Abstract
The degradation of cholesterol and related steroids by microbes follows fundamentally different strategies in aerobic and anaerobic environments. In anaerobic bacteria, the primary C26 of the isoprenoid side chain is hydroxylated without oxygen via a three-step cascade: (i) water-dependent hydroxylation at the tertiary C25, (ii) ATP-dependent dehydration to form a subterminal alkene, and (iii) water-dependent hydroxylation at the primary C26 to form an allylic alcohol. However, the enzymes involved in the ATP-dependent dehydration have remained unknown. Here, we isolated an ATP-dependent 25-hydroxy-steroid kinase (25-HSK) from the anaerobic bacterium Sterolibacterium denitrificans. This highly active enzyme preferentially phosphorylated the tertiary C25 of steroid alcohols, including metabolites of cholesterol and sitosterol degradation or 25-OH-vitamin D3. Kinetic data were in agreement with a sequential mechanism via a ternary complex. Remarkably, 25-HSK readily catalyzed the formation of γ-(18O)2-ATP from ADP and the C25-(18O)2-phosphoester. The observed full reversibility of 25-HSK with an equilibrium constant below one can be rationalized by an unusual high phosphoryl transfer potential of tertiary steroid C25-phosphoesters, which is ≈20 kJ mol−1 higher than that of standard sugar phosphoesters and even slightly greater than the β,γ-phosphoanhydride of ATP. In summary, 25-HSK plays an essential role in anaerobic bacterial degradation of zoo- and phytosterols and shows only little similarity to known phosphotransferases.
Highlights
Cholesterol and related zoo, phyto, and mycosterols share a common architecture comprising a tetracyclic sterane core structure linked to an isoprenoid side chain
We demonstrated in extracts of S. denitrificans grown with cholesterol and nitrate that the formation of a tertiary alcohol at the steroid side chain initiates an enzymatic reaction cascade that results in the formation of a C26-carboxylate
It comprises the ATP-dependent dehydration of 25-OH-CDO to the Δ24 alkene desmost-1,4-diene-3-one (DDO), followed by a second water-dependent hydroxylation at the allylic C26 giving (E)-26-OH-DDO (Fig. 1) [15]. The latter step is catalyzed by steroid C26 dehydrogenase 1 (S26 steroid C25 dehydrogenase 1 (DH1)), a further molybdenumdependent enzyme of the DMSO reductase family that catalyzed the subsequent dehydrogenation of the allylic alcohol to the corresponding aldehyde
Summary
Cholesterol and related zoo-, phyto-, and mycosterols share a common architecture comprising a tetracyclic sterane core structure linked to an isoprenoid side chain. We identified a highly active and specific 25-hydroxysteroid kinase (25-HSK) that ATP-dependently formed the 25phosphoester of CDO and related 25-OH-steroids with an extremely high phosphoryl group transfer potential.
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