|
Cytochrome P450 monooxygenases are heme protein-dependent mixed function oxidase systems that utilize NADPH and/or NADH to reductively cleave atmospheric dioxygen to produce a functionalized organic substrate and a molecule of water. In many cases, the products represent hydroxylated derivatives of the substrate at one of its carbon moieties. In other cases, P450s mediate hydroxylations at nitrogen and sulfur heteroatoms, dehalogenations, dealkylations, deaminations and epoxidations. Although no single P450 is capable of carrying out this vast array of oxygenations as part of its natural function, in mammalian systems it is not uncommon for a single substrate to be metabolized at alternate positions by a group of different P450s or for a single P450 to metabolize multiple substrates. These enzymes are present in prokaryotes, lower eukaryotes, and probably all higher eukaryotes, having been identified in plants, insects, birds and fish, as well as in numerous mammalian tissues and cell types. In eukaryotic cells, P450s occur predominantly in microsomal membranes after synthesis by cytosolic ribosomes and co-translational transport into the endoplasmic reticular system. In their final form, microsomal P450s retain their N-terminal hydrophobic signal sequence of approximately 50 amino acids anchored within the microsomal membrane and have their catalytic domain exposed on the cytosolic side of the membrane. Catalytic activities for these cytosolic P450s are mediated by association of each P450 with a single membrane-anchored NADPH-dependent P450 reductase. Occasionally, soluble P450s occur in other subcellular locations (e.g., mitochondria, chloroplasts) but these utilize other flavoproteins as their electron transfer partners.
Reflecting a wide diversity of reactions that they carry out, P450s are encoded by a highly divergent gene superfamily which maintains a diagnostic P450 signature motif (F--G-R-C-G) surrounding the protoporphyrin IX heme cysteine ligand Within individual organisms, this superfamily contains a spectrum of P450 gene families that differ substantially in their primary sequence, substrate specificities, genomic organization and inducibilities. The structurally-related monooxygenase proteins encoded by these P450 families (designated CYP1, CYP2, etc.) share 40% amino acid identity with one another, those encoded by P450 subfamilies (designated A,B,C, etc.) share 55% amino acid identity and those encoded by P450 allelic variants (designated v1, v2, etc.) share 55-99% amino acid identity unless evidence exists for the existence of two independent loci. Further details on the superfamily of P450 genes are available at http://drnelson.utmem.edu.
Analysis of substrate-product relationships in many plants have attributed the metabolism of several hundred different substrates to the cytochrome P450s. These activities can be broadly separated into two classes - those that exist in biosynthetic pathways and those that exist in detoxificative pathways. Biosynthetic P450s exist in the pathways leading to the synthesis of UV protectants (flavonoids), pigments (anthocyanins), defense compounds (isoflavonoids, phytoalexins,hydroxamic acids), fatty acids, hormones (gibberellins, brassinosteroids), signaling molecules (salicylic acid, jasmonic acid), accessory pigments (carotenoids), defense compounds (terpenes) and structural polymers (lignins). Catabolic P450s participate in the breakdown of some of these endogenous signaling molecules as well as exogenous compounds encountered in the environment (herbicides, insecticides, pollutants). Classification of these P450s based solely on a predominant biosynthetic activity provides an overly-simplified view of the catalytic activities of individual monooxygenases given the possibility that some of these P450s metabolize both endogenous and exogenous compounds.
The Arabidopsis thaliana genome contains 272 sequences with the F--G-R-C-G P450 signature motif. Annotation of these P450 sequences based on their degree of amino acid identity with one another indicates that these P450 sequences are distributed among 45 P450 families (sharing 40% identity) and 72 P450 subfamilies (sharing 55% identity). Twenty-six of these sequences represent pseudogenes that lack a complete open reading frame or contain frameshifts or in-frame stop codons. Sequence comparisons of the intact genes within this superfamily indicate that they share only 10-15% amino acid identity primarily localized in the region surrounding the heme cysteine axial ligand and in a short region immediately upstream from it. For those Arabidopsis P450s analyzed, significantly more conservation exists at the secondary structure level with the same number and placement of major alpha-helices and beta-pleated sheets as in the crystal structures defined for several bacterial and a single mammalian P450.
Related plant P450 references:
- Schuler, M.A. and Werck-Reichhart, D. (2003) Functional Genomics of P450s. Annu. Rev. Plant Biol. 54, 629-637.
- Chapple, C. (1998) Molecular-genetic analysis of plant cytochrome P450-dependent monooxygenases. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 311-343.
- Schuler, M.A. (1996) The role of cytochrome P450 monooxygenases in plant-insect interactions. Plant Physiol. 112, 1411-1419.
- Werck-Reichhart, D., Hehn, A. and Didierjean, L. (2000) Cytochromes P450 for engineering herbicide tolerance. Trends Plant Sci. 5, 116-123.
- Werck-Reichhart, D., Bak, S. and Paquette, S. (2002) Cytochrome P450. In: The Arabidopsis Book. Edited by Somerville, C. and Meyerowitz, E. Published by American Society of Plant Biologists. Available at http://www.aspb.org/downloads/arabidopsis/werckfinal.pdf.