THE PROXIMAL PATHWAY OF METABOLISM OF THE CHLORINATED SIGNAL MOLECULEDIFFERENTIATION-INDUCING FACTOR-I (DIF-1) IN THE CELLULAR SLIME-MOLD DICTYOSTELIUM

Stalk cell differentiation during development of the slime mould Dicty ostelium is induced by a chlorinated alkyl phenone called differentiat ion-inducing factor-1 (DIF-1). Inactivation of DIF-1 is likely to be a key element in the DIF-1 signalling system, and we have shown previou sly that this is accomplished by a dedicated metabolic pathway involvi ng up to 12 unidentified metabolites. We report here the structure of the first four metabolites produced from DIF-1, as deduced by m.s, n.m .r. and chemical synthesis. The structures of these compounds show tha t the first step in metabolism is a dechlorination of the phenolic rin g, producing DIF metabolite 1 (DM1). DM1 is identical with the previou sly known minor DIF activity, DIF-3. DIF-3 is then metabolized by thre e successive oxidations of its aliphatic side chain: a hydroxylation a t omega-2 to produce DM2, oxidation of the hydroxy group to a ketone g roup to produce DM3 and a further hydroxylation at omega-1 to produce DM4, a hydroxyketone of DIF-3. We have investigated the enzymology of DIF-1 metabolism. It is already known that the first step, to produce DIF-3, is catalysed by a novel dechlorinase. The enzyme activity respo nsible for the first side-chain oxidation (DIF-3 hydroxylase) was dete cted by incubating [H-3]DIF-3 with cell-free extracts and resolving th e reaction products by t.l.c. DIF-3 hydroxylase has many of the proper ties of a cytochrome P-450. It is membrane-bound and uses NADPH as co- substrate. It is also inhibited by CO, the classic cytochrome P-450 in hibitor, and by several other cytochrome P-450 inhibitors, as well as by diphenyliodonium chloride, an inhibitor of cytochrome P-450 reducta se. DIF-3 hydroxylase is highly specific for DIF-3: other closely rela ted compounds do not compete for the activity at 100-fold molar excess , with the exception of the DIF-3 analogue lacking the chlorine atom. The K-m for DIF-3 of 47 nM is consistent with this enzyme being respon sible for DIF-3 metabolism in vivo. The two further oxidations necessa ry to produce DM4 are also performed in vitro by similar enzyme activi ties. One of the inhibitors of DIF-3 hydroxylase, ancymidol (IC50 67 n M) is likely to be particularly suitable for probing the function of D IF metabolism during development.