Structural Characterization of the Complex between α-Naphthoflavone and Human Cytochrome P450 1B1

Abstract

The atomic structure of human P450 1B1 was determined by x-ray crystallography to 2.7 Å resolution with ␣-naphthoflavone (ANF) bound in the active site cavity. Although the amino acid sequences of human P450s 1B1 and 1A2 have diverged significantly, both enzymes exhibit narrow active site cavities, which underlie similarities in their substrate profiles. Helix I residues adopt a relatively flat conformation in both enzymes, and a characteristic distortion of helix F places Phe 231 in 1B1 and Phe 226 in 1A2 in similar positions forstacking with ANF. ANF binds in a distinctly different orientation in P450 1B1 from that observed for 1A2. This reflects, in part, divergent conformations of the helix B-C loop that are stabilized by different hydrogen-bonding interactions in the two enzymes. Additionally, differences between the two enzymes for other amino acids that line the edges of the cavity contribute to distinct orientations of ANF in the two active sites. Thus, the narrow cavity is conserved in both P450 subfamily 1A and P450 subfamily 1B with sequence divergence around the edges of the cavity that modify substrate and inhibitor binding. The conservation of these P450 1B1 active site amino acid residues across vertebrate species suggests that these structural features are conserved.Enzymes of the cytochrome P450 (CYP) superfamily play a significant physiologic role in the detoxication of foreign compounds and the biosynthesis of endogenous compounds, including steroid hormones, bile acids, and cholesterol. P450 3 families 1, 2, 3, and 4 contribute most extensively to the transformation of xenobiotics to more polar metabolites that can be better excreted. In humans and most mammals, family 1 comprises three well studied enzymes: P450s 1A1, 1A2, and 1B1. P450 1A2 is expressed mainly in liver, whereas P450s 1B1 and 1A1 are expressed in many extrahepatic organs. Transcription of the corresponding genes is activated by aryl hydrocarbon receptor (AhR), a transcription factor that binds planar aromatic hydrocarbons, and P450s 1A and 1B oxidize a variety of polycyclic aromatic hydrocarbons (1). Some of their metabolites are carcinogenic, and P450 1B1 is uniquely associated with the carcinogenicity of dimethylbenzanthracene in a mouse model of tumorigenesis (2).Additionally, P450 family 1 enzymes contribute to metabolism of endogenous compounds that include 17␤-estradiol, retinals, arachidonic acid, and melatonin. In contrast to P450 1A enzymes, 1B1 is highly conserved throughout vertebrate evolution, suggesting an important physiologic function. Consistent with this notion, allelic variants of the CYP1B1 gene are associated with an increased risk for glaucoma (3, 4), but the underlying mechanism is unclear. Expression of the CYP1B1 gene is also regulated conditionally by the estrogen receptor (ER) and cAMP-response element-binding protein (CREB) (5, 6). Elevated expression of P450 1B1 has been reported for a number of tumors of breast, prostate, ovary, lung, and brain origin, and it has been suggested that selective inh...

Correlating Structure and Function of Drug-Metabolizing Enzymes: Progress and Ongoing Challenges

“…Of these six P450s, 1A2 exhibits the smallest and narrowest active site (Sansen et al, 2007). This architecture is conserved in structures of P450s 1A1 (Walsh et al, 2013) and 1B1 (Wang et al, 2011), and the narrow hydrophobic cavity is consistent with the roles of these enzymes in metabolism of polycyclic aromatic hydrocarbons. This narrow cavity is reinforced by the passage of helix F below helix G, and this redirection of the F helix is associated with a distortion of the F helix as it passes over the active site cavity (Fig.…”

CYP63A2, a Catalytically Versatile Fungal P450 Monooxygenase Capable of Oxidizing Higher-Molecular-Weight Polycyclic Aromatic Hydrocarbons, Alkylphenols, and Alkanes

“…To make this follow-up experimental functional characterization more efficient, we first utilized a computational modeling and docking strategy to estimate the substrate range and catalytic potential of CYP63A2. Comparison of the CYP63A2 3D model with the crystal structures of P450s from prokaryotes (42,43) and higher eukaryotes (44)(45)(46) indicated that CYP63A2 is the largest among the analyzed P450s (Fig. 2 and Table 2).…”

Cytochrome P450 Cyp1b1*2 Gene and Its Association With T2d in Tabuk Population, Northwestern Region of Saudi Arabia

“…This change in the amino acids sequences of the protein may alter its expression, or the catalytic activity, which may induce diseases as a phenotype [6]. The crystal structure of the CYP1B1 was determined [7], it has a narrow slot-like active site that is similar to that of CYP1A1; however, the residues around the edge of the active sites are different for specific binding of substrates and inhibitors [7]. Using the reverse transcriptase-coupled polymerase chain reaction (RT-PCR), CYP1B1 mRNA was detected in hepatocytes, lymphocytes, and uterus [8].…”