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Originally published In Press as doi:10.1074/jbc.M702361200 on April 11, 2007

J. Biol. Chem., Vol. 282, Issue 23, 17306-17313, June 8, 2007
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Structure of the Human Lung Cytochrome P450 2A13*Formula

Brian D. Smith{ddagger}, Jason L. Sanders{ddagger}, Patrick R. Porubsky{ddagger}, Gerald H. Lushington§, C. David Stout, and Emily E. Scott{ddagger}1

From the {ddagger}Department of Medicinal Chemistry and §Molecular Graphics & Modeling Laboratory, University of Kansas, Lawrence, Kansas 66045 and the Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

Received for publication, March 19, 2007 , and in revised form, April 2, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
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 Km values than does CYP2A6. To investigate the structural differences between these two enzymes the structure of CYP2A13 was determined to 2.35Å 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 0.5Å for the C{alpha} 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 Asn297 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The two functional members of the human P450 2A gene family, CYP2A6 and CYP2A13, differ in only 32 of their 494 amino acids (supplementary Fig. 1). Both metabolize substrates including coumarin, nicotine, and the nicotine-derived procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK),2 the most abundant and potent lung carcinogen in tobacco. Additionally, CYP2A enzymes are reported to metabolize the mycotoxin aflatoxin B1 (1, 2). Both NNK and aflatoxin B1 require activation to generate the reactive intermediates that form DNA adducts and ultimately result in carcinogenicity.

Several key findings indicate that 2A13, rather than 2A6, plays an important role in in situ metabolism of NNK in the human respiratory tract. First, substantial amounts of CYP2A13 mRNA are generated in the respiratory tract, especially the nasal mucosa, trachea, and lung (3). By comparison, several studies reported either significantly lower CYP2A6 mRNA levels in respiratory tract tissues (3-5) or were unable to detect CYP2A6 mRNA at all (6). Recently a CYP2A13-specific antibody was used to confirm the presence of CYP2A13 protein in the epithelia of bronchus and trachea (7). Second, although CYP2A6 and CYP2A13 act on common substrates, rates of metabolism can differ significantly. Studies with recombinant enzymes have demonstrated that CYP2A13 is 30-215 times more efficient at activating NNK into its carcinogenic metabolites than CYP2A6 (3, 8, 9). Although cytochromes P450 2E1, 2D6, and 3A4 have also been shown to metabolize NNK in vitro, their Km values are much higher than Km values for the 2A enzymes (10). Finally, a substantial reduction in lung adenocarcinoma has been associated with a genetic polymorphism of CYP2A13 (11) that is reported to cause a 2-3-fold reduction of catalytic efficiency for NNK (12). Thus, activation of NNK by CYP2A13 in the lung may be a potent determinant for lung adenocarcinoma in smokers, which is the leading cause of cancer death. CYP2A13 is also known to preferentially activate a number of other xenobiotics. CYP2A13 is more than four times more active than CYP2A6 in the metabolism of hexamethylphosphoramide, N,N-dimethylaniline, and 2'-methoxyacetophenone (3). Additionally, a recent report demonstrated CYP2A13 metabolic activation of aflatoxin B1 into both aflatoxin B1 and aflatoxin M1 8,9-epoxides, whereas CYP2A6 produced neither carcinogenic metabolite (1).

To better define the structural features of CYP2A enzymes that determine their substrate metabolism, and to specifically investigate the structural differences between human CYP2A6 and CYP2A13 enzymes, we have determined a structure of human CYP2A13 by x-ray crystallography. Careful comparison of the CYP2A13 active site with previously determined structures of CYP2A6 (13) allows identification of a set of amino acid residues that are likely to modulate the observed differences in human CYP2A function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Protein Design, Expression, and Purification—The cDNA for CYP2A13 was a gift from Dr. X. Ding (Wadsworth Center, Albany, NY). The full-length gene was altered to yield protein (2A13dH) that lacks the N-terminal transmembrane sequence ({Delta}2-23), has several charged residues substituted at the modified N terminus (24WRQRKSR30 to 24AKKTSSK30) and has four histidine residues added at the C terminus (supplementary Fig. 1). This altered gene was inserted into the pKK233-2 plasmid (GE Healthcare). 2A13dH was then expressed in Escherichia coli TOPP-3 cells (Stratagene, La Jolla, CA) with an induction time of 48 h as described (14), but without the addition of imidazole.

E. coli cells were harvested and disrupted as described (15). After centrifugation to pellet cell debris, 4.8 mM Cymal-5 detergent (Anatrace, Maumee, OH) and 0.3 M NaCl were added to the supernatant, followed by ultracentrifugation at 80,000 x g for 60 min. The solubilized CYP2A13 lysate was applied to Ni2+-agarose resin (Qiagen, Valencia, CA), which was subsequently washed with the loading buffer. The resin was washed first with 100 mM potassium phosphate buffer (100 mM potassium phosphate, pH 7.4, 20% glycerol, 200 mM NaCl, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethanesulfonyl fluoride, and 4.8 mM Cymal-5) and then with the same buffer including 6 mM histidine. CYP2A13 was eluted using 10 mM potassium phosphate buffer with NaCl reduced to 100 mM, and supplemented with 2 mM EDTA and 40 mM histidine. 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. Purified CYP2A13 was eluted using the same buffer with potassium phosphate increased to 50 mM and including 500 mM NaCl. Purified protein was concentrated using centrifugal ultrafiltration.

Protein Crystallization, Data Collection, and Structure Determination—Crystals were grown using the hanging drop vapor diffusion equilibration. A 50 mg/ml solution of purified protein in 50 mM potassium phosphate buffer, pH 7.4, 20% glycerol, 1 mM phenylmethanesulfonyl fluoride, 0.2 mM dithiothreitol, 1 mM EDTA, 0.5 M NaCl, and 0.066 mM n-tridecyl-beta-D-maltopyranoside was equilibrated against 0.3 M (NH4)2SO4, 0.15 M sodium HEPES, pH 7.2, and 25% polyethylene glycol 2000 monomethyl ether at 20 °C. Plate-like crystals that grew in 10 days were immersed in mother liquor supplemented with 30% glycerol, and frozen in liquid nitrogen prior to data collection. A single native data set was collected on beamline 11-1 at the Stanford Synchrotron Radiation Laboratory (Stanford, CA). Data were recorded in two passes, a high resolution pass (180°, 1° oscillations, 10-s exposures) and a low resolution pass (90°, 1° oscillations, 2-s exposures) using an ADSC Q315 detector. The data to 2.35 Å were integrated and processed using the programs Mosflm and Scala (16). Statistics are shown in Table 1.


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  		TABLE 1
Data collection and refinement statistics

a Values for the highest-resolution shell in parentheses.

b B values were defined as the average of the B values for the amino acid residues immediately surrounding the ligands.

c Position A is defined as direct hydrogen bonding of the indole to Asn297. Position B is defined as hydrogen bonding of indole to Asn297 via a bridging water molecule.

 
The 2A13dH structure was solved by molecular replacement using the 4-(4-chlorophenyl)imidazole complex of cytochrome P450 2B4 (Protein Data Bank 1SUO) as a search model and Phaser (17). The space group is P2, a very rare space group for proteins, but this assignment was based on the presence of 0k0 reflections with k odd. In addition, molecular replacement calculations gave the highest log likelihood (1443) for a solution with six molecules in the asymmetric unit that packed well without space for additional molecules. The initial experimental electron density map reinforced the space group assignment and the molecular replacement solution. The model was refined using CNS (18). Rigid body refinement was followed by iterative rounds of positional and isotropic B-factor minimization with constrained NCS. Xtalview (19) was used for model building into {sigma}A-weighted 2Fo - Fc electron density maps. During the last several iterations of refinement, NCS was constrained with decreasing weights, as validated by decreases in Rfree.

The final model contains residues 31-494, heme, indole, and 591 water molecules. The crystallographic R-factor is 21.8 and the Rfree is 27.6. Residues in most favored regions of the Ramachandran plot include 86.8% of the structure, with 12.2% in additionally allowed regions, 0.8% in generously allowed regions, and 0.2% in disallowed regions. Coordinates have been deposited in the Protein Data Bank (PDB code 2P85).

Docking of NNK—Ligand structures were sketched and protonated in SYBYL3 and were then structurally optimized using the Tripos Force Field (21) (Gasteiger-Marsili electrostatics (22) with an 8.0-Å nonbonding interaction cutoff; default convergence thresholds). The receptor structures were prepared from crystallographic coordinates of CYP2A6 (13) and CYP2A13 (reported herein) by protonating the structures and adding Gasteiger-Marsili charges in SYBYL, and aligning the two according to conserved backbone residues. The coumarin ligand in the CYP2A6 structure was then transposed into the CYP2A13 receptor, and the three crystallographic waters with close contacts to the transposed coumarin were deleted. Both CYP2A6 and CYP2A13 were then conditioned in the presence of bound coumarin via short (1 ps; 300 K; Tripos Force Field) molecular dynamics simulations. The coumarin ligands were then removed from the receptor. Coumarin was then redocked into these two receptors for validation purposes via the FlexX program (23) taking into explicit consideration all receptor residues within 8.0 Å of the ligand (including the heme and remaining crystallographic waters, but neglecting the co-crystallized ligand) and using Gasteiger-Marsili charges for both ligand and receptor. Generation of docked conformers or poses was accomplished via the ligand fragment search and reassembly algorithm implemented in FlexX. The Asn297 side chain amide was specified as a critical H-bond acceptor site. No other preconditions were set on the ligand position or orientation. 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 <1.2 Å), with the carbonyl oxygen forming a hydrogen bond with the Asn297 side chain and the ring oxygen point up away from the heme. In the remaining 15 structures, the above H-bond was preserved, however, the ring oxygen was found to point down toward the heme. The same docking methodology was then applied to NNK binding to CYP2A6 and CYP2A13.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
X-ray Structure Determination and Overview—To better understand the differences between CYP2A6 and CYP2A13 ligand binding and metabolism, the structure of CYP2A13 was determined by x-ray crystallography. Truncated and His-tagged cytochrome P450 2A13dH crystallized in the P2 space group with six molecules in the asymmetric unit. The initial experimental maps were easily interpreted for most of the protein and the model refined using iterative model adjustment and refinement. Statistics for data collection and refinement are shown in Table 1.


Figure 1
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  		FIGURE 1.
CYP2A13 structure (blue) overlaid with the CYP2A6 structure (gray). Heme is shown as red sticks and sphere (iron).

 
The CYP2A13 structure follows the typical fold for mammalian cytochromes and is composed of 20 {alpha}-helices and 4 beta-sheets enclosing the heme prosthetic group (Fig. 1), with no apparent open channels. The six molecules in the asymmetric unit are very similar (average C{alpha} root mean square deviation 0.4 Å). The overall CYP2A13 structure is very similar to that of the closely related human liver cytochrome P450 2A6 (Fig. 1). The root mean square deviation for C{alpha} atoms in CYP2A13 versus CYP2A6 (PDB 1Z10) is 0.5 Å, only slightly higher than the differences among the six different CYP2A13 molecules in the asymmetric unit or the four CYP2A6 molecules present in that asymmetric unit. Four of the six CYP2A13 molecules have the same packing as the four molecules in the CYP2A6 asymmetric unit. The secondary structure units are essentially identical between the two CYP2A enzymes (supplementary Fig. 1).

Active Site—The CYP2A13 active site is very hydrophobic and tightly packed. The cavity is small and planar, complementing the size and shape of many CYP2A substrates. The planar active site is oriented edge-on to the heme group. The active site of CYP2A13 is 307 Å3, some 15-20% larger that that of the CYP2A6 structures and proportioned differently as a result of individual amino acid differences lining the cavity (Fig. 2). The two CYP2A enzymes have the smallest active sites of the human cytochromes P450 whose structures have been determined. Amino acids lining the CYP2A13 active site include Phe107, Phe111, Ala117, Phe118, Phe209, Leu296, Asn297, Phe300, Ala301, Glu304, Thr305, Met365, Leu366, Leu370, and Phe480.

Notably, six of the amino acids lining the active site are phenylalanine residues: Phe107, Phe111, Phe118, Phe209, Phe300, and Phe480 (Fig. 2). These phenylalanines cluster to form the "roof" of the active site. This cluster is even larger than the phenylalanine cluster in CYP2A6, as Ile300 in CYP2A6 is Phe300 in CYP2A13. The aromatic side chains substantially fill the active site and provide multiple opportunities for parallel-displaced {pi}-{pi} stacking and edge-to-face interactions with aromatic ligands and with each other to stabilize the tertiary protein structure. Negishi and co-workers (24) demonstrated that mutation at positions 209 or 481 substantially altered steroid hydroxylase activity in mouse 2A enzymes. Although Phe209 interacts directly with substrates in the active site cavity, residue 481 does not line the CYP2A13 active site. However, in CYP2A13, the side chain of Ala481 projects directly toward Phe209. In the CYP2A4 and CYP2A5 studies, mutation at 481 likely altered the packing of Phe209 and/or the adjacent Phe480, both of which do line the active site cavity, to alter metabolism indirectly.

Although no exogenous ligands were added to the purified CYP2A13, initial experimental maps indicated the presence of a ligand in the active sites of all six molecules of the asymmetric unit. During refinement, subsequent electron density maps reinforced the presence of this strong planar density. The modest resolution of the x-ray diffraction experiment did not allow identification of the bound molecule in the crystals. However, we have independently determined that the ligand is indole. CYP2A6 is one of several cytochromes P450 reported to metabolize indole into various indigoid pigments (25). In fact, when human CYP2A6 and the natural redox partner are overexpressed in E. coli, the bacterial cultures turn blue as a result of indigoid production from indole normally present in the E. coli cells. Likewise, when CYP2A13 is expressed in E. coli, the cultures also generate indigoid pigments, likely by accepting electrons from an endogenous E. coli reductase. We suspected that indole co-purified with the protein and constituted the ligand in the active site. First, early in the purification when large amounts of indigoid pigments are still present, UV-visible spectra indicate Soret peaks at both 393 and 418 nm, indicating a mixture of CYP2A13 molecules, some with a ligand and some with water in the active site, respectively. The spectrum of the purified protein has an absorbance maximum at 418 nm, with only a slight shoulder toward 393 nm, but indole does not cause a significant shift in the Soret at concentrations less than 40-fold over the CYP2A13 protein concentration. Second, the overall size, shape, and planarity of the bicyclic indole ring system correlate well with the observed electron density in the active site (Fig. 3). Third, CYP2A13 purified for crystallization reacts with p-dimethylaminocinnamaldehyde to yield a product with an absorbance at 625 nm, indicative of the presence of indole and/or certain indole metabolites (26). Finally, we were able to extract the 625 nm p-dimethylaminocinnamaldehyde product from purified CYP2A13 protein and identify it as a complex with unsubstituted indole by mass spectrometry. On the basis of these results, we have modeled the electron density in the present CYP2A13 structure as indole.


Figure 2
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  		FIGURE 2.
Comparison of active site volumes of CYP2A6 (yellow mesh) and CYP2A13 (blue mesh) surrounded by active site residues of CYP2A13 (blue ribbons and sticks). Heme, red sticks. The solvent-occupied surface of the active site cavities were calculated using VOIDOO (38) with a 1.4-Å probe and a mesh of 0.33.

 
This strongly planar indole density in the CYP2A13 structure appears to adopt two alternate orientations in the active site. For most molecules, the predominant orientation overlaps the location of the coumarin molecule in CYP2A6 and similarly forms a direct hydrogen bond to Asn297 (indole position A, Fig. 3A). However, one of the active sites has very strong density supporting a second orientation of indole with the benzene ring in the same location, but with the fused pyrrole ring more perpendicular to the heme plane. In this orientation indole also binds to Asn297, this time via a bridging water molecule (indole position B, Fig. 3B). In addition to the electron density, this second orientation is also supported by molecular dynamics simulations at 100 K that indicate the bridging arrangement is a stable one. Subsequent refinement of the occupancies of both ligands in each active site indicated that all active sites likely contain a mixture of indole in orientations A and B ranging from 60/40 to 40/60 (Fig. 3C). Dual ligand orientations have been observed previously in a complex of CYP2C5 with 4-methyl-N-methyl-N-(2-phenyl-2H-pyrazol-3-yl)benzene-sulfonamide (27). In both the CYP2A6 and CYP2A13 structures, Asn297 is the only polar residue in the active site and this amino acid likely plays a key role in ligand orientation. Asn297 is stabilized by a hydrogen bonding network including a water molecule and the backbone of residues in the loop between helices B' and C in CYP2A13 as well as CYP2A6. Asparagine is highly conserved at position 297 in CYP2A enzymes from many different organisms, but not in other subfamilies of human cytochromes P450. Thus, this hydrogen bond interaction may be a hallmark of CYP2A enzyme interactions with their ligands and responsible for orientation of many CYP2A ligands in the active site.

Although the indole in CYP2A13 binds in nearly the same location as coumarin in CYP2A6 with respect to the heme and Asn297, the plane of coumarin and indole differ by ~30°. When viewed end-on from opposite Asn297, coumarin is oriented along a northwest/southeast axis, whereas both of the CYP2A13 indole ligands are oriented along a northeast/south-west axis (Fig. 4). Structural proximity implicates four amino acid residues in orientation of the ligand plane. In CYP2A6, Ile300 projects into the active site cavity from the northeast, limiting space available to the ligand in this quadrant. In CYP2A13, residue 300 is phenylalanine, but the larger side chain is torsioned away from the ligand, resulting in more space in the north-east quadrant of the CYP2A13 active site. Rotation of the aromatic ring away from the active site is likely permitted because of a "second shell" residue difference between CYP2A6 and CYP2A13 at the C-terminal end of the F helix. Located directly above Phe300 in the active site, the reduction in size from Ile208 in CYP2A6 to Ser208 in CYP2A13 both provides space for the aromatic ring torsion in CYP2A13 and repositions the aromatic ring of Phe209. The CYP2A13 S208I mutation has been shown to significantly decrease NNK metabolism to both metabolites (9), suggesting it might also be key in orienting other ligands. The identity of the residue at position 209 is key for determining metabolism with coumarin, steroids, and aflatoxin B1 in mouse CYP2A enzymes (2, 28, 29).

At the base of the active site near the heme, residues 301 and 117 also act in concert to control the ligand orientation. In CYP2A13, the Ala301 side chain methyl occupies space that is available to ligands in CYP2A6, which has glycine at this position. On the opposite side of the active site, residue 117 is an alanine in CYP2A13 and occupies less space than the corresponding valine at this position in CYP2A6. Although there has been little exploration of the role of residue 301 in CYP2A enzymes, mutation at position 117 has been demonstrated to substantially impact the catalytic efficiency of CYP2A13 for coumarin (30), NNK (9), and aflatoxin B1 (1). For coumarin and NNK the effects are primarily reflected in changes in Vmax rather than Km, suggesting orientation of the ligand may be related to catalysis rather than ligand affinity. Additionally, variants at this position are responsible for significant differences in coumarin hydroxylation in mouse 2A enzymes (31).

Additional active site differences between CYP2A6 and CYP2A13 occur at positions 365 and 366. These residues, along with Thr305, are located at the end of the active site opposite Asn297. In CYP2A6, the side chains of Thr305 and Ile366 approach each other to close off the end of the active site, with Val365 just behind and shielded from the active site cavity. In CYP2A13, the larger methionine at 365 extends much closer toward the ligand and the flanking residues Thr305 and Leu366 are farther separated. As a result, the terminal atoms of Met365 make up part of the active site wall. Although these residues are more distant from the indole ligand in this structure, they are likely to play a role in orienting slightly larger ligands. The identity of the side chain at position 365 in mouse CYP2A4 and CYP2A5 has a role in metabolism of coumarin, testosterone, and aflatoxin B1 (2, 32).


Figure 3
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  		FIGURE 3.
Indole binding in the CYP2A13 active site. Panel A, indole in position A in CYP2A13 molecule C of the asymmetric unit. Panel B, indole in position B in CYP2A13 molecule F of the asymmetric unit. Panel C, the indole density could be best fit by partial occupancy of indole in two positions with the benzene rings overlapping (CYP2A13 molecule A shown). Indole in position A, pink sticks; indole in position B, cyan sticks; composite omit {sigma}A-weighted 2|Fo| - |Fc| electron density contoured at 1.0 {sigma} around indoles, blue mesh; heme, red sticks and spheres; components of active site walls and active site residues colored by tertiary structure component: B' helix and adjacent loops, blue; I helix, yellow; F helix, orange; loop between K helix and beta1-4, green; beta4-1/beta4-2 turn, magenta.

 


Figure 4
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  		FIGURE 4.
The CYP2A13 protein (blue ribbons and sticks), heme (red sticks), and indole A (cyan sticks) are shown overlaid with selected CYP2A6 residues (green sticks) that reorient the plane of coumarin (green sticks) in the active site. CYP2A13 indole B is not shown for clarity, but adopts a very similar plane to indole A.

 
Thus, both structure and function studies support the idea that a combination of residues at positions 117, 208, 300, 301, 365, and possibly 366 modulate ligand binding and metabolism in CYP2A enzymes. Analysis of these residues in different CYP2A enzymes may help understand phenacetin metabolism. CYP2A13 has recently been reported (33) to metabolize phenacetin, normally a CYP1A2 marker substrate, more efficiently than CYP1A2 itself. Rabbit CYP2A10 and CYP2A11 also have this capacity, but CYP2A6 does not. Comparison of the active site residues at key positions where CYP2A13 and CYP2A6 differ (Table 2) suggests that in 2A enzymes residues Ser208, Phe300, Ala301, and Met365 are compatible with phenacetin metabolism.


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  		TABLE 2
Comparison of key active site residues in 2A enzymes

 
Heme Binding—CYP2A6 and CYP2A13 also differ in two of the residues responsible for interaction with the heme. In CYP2A6, Ser369 hydrogen bonds through its side chain hydroxyl group to the A ring propionate. Hydrogen bonding residues are generally conserved at this position in microsomal CYPs. CYP2A13, however, has a glycine at this position and is thus incapable of the hydrogen binding interaction. In the place of the serine side chain, CYP2A13 has a water molecule ideally positioned to replace this interaction. The water molecule is stabilized by strong hydrogen bonds to both the backbone amine and carbonyl of residue 370.

The residue at position 372 also interacts with the A ring propionate and differs between the two CYP2A proteins. This residue is histidine in 70% of the family 1 and 2 cytochromes P450, including CYP2A13. The imidazole ring of His372 hydrogen bonds to the terminal propionate oxygens. In CYP2A6 and the remaining 30% of the family 1 and 2 enzymes, this residue is an arginine. In CYP2A6, the N{epsilon} of Arg372 takes the place of the imidazole nitrogen. From the structures, it appears that the two arrangements might be equivalent. Three site-directed mutagenesis studies, however, indicate that this residue is functionally important. The CYP2A13 mutation H372R disrupts the ability of the enzyme to form either of the carcinogenic aflatoxin 8,9-epoxide metabolites and substantially increased formation of the detoxification products aflatoxin P1 (1). In metabolism of NNK, the H372R mutation obliterates the ability to form the keto alcohol metabolite, and reduces the kcat for formation of the keto aldehyde by almost 130-fold compared with the CYP2A13 wild type enzyme (9), essentially converting the metabolite profile for NNK to that of CYP2A6. Additionally, CYP2A13 H372R has an 8-fold increase in the kcat for coumarin to near 2A6 levels, whereas introduction of the R372H mutation in CYP2A6 reduces its coumarin kcat 5-fold to ~2A13 levels (30).

Docking of NNK into the Active Site—Of all human cytochrome P450s, the human lung-specific CYP2A13 has been reported as the most effective activator of nicotine-derived NNK. 2A13 activates NNK at both the methyl and methylene positions to form DNA pyridyloxobutylating and methylating intermediates that are ultimately detected as a keto alcohol and a keto aldehyde, respectively (32-34). CYP2A6 is much less efficient at NNK activation (Table 3). When NNK was docked into the CYP2A6 structure, all 30 docked conformers predicted a hydrogen bond between the NNK carbonyl oxygen and the Asn297 side chain and resulted in an orientation with the pyridine ring too close to the heme iron to permit close approach by either the methylene and methyl carbons (no approaches closer than 7.0 and 5.5 Å, respectively, Fig. 5A). This orientation is incompatible with generation of either carcinogenic metabolite. In contrast, when NNK was docked into the CYP2A13 structure, NNK generally oriented with the pyridine ring oriented away from the heme iron (26/30 docked conformers) with its nitrogen hydrogen bonded to Asn297. Nineteen of these docked conformers placed the methyl carbon nearest to the heme iron (as close as 3.9 Å, Fig. 5B). Another seven of the docked conformers placed the methylene carbon nearest to the heme iron (as close as 4.9 Å, Fig. 5C). These orientations are suitable for hydroxylation by the activated oxygen intermediate into the keto alcohol and keto aldehyde metabolites observed experimentally. In the remaining four CYP2A13 docked conformers the nitroso oxygen hydrogen bonded to Asn297 and the pyridine ring was pointed toward the heme, similar to that observed in all of the CYP2A6 conformers. The energies of all CYP2A6 and CYP2A13 docking conformers were similar. Thus, subtle differences in the structures modify ligand orientation in docking experiments to correctly predict the metabolites observed (Table 3). In particular, residues at positions 366 and 365 differ between CYP2A13 and CYP2A6 and have very different interactions with NNK in CYP2A6 compared with CYP2A13. The combination and positioning of Met365-Leu366 in CYP2A13 creates a slight additional space adjacent to these residues that is not available adjacent to Val365-Ile366 in CYP2A6 (Fig. 2). When NNK is positioned for methyl hydroxylation in CYP2A13, the terminal nitrosamine functional group occupies this small pocket (Fig. 5B). Rotation about the C-C bond adjacent to the nitrosamine would then yield the orientation of NNK suitable for methylene hydroxylation (Fig. 5C). Neither of these two orientations appears to be available to NNK in CYP2A6, potentially due to the replacement of Met365-Leu366 with Val365-Ile366.


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  		TABLE 3
Comparison of CYP2A13 and CYP2A6 kinetic parameters and docking orientations

 

   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Because lung cancer is the most common cancer and the leading cause of cancer-related death (37), understanding in situ activation of nicotine-derived NNK into carcinogens by CYP2A13 is an important goal. Comparison of the human CYP2A13 and CYP2A6 structures reveals several common features that may be hallmarks of CYP2A enzymes: 1) a relatively small, planar active site; 2) numerous active site phenylalanine residues for {pi}-{pi} interactions with aromatic ligands; and 3) Asn297 oriented for hydrogen bonding with ligands. Differences between the two structures indicate that residues 117, 208, 300, and 301 act in concert to define the plane of ligands in the active site. Residue differences at positions 365 and/or 366 are also likely determinants of ligand binding and catalysis, especially for NNK, whereas the structure suggests residues at 369 and 372 might be more directly involved in heme binding. Most of the remaining differences between CYP2A6 and CYP2A13 are located outside the active site either in the N-terminal transmembrane region or on the surface of the protein. Mutagenesis results support the effects of residues at 117, 208, and 365, whereas the roles of residues at positions 297, 300, and 301 should be further explored by mutagenesis and functional studies.


Figure 5
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  		FIGURE 5.
NNK docking into CYP2A6 and CYP2A13. Panel A, NNK docked into the CYP2A6 structure assumes a nonproductive orientation with the methylene and methyl carbons no closer than 7.0 and 5.5 Å from the heme iron, respectively. In contrast, NNK docked into the CYP2A13 structure assumes an orientation with either the methyl (panel B) or methylene (panel C) oriented for hydroxylation into the two carcinogenic metabolites observed experimentally. Black circles highlight methyl and methylene carbons hydroxylated to form carcinogenic metabolites. Residues and helices shown are as described in the legend to Fig. 3.

 

   FOOTNOTES
 
* This work was supported by the National Institutes of Health Grants RR016475 (to J. L. S. and P. R. P.), GM61545 (to C. D. S.), RR017708 (to E. E. S.), and GM076343 (to E. E. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 To whom correspondence should be addressed: 1251 Wescoe Hall Dr., Lawrence, KS 66045. Tel.: 785-864-5559; Fax: 785-864-5326; E-mail: eescott{at}ku.edu.

2 The abbreviation used is: NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Back

3 SYBYL 7.2, Tripos, Inc., St. Louis, MO. Back


   ACKNOWLEDGMENTS
 
We thank Dr. X. Ding (Wadsworth Center, Albany, NY) for the gift of CYP2A13 cDNA. We acknowledge access to the facilities and support staff of SSRL. The SSRL is operated by the Department of Energy, Office of Basic Energy Sciences. The SSRL Biotechnology Program is supported by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program, and by the Department of Energy, Office of Biological and Environmental Research. We also thank Dr. Audrey Lamb for assistance with Phaser.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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