, and Arg52 as an essential Arg. Comparison of the sequences of complementary and genomic DNAs found that protein-encoding DNA is interrupted by 14 intervening sequences. The ARP cDNA was expressed in the yeast Saccharomyces cerevisiae under the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene, yielding 0.02 units/ml of a secreted active peroxidase.
Sawai-Hatanaka
H
H
Suntory Research Center, Suntory Ltd., Osaka, Japan.
Ashikari
T
T
Tanaka
Y
Y
Asada
Y
Y
Nakayama
T
T
Minakata
H
H
Kunishima
N
N
Fukuyama
K
K
Yamada
H
H
Shibano
Y
Y
D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000032:analysis; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal / Q000502:physiology*; D015723:Gene Library; D007438:Introns; D003904:Mitosporic Fungi / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000378:metabolism
D004271:DNA, Fungal; D005656:Fungal Proteins; D010544:Peroxidases