Morandini P, Offer J, Traynor D, Nayler O, Neuhaus D, Taylor G W, Kay R R
MRC Laboratory of Molecular Biology, Cambridge, U.K.
Biochem J. 1995 Mar 15;306 ( Pt 3)(Pt 3):735-43. doi: 10.1042/bj3060735.
Stalk cell differentiation during development of the slime mould Dictyostelium is induced by a chlorinated alkyl phenone called differentiation-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 previously that this is accomplished by a dedicated metabolic pathway involving 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 that the first step in metabolism is a dechlorination of the phenolic ring, producing DIF metabolite 1 (DM1). DM1 is identical with the previously known minor DIF activity, DIF-3. DIF-3 is then metabolized by three successive oxidations of its aliphatic side chain: a hydroxylation at omega-2 to produce DM2, oxidation of the hydroxy group to a ketone group 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 responsible for the first side-chain oxidation (DIF-3 hydroxylase) was detected by incubating [3H]DIF-3 with cell-free extracts and resolving the reaction products by t.l.c. DIF-3 hydroxylase has many of the properties 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 inhibitor, and by several other cytochrome P-450 inhibitors, as well as by diphenyliodonium chloride, an inhibitor of cytochrome P-450 reductase. DIF-3 hydroxylase is highly specific for DIF-3: other closely related 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 Km for DIF-3 of 47 nM is consistent with this enzyme being responsible for DIF-3 metabolism in vivo. The two further oxidations necessary to produce DM4 are also performed in vitro by similar enzyme activities. One of the inhibitors of DIF-3 hydroxylase, ancymidol (IC50 67 nM) is likely to be particularly suitable for probing the function of DIF metabolism during development.
黏菌盘基网柄菌发育过程中的柄细胞分化由一种名为分化诱导因子-1（DIF-1）的氯化烷基苯酮诱导。DIF-1的失活可能是DIF-1信号系统中的关键要素，我们之前已经表明，这是通过一条涉及多达12种未鉴定代谢物的专门代谢途径实现的。我们在此报告通过质谱、核磁共振和化学合成推导得出的由DIF-1产生的前四种代谢物的结构。这些化合物的结构表明，代谢的第一步是酚环的脱氯，产生DIF代谢物1（DM1）。DM1与先前已知的次要DIF活性物质DIF-3相同。然后，DIF-3通过其脂肪族侧链的三次连续氧化进行代谢：在ω-2位羟基化产生DM2，羟基氧化为酮基产生DM3，以及在ω-1位进一步羟基化产生DM4，即DIF-3的羟基酮。我们研究了DIF-1代谢的酶学。已知产生DIF-3的第一步由一种新型脱氯酶催化。通过将[³H]DIF-3与无细胞提取物一起温育并用薄层层析解析反应产物，检测到负责第一次侧链氧化的酶活性（DIF-3羟化酶）。DIF-3羟化酶具有许多细胞色素P-450的特性。它与膜结合并使用NADPH作为共底物。它也受到一氧化碳（经典的细胞色素P-450抑制剂）、其他几种细胞色素P-450抑制剂以及细胞色素P-450还原酶抑制剂氯化二苯基碘鎓的抑制。DIF-3羟化酶对DIF-3具有高度特异性：其他密切相关的化合物在100倍摩尔过量时不会竞争其活性，但缺少氯原子的DIF-3类似物除外。DIF-3的Km为47 nM，这与该酶在体内负责DIF-3代谢一致。产生DM4所需的另外两次氧化在体外也由类似的酶活性进行。DIF-3羟化酶的一种抑制剂嘧啶醇（IC50为67 nM）可能特别适合用于探究发育过程中DIF代谢的功能。
