J.K. Yano1, M.-H. Hsu1, K.J. Griffin1, C.D. Stout2, and E.F. Johnson1
1Department of Molecular and Experimental Medicine, The Scripps
Research Institute, La Jolla,
Figure 1.
Stereo views of sA-weighted 2|Fo|-|Fc|
composite omit electron density maps contoured at 1 s and rendered within 1 Å of
the heme and substrate for the P450 2A6 (a) coumarin or (b) methoxsalen
complexes. Coumarin and methoxsalen are stabilized by hydrogen bonding with
Asn297, which places the carbon atom to be oxidized 3.2 ± 0.13 Å (coumarin) or
3.8 ± 0.09 Å (methoxsalen) from the heme iron. The distances are shown as a red
dotted line and the values quoted for distances are the mean and standard
deviation for the four molecules in the asymmetric unit. The peptide backbone
is represented as a thin gray coil, and side chains are rendered as stick
figures with the following colors for atoms: carbons are colored gray for the
protein or yellow for substrates, oxygens are red, and nitrogens are blue. The
heme group is colored salmon. These molecular graphics images were generated
using PyMOL (pymol.sourceforge.net).
There are over 50 mammalian cytochrome P450 genes in at least 17 families.
Microsomal cytochrome P450 enzymes catalyze specific steps in the biosynthesis
of steroid hormones, cholesterol, prostanoids and bile acids, participate in
the catabolism of endogenous compounds, including fatty acids and steroids, and
are involved in the degradation of exogenous compounds, including a wide
variety of structurally diverse drugs and carcinogens. The human cytochrome
P450 2A6 is principally involved in the break down of nicotine in the
bloodstream as it circulates through the liver. Oxidation of nicotine by this
P450 leads to detoxication, but the enzyme also activates tobacco-specific
procarcinogens to mutagenic products (1-4).
Using x-ray diffraction data sets collected on BL 11-1 and BL 9-1 at SSRL, a
team of scientists at The Scripps Research Institute has solved the crystal
structure of human cytochrome P450 2A6. The resources of these beam lines were
employed for automated screening of many crystals, in order to identify
crystals that diffracted well and at the same time had substrate or inhibitor
bound to the enzyme. For the crystals that did diffract well, the parallel
x-ray beams and large area detectors available on these beam lines were
required to record the data, as the protein crystals not only diffracted to
high resolution, but also exhibited a large unit cell. P450 2A6 structures were
solved with the alternative substrate, coumarin, and with the inhibitor,
methoxsalen, bound in the active site of the enzyme, adjacent to its iron
containing heme group. The compact, hydrophobic active site contains one
hydrogen bond donor, Asn297, which orients coumarin for regioselective
oxidation (Fig. 1a). Methoxsalen also interacts with Asn297 and effectively
fills the active site cavity without significantly perturbing the structure
(Fig. 1b). Precise knowledge of these molecular details and interactions is
critical for the rational design of new inhibitors. This structural information
is being used in on-going high resolution experiments at SSRL to probe the
active site of P450 2A6 with additional small molecule compounds. An effective
inhibitor of P450 2A6 could be used to diminish smoking and tobacco-related
cancers by reducing dependence on nicotine and by blocking formation of
carcinogens.
This work was supported by National Institutes of Health Grant GM031001 (to
E.F.J.) and fellowship 12FT-0185 from The Tobacco-Related Disease Research
Program (J.K.Y.)
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Last Updated: | 21 DEC 2005 |
Content Owner: | Dave Stout |
Page Editor: | Lisa Dunn |