A fully reversible 25-hydroxy steroid kinase involved in oxygen-independent cholesterol side-chain oxidation

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 denitrcans. This highly active enzyme preferentially phosphorylated the tertiary C25 of steroid alcohols, including metabolites of cholesterol and sitosterol degradation or 25-OHvitamin D3. Kinetic data were in agreement with a sequential mechanism via a ternary complex. Remarkably, 25-HSK readily catalyzed the formation of gamma-(O-18)(2)-ATP from ADP and the C25-(O-18)(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 approximate to 20 kJ mol(-1) higher than that of standard sugar phosphoesters and even slightly greater than the beta,gamma-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.

Automated summary

The study identified an ATP-dependent 25-hydroxy-steroid kinase (25-HSK) from anaerobic bacteria, which plays a crucial role in the degradation of zoo- and phytosterols. This enzyme shows little similarity to known phosphotransferases and has a high phosphoryl transfer potential for tertiary steroid C25-phosphoesters.