The gene PDAT9 from the fungus Nectria haematococca encodes
pisatin demethylase, an
enzyme that detoxifies the phytoalexin
pisatin, an antimicrobial compound produced by pea in response to
infection by this plant pathogen. PDAT9 was found to contain an open reading frame (ORF) encoding 515
amino acids and four introns of 52-58
nucleotides each within its coding region. The amino acid sequence F-G-A-G-S-R-S-C-I-G, indicative of the "fifth
ligand binding site" present in all
cytochrome P450s, occurs as residues 446 to 455, confirming that PDAT9 is a
cytochrome P450. The deduced amino acid sequence is distinct from all other reported
cytochrome P-450s, and PDAT9 has been assigned to a new
cytochrome P450 family, CYP57. A 1.3 kb SacI fragment of the PDAT9 ORF that lacked the fifth
ligand binding site, hybridized to unique
DNA fragments in N. haematococca isolates known to possess PDA genes that encode different whole cell phenotypes for
pisatin demethylating activity. These genes were also tentatively identified as
cytochrome P450s by the hybridization of the same fragments to separate subclones of PDAT9, one of which contained the fifth
ligand sequence. That probe also hybridized to
DNA other than that attributed to
pisatin demethylase genes; these other DNAs are presumed to represent other
cytochrome P450s.