LAUSR

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. Monooxygenases catalyse the hydroxylation of C-H bonds using oxygen as a co-substrate, which, in turn, is unavailable for anaerobic bacteria. Here, the authors report a three-step reaction cascade involving two hydroxylases and one dehydratase which hydroxylate the C26 methyl group of cholesterol with water as a co-substrate.