5 mM together with cinnamic acid at a range of concentrations, and decarboxylation was determined at 6 h. Low concentrations of cinnamic acid (0.01 mM) were sufficient

to induce the decarboxylase, which then acted on both of the acids but predominantly against the more numerous 2,3,4,5,6-pentafluorocinnamic acid molecules, forming a mixture of pentafluorostyrene and styrene ( Fig. 4). Increased concentrations of cinnamic acid progressively increased decarboxylase induction. At equimolar (0.5 mM) acid concentrations, more styrene was formed than 2,3,4,5,6-pentafluorostyrene, indicating acid competition for the active site and greater affinity of the enzyme for cinnamic acid than 2,3,4,5,6-pentafluorocinnamic acid. Higher LGK-974 order INCB024360 molecular weight concentrations of cinnamic acid progressively reduced decarboxylation but affected the decarboxylation of 2,3,4,5,6-pentafluorocinnamic acid to a greater extent ( Fig. 4). From this experiment, it was confirmed that the concentration of

cinnamic acid required to induce decarboxylation was low (< 0.01 mM) but that induction progressively increased up to 1.5 mM. 2,3,4,5,6-Pentafluorocinnamic acid was therefore a substrate for decarboxylation only, not an inducer, a fact confirmed by the lack of transcription of either padA1 or ohbA1 ( Fig. 2). Thus, 2,3,4,5,6-pentafluorocinnamic acid could be used as a reporter to detect activity of Pad-decarboxylation and padA1 induction by other compounds, which in themselves may not be substrates for decarboxylation. Detailed

probing of the decarboxylase system and the structural requirements for transcriptional induction of padA1 were then carried out using 1 mM substrate concentrations against whole conidia, 1 mM substrate concentrations against cell-free extracts after 6 h induction, and 0.5 mM Cyclooxygenase (COX) substrate + 0.5 mM 2,3,4,5,6-pentafluorocinnamic acid against whole conidia. Those compounds decarboxylated by whole conidia were both substrates and inducers, whereas those decarboxylated by cell-free extracts were substrates, and those liberating 2,3,4,5,6-pentafluorostyrene were inducers. A substantial number of potential substrates are listed in Supplementary data Table 1 in order of molecular mass and listed according to the entry number (referred subsequently as SD entry followed by the relevant number, e.g. acrylic acid in SD entry 1 and 2,3,4,5,6-pentafluorocinnamic acid is SD entry 121). These compounds were used to determine the important structural features required of successful substrates for decarboxylation by the Pad system. The carboxylic acid group at C1 in both sorbic acid and cinnamic acid is the hydrophilic head-group of these amphipathic compounds, whereas the remainder of their structures are substantially hydrophobic. As anticipated, any changes made in the level of oxidation at C1 completely removed all decarboxylase activities in A. niger conidia.