Posttranslational modification by an isolevuglandin diminishes activity of the mitochondrial cytochrome P450 27A1

Abstract

Posttranslational modifi cation by isolevuglandins (isoLGs), arachidonate oxidation products, is an important yet understudied process associated with altered protein properties. This type of modifi cation is detected in cytochrome P450 27A1 (CYP27A1), a multifunction enzyme expressed in almost every cell and involved in the metabolism of cholesterol and other sterols. Previously, the CYP27A1 Lys358 -isoLG adduct was found in human retina affl icted with age-related macular degeneration. Yet, the effect of Lys358 modifi cation on enzyme activity was not investigated. Herein, we characterized catalytic properties of Lys358 as well as Lys 476 CYP27A1 mutants before and after isoLG treatment and quantifi ed the extent of modifi cation by multiple reaction monitoring. The K358R mutant was less susceptible to isoLG-induced loss of catalytic activity than the wild type (WT), whereas the K476R mutant was nearly as vulnerable as the WT. Both mutants showed less isoLG modifi cation than WT. Thus, modifi cation of Lys358 , a residue involved in redox partner interactions, is the major contributor to isoLG-associated loss of CYP27A1 activity. Our data show the specifi city of isoLG modifi cation, provide direct evidence that isoLG adduction impairs enzyme activity, and support our hypothesis that isoLG modifi cation in the retina is detrimental to CYP27A1 enzyme activity, potentially disrupting cholesterol homeostasis. Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc.