Technical Abstract: Nonheme haloperoxidase (HPO-P) isolated from Pseudomonas pyrrocinia catalyzed the peroxidation of alkyl acids to peracids. Among alkyl acids tested as substrates, acetate was most readily peroxidized. The reaction product, peracetate, possessed potent antifungal activity: 50% killing (LD50) of Aspergillus flavus occurred at 25 muM peracetate. To determine whether HPO-P could enhance antifungal activity in plants transgenically, tobacco was transformed with the HPO-P gene isolated from P. pyrrocinia. Leaf extracts of transgenic tobacco inhibited A. flavus growth by up to 80%. In order to elucidate if peracid formation by HPO-P could be the basis for increased fungal resistance, effects of hydrogen peroxide-HPO-P mixtures on the lethality of acetate were determined. LD50 of A. flavus exposed to acetate in the presence of these enzyme mixtures occurred at 30 mM acetate. Since this value was identical to that obtained without the eenzyme mixtures (also 30 mM acetate) and was too low to account for enhanced antifungal activity in transgenic plants, kinetics of the enzyme reaction were examined for clarification. The concentration of acetate needed for enzyme saturation (Km = 250 mM) was found to be lethal prior to oxidation to peracetate. Thus, peracid generation by HPO-P is not the basis for enhanced antifungal activity in plants transformed with the HPO-P gene.