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Journal of Lipid Research, Vol. 45, 2345-2353, December 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

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

Dilyara A. Murtazina^*, Ulla Andersson, In-Su Hahn, Ingemar Bjorkhem, G. A. S. Ansari^** and Irina A. Pikuleva^1,*

^* Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555-1031
Biomolecular Resource Facility, University of Texas Medical Branch, Galveston, TX 77555-1031
^** Department of Human Biological Chemistry, University of Texas Medical Branch, Galveston, TX 77555-1031
Department of Clinical Chemistry, Karolinska Institute, Huddinge Hospital, S-141 88, Huddinge, Sweden

¹ To whom correspondence should be addressed. e-mail: irpikule{at}utmb.edu

Cytochrome P450 27A1 (P450 27A1) is an important metabolic enzyme involved in bile acid biosynthesis and the activation of vitamin D₃ 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 ionization mass spectrometry identified phosphatidylglycerol (PG) as the major PL copurified with P450 27A1. Subsequent reconstitution of P450 into exogenous PG vesicles assessed the effect of this contamination on substrate binding and enzyme activity. Two other PLs, phosphatidylethanolamine (PE) and phosphatidylserine (PS), were also tested. PG and PE increased the K_d for 5ß-cholestane-3,7,12-triol and cholesterol binding, whereas PS had no effect on either substrate binding. PG and PE did not significantly alter 5ß-cholestane-3,7,12-triol hydroxylase activity and even stimulated cholesterol hydroxylase activity. PS inhibited 5ß-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.

Abbreviations: CAD, collisionally activated dissociation; P450 27A1, cytochrome P450 27A1; ES-MS/MS, electrospray ionization tandem mass spectrometry; MW, molecular weight; PBS, potassium phosphate buffer; PE, phosphatidylethanolamine; PL, phospholipid; PG, phosphatidylglycerol; PS, phosphatidylserine; WT, wild type

Supplementary key words cytochrome P450 27A1 • heterologous expression • Escherichia coli • bile acid biosynthesis

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