Biochemical properties of the products of cytochrome P450 genes (PDA) encoding pisatin demethylase activity in Nectria haematococca

Abstract

Pea plants produce the antibiotic (+)pisatin in response to infection by the fungus Nectria haematococca, which can detoxify pisatin utilizing a cytochrome P450 monooxygenase called pisatin demethylase. Genes (PDA) have been identified that encode different whole-cell Pda phenotypes that can be distinguished by the length of the lag period and the resulting amount of enzyme activity produced: Pda^SH = short lag, high activity; Pda^SM = short lag, moderate activity; and Pda^LL = long lag, low activity. Only the Pda^SH and Pda^SM phenotypes have been correlated with pathogenicity on pea. In this study, we utilize heterologous expression of the PDA ^LL gene PDA6-1 in Aspergillus nidulans to compare the biochemical properties of the product of this gene with the products of the PDA ^SH gene PDA1 expressed in N. haematococca. Preliminary measurements were also done on the PDA ^SM gene PDA5 expressed in N. haematococca. The PDA gene products differed somewhat in their substrate specificity and in their sensitivity to a few cytochrome P450 inhibitors. However, the enzymes produced by PDA6-1 and PDA1 both had low apparent K _m values toward (+)pisatin (< 0.25 μM) and a common high degree of insensitivity to most P450 inhibitors, suggesting similar shared biochemical traits as would be expected of products of a highly homologous gene family. Our results indicate that the different whole-cell phenotypes of N. haematococca are not due to significant differences in the biochemical properties of the gene products and are consistent with recent results that indicate that the phenotypic differences are due to different degrees of expression of the genes.