Phospholipids modify substrate binding and enzyme activity of human cytochrome P450 27A1

Cytochrome P450 27A1 (P450 27A1) is an impor- tant metabolic enzyme involved in bile acid biosynthesis and the activation of vitamin D 3 in mammals. Recombinant P450 27A1 heterologously expressed in Escherichia coli was found to be copurified with phospholipids (PLs). The PL content varied in different preparations and was dependent on the purification protocol. A link between the increased amounts of PLs and deterioration of the enzyme substrate binding properties was also observed. Tandem negative ion- ization mass spectrometry identified phosphatidylglycerol (PG) as the major PL copurified with P450 27A1. Subse- quent reconstitution of P450 into exogenous PG vesicles as- sessed the effect of this contamination on substrate binding and enzyme activity. Two other PLs, phosphatidylethanola- mine (PE) and phosphatidylserine (PS), were also tested. PG and PE increased the K d for 5 � -cholestane-3 � ,7 � ,12 � -triol and cholesterol binding, whereas PS had no effect on either substrate binding. PG and PE did not significantly alter 5 � - cholestane-3 � ,7 � ,12 � -triol hydroxylase activity and even stimulated cholesterol hydroxylase activity. PS inhibited 5 � - cholestane-3 � ,7 � ,12 � -triol hydrolyase activity and had no effect on cholesterol hydroxylase activity. Our study shows the potential for PLs to regulate the activity of P450 27A1 in vivo and alter the amount of cholesterol degraded through the "classical" and "alternative" bile acid biosynthetic pathways. — Murtazina, D. A., U. Andersson, I-S. Hahn, I. Bjorkhem, G. A. S. Ansari, and I. A. Pikuleva. Phospholipids modify substrate binding and enzyme activity of human cytochrome P450 27A1. J. Lipid Res. 2004. 45: 2345-2353.