Abstract
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.
Highlights
Enzymatic hydroxylation of unactivated primary carbons is generally associated with the use of molecular oxygen as co-substrate for monooxygenases
In aerobic cholesterol degrading bacteria, side chain oxidation is initiated by the hydroxylation of the unactivated primary C26 to a primary alcohol by cytochrome P450 enzymes that is further oxidised to a C26carboxylate[20,21]
ultra-fast performance liquid chromatography (UPLC)- high resolution mass spectrometry (HRMS) analysis of 2 did not allow a clear assignment to a molecular mass, whereas analysis of compound 3 revealed a [M + H]+ ion with m/z = 381.3161 ± 0.4 Da suggesting the loss of H2O from the substrate (m/z = 399.3274 ± 1.1; for MS data of all steroid intermediates analysed in this work, see Supplementary Table 1)
Summary
Enzymatic hydroxylation of unactivated primary carbons is generally associated with the use of molecular oxygen as co-substrate for monooxygenases. 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. 1234567890():,; The selective oxidation of unactivated C–H bonds at alkyl functionalities to alcohols is of great importance for a plethora of synthetic processes Enzymatic solutions for these challenging reactions have continuously motivated organic chemists for developing bioinspired strategies[1,2,3,4,5]. In aerobic cholesterol degrading bacteria, side chain oxidation is initiated by the hydroxylation of the unactivated primary C26 (or equivalent C27) to a primary alcohol by cytochrome P450 enzymes that is further oxidised to a C26carboxylate[20,21]. In the denitrifying β-proteobacterial model organism Sterolibacterium denitrificans Chol-1S, anaerobic cholesterol degradation is initiated by the periplasmic isomerization/
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