Guzov V M, Unnithan G C, Chernogolov A A, Feyereisen R
Department of Entomology and Center for Toxicology, University of Arizona, Forbes 410, Tucson, Arizona, 85721, USA.
Arch Biochem Biophys. 1998 Nov 15;359(2):231-40. doi: 10.1006/abbi.1998.0901.
Eukaryotic P450 proteins are membrane proteins found predominantly in the endoplasmic reticulum. In vertebrates, several biosynthetic P450s are found in mitochondria as well. We cloned three putative insect mitochondrial P450s from larval house fly cDNA. These P450s are members of a new P450 family, CYP12. The CYP12 proteins are most closely related to the mammalian mitochondrial P450 of the CYP11, CYP24, and CYP27 families. The most abundant cDNA, CYP12A1, was expressed in Escherichia coli and purified. NADPH-dependent reduction of CYP12A1 was rapid and efficient with the bovine mitochondrial proteins adrenodoxin reductase and adrenodoxin as electron transfer partners. In contrast, house fly microsomal NADPH cytochrome P450 reductase reduced CYP12A1 only poorly. In a reconstituted system with the bovine mitochondrial electron donors, CYP12A1 metabolized a variety of insecticides and other xenobiotics, but did not metabolize ecdysteroids, juvenoids, or fatty acids. Subcellular localization of CYP12A1 by immunogold histochemistry established the mitochondrial nature of this protein. CYP12A1 mRNA levels are constitutively higher in an insecticide-resistant strain than in a susceptible strain, and this trait maps to chromosome II in the house fly, where the constitutive overexpression of the pesticide-metabolizing microsomal CYP6A1 also maps. Multiple mitochondrial P450s have evolved in insects and may play a role in the metabolism of xenobiotics in addition to their possibly ancestral functions in steroidogenesis.
真核生物的细胞色素P450蛋白是主要存在于内质网中的膜蛋白。在脊椎动物中，一些生物合成的细胞色素P450也存在于线粒体中。我们从家蝇幼虫的cDNA中克隆了三种假定的昆虫线粒体细胞色素P450。这些细胞色素P450是一个新的细胞色素P450家族CYP12的成员。CYP12蛋白与CYP11、CYP24和CYP27家族的哺乳动物线粒体细胞色素P450关系最为密切。最丰富的cDNA，即CYP12A1，在大肠杆菌中表达并纯化。以牛线粒体蛋白肾上腺皮质铁氧化还原蛋白还原酶和肾上腺皮质铁氧化还原蛋白作为电子传递伙伴，CYP12A1的NADPH依赖性还原反应迅速且高效。相比之下，家蝇微粒体NADPH细胞色素P450还原酶对CYP12A1的还原作用很差。在与牛线粒体电子供体组成的重组系统中，CYP12A1代谢多种杀虫剂和其他外源性物质，但不代谢蜕皮甾体、保幼激素或脂肪酸。通过免疫金组织化学对CYP12A1进行亚细胞定位，确定了该蛋白的线粒体性质。在抗杀虫剂品系中，CYP12A1的mRNA水平始终高于敏感品系，这一特性在家蝇的二号染色体上定位，而代谢杀虫剂的微粒体CYP6A1的组成型过表达也定位在二号染色体上。昆虫中已经进化出多种线粒体细胞色素P450，它们除了在类固醇生成中可能具有的祖先功能外，还可能在外源性物质的代谢中发挥作用。
