Microbiology, Faculty of Biology, Albert-Ludwigs-Universität Freiburg, Schänzlestr. 1, 79104, Freiburg, Germany.
Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstrasse 25, 79104, Freiburg, Germany.
Nat Commun. 2020 Aug 6;11(1):3906. doi: 10.1038/s41467-020-17675-7.
Enzymatic hydroxylation of unactivated primary carbons is generally associated with the use of molecular oxygen as co-substrate for monooxygenases. However, in anaerobic cholesterol-degrading bacteria such as Sterolibacterium denitrificans the primary carbon of the isoprenoid side chain is oxidised to a carboxylate in the absence of oxygen. Here, we identify an enzymatic reaction sequence comprising two molybdenum-dependent hydroxylases and one ATP-dependent dehydratase that accomplish the hydroxylation of unactivated primary C26 methyl group of cholesterol with water: (i) hydroxylation of C25 to a tertiary alcohol, (ii) ATP-dependent dehydration to an alkene via a phosphorylated intermediate, (iii) hydroxylation of C26 to an allylic alcohol that is subsequently oxidised to the carboxylate. The three-step enzymatic reaction cascade divides the high activation energy barrier of primary C-H bond cleavage into three biologically feasible steps. This finding expands our knowledge of biological C-H activations beyond canonical oxygenase-dependent reactions.
未活化的伯碳原子的酶促羟化通常与单加氧酶将分子氧用作共底物相关。然而，在诸如甾醇杆菌（Sterolibacterium denitrificans）的厌氧胆固醇降解细菌中，异戊烯侧链的伯碳原子在无氧条件下被氧化为羧酸盐。在这里，我们确定了一个包含两个钼依赖的羟化酶和一个 ATP 依赖的脱水酶的酶促反应序列，该序列能够用水实现胆固醇未活化的 C26 伯甲基的羟化：（i）C25 的羟化生成叔醇，（ii）通过磷酸化中间产物的 ATP 依赖脱水生成烯烃，（iii）C26 的羟化生成烯丙醇，随后被氧化为羧酸盐。三步酶促反应级联将伯 C-H 键断裂的高活化能垒分为三个在生物学上可行的步骤。这一发现扩展了我们对生物 C-H 活化的认识，超越了典型的氧依赖反应。
