In enzymology, a cholestanetriol 26-monooxygenase (EC 1.14.13.15) is an enzyme that catalyzes the chemical reaction

5beta-cholestane-3alpha,7alpha,12alpha-triol + NADPH + H⁺ + O₂ ${\displaystyle \rightleftharpoons }$ (25R)-5beta-cholestane-3alpha,7alpha,12alpha,26-tetraol + NADP⁺ + H₂O

The 4 substrates of this enzyme are 5beta-cholestane-3alpha,7alpha,12alpha-triol, NADPH, H⁺, and O₂, whereas its 3 products are (25R)-5beta-cholestane-3alpha,7alpha,12alpha,26-tetraol, NADP⁺, and H₂O.

This enzyme belongs to the family of oxidoreductases, specifically those acting on paired donors, with O2 as oxidant and incorporation or reduction of oxygen. The oxygen incorporated need not be derived from O2 with NADH or NADPH as one donor, and incorporation of one atom o oxygen into the other donor. The systematic name of this enzyme class is 5beta-cholestane-3alpha,7alpha,12alpha-triol,NADPH:oxygen oxidoreductase (26-hydroxylating). Other names in common use include 5beta-cholestane-3alpha,7alpha,12alpha-triol 26-hydroxylase, 5beta-cholestane-3alpha,7alpha,12alpha-triol hydroxylase, cholestanetriol 26-hydroxylase, sterol 27-hydroxylase, sterol 26-hydroxylase, cholesterol 27-hydroxylase, CYP27A, CYP27A1, and cytochrome P450 27A1'. This enzyme participates in bile acid biosynthesis and ppar signaling pathway.

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