Structure of the Human Lung Cytochrome P450 2A13

Brian D. Smith,Jason L. Sanders,C. David Stout,Emily E. Scott,Gerald H. Lushington,Patrick R. Porubsky

doi:10.1074/jbc.m702361200

Abstract

The human lung cytochrome P450 2A13 (CYP2A13) activates the nicotine-derived procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) into DNA-altering compounds that cause lung cancer. Another cytochrome P450, CYP2A6, is also present in human lung, but at much lower levels. Although these two enzymes are 93.5% identical, CYP2A13 metabolizes NNK with much lower K(m) values than does CYP2A6. To investigate the structural differences between these two enzymes the structure of CYP2A13 was determined to 2.35A by x-ray crystallography and compared with structures of CYP2A6. As expected, the overall CYP2A13 and CYP2A6 structures are very similar with an average root mean square deviation of 0.5A for the Calpha atoms. Like CYP2A6, the CYP2A13 active site cavity is small and highly hydrophobic with a cluster of Phe residues composing the active site roof. Active site residue Asn(297) is positioned to hydrogen bond with an adventitious ligand, identified as indole. Amino acid differences between CYP2A6 and CYP2A13 at positions 117, 300, 301, and 208 relate to different orientations of the ligand plane in the two protein structures and may underlie the significant variations observed in binding and catalysis of many CYP2A ligands. In addition, docking studies suggest that residues 365 and 366 may also contribute to differences in NNK metabolism.

Highlights

Several key findings indicate that 2A13, rather than 2A6, plays an important role in in situ metabolism of NNK in the human respiratory tract
A substantial reduction in lung adenocarcinoma has been associated with a genetic polymorphism of cytochrome P450 2A13 (CYP2A13) [11] that is reported to cause a 2–3-fold reduction of catalytic efficiency for NNK [12]
To better define the structural features of CYP2A enzymes that determine their substrate metabolism, and to investigate the structural differences between human CYP2A6 and CYP2A13 enzymes, we have determined a structure of human CYP2A13 by x-ray crystallography

Summary

EXPERIMENTAL PROCEDURES

Protein Design, Expression, and Purification—The cDNA for CYP2A13 was a gift from Dr X. The purest P450-containing fractions were pooled, diluted 10 times with 5 mM potassium phosphate, pH 7.4, 20% glycerol, 1 mM EDTA, 0.2 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, and 4.8 mM Cymal-5, and loaded onto a CM-Sepharose CL-6B column This column was washed with the previous buffer omitting the detergent. Among the 30 ligand docked conformers requested per ligand, 15 of the coumarin structures bound to CYP2A6 were in close qualitative agreement with the crystallographically determined position (root mean squared deviation Ͻ1.2 Å), with the carbonyl oxygen forming a hydrogen bond with the Asn297 side chain and the ring oxygen point up away from the heme. The same docking methodology was applied to NNK binding to CYP2A6 and CYP2A13

RESULTS AND DISCUSSION

CONCLUSIONS

No compatible docking conformers