Adaptations for the Oxidation of Polycyclic Aromatic Hydrocarbons Exhibited by the Structure of Human P450 1A2
Microsomal cytochrome P450 family 1 enzymes play prominent roles in xenobiotic detoxication and procarcinogen activation. P450 1A2 is the principal cytochrome P450 family 1 enzyme expressed in human liver and participates extensively in drug oxidations. This enzyme is also of great importance in the bioactivation of mutagens, including the N-hydroxylation of arylamines. P450-catalyzed reactions involve a wide range of substrates, and this versatility is reflected in a structural diversity evident in the active sites of available P450 structures. Here, we present the structure of human P450 1A2 in complex with the inhibitor α-naphthoflavone, determined to a resolution of 1.95 Aå. α-Naphthoflavone is bound in the active site above the distal surface of the heme prosthetic group. The structure reveals a compact, closed active site cavity that is highly adapted for the positioning and oxidation of relatively large, planar substrates. This unique topology is clearly distinct from known active site architectures of P450 family 2 and 3 enzymes and demonstrates how P450 family 1 enzymes have evolved to catalyze efficiently polycyclic aromatic hydrocarbon oxidation. This report provides the first structure of a microsomal P450 from family 1 and offers a template to study further structure-function relationships of alternative substrates and other cytochrome P450 family 1 members. Microsomal cytochrome P450 family 1 enzymes play prominent roles in xenobiotic detoxication and procarcinogen activation. P450 1A2 is the principal cytochrome P450 family 1 enzyme expressed in human liver and participates extensively in drug oxidations. This enzyme is also of great importance in the bioactivation of mutagens, including the N-hydroxylation of arylamines. P450-catalyzed reactions involve a wide range of substrates, and this versatility is reflected in a structural diversity evident in the active sites of available P450 structures. Here, we present the structure of human P450 1A2 in complex with the inhibitor α-naphthoflavone, determined to a resolution of 1.95 Aå. α-Naphthoflavone is bound in the active site above the distal surface of the heme prosthetic group. The structure reveals a compact, closed active site cavity that is highly adapted for the positioning and oxidation of relatively large, planar substrates. This unique topology is clearly distinct from known active site architectures of P450 family 2 and 3 enzymes and demonstrates how P450 family 1 enzymes have evolved to catalyze efficiently polycyclic aromatic hydrocarbon oxidation. This report provides the first structure of a microsomal P450 from family 1 and offers a template to study further structure-function relationships of alternative substrates and other cytochrome P450 family 1 members. Enzymes of the cytochrome P450 (CYP) 5CYP and P450 are generic terms for a cytochrome P450 enzyme. Individual P450s are identified using a number-letter-number format based on amino acid sequence relatedness. 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. The enzymes comprising P450 families 1, 2, and 3 contribute most extensively to the biotransformation of xenobiotics to more polar metabolites that are more readily excreted. In humans and most mammals, family 1 contains three well characterized P450 monooxygenases; 1A1, 1A2, and 1B1. These enzymes are generally distinguished from P450s in other families by their capacity to oxidize a variety of polynuclear aromatic hydrocarbons (PAHs). 6The abbreviations used are: PAH, polynuclear aromatic hydrocarbon; ANF, α-naphthoflavone; PEG, polyethylene glycol; PDB, Protein Data Bank. Moreover, the expression levels of the three enzymes are induced by exposure to PAHs (1Nebert D.W. Dalton T.P. Okey A.B. Gonzalez F.J. J. Biol. Chem. 2004; 279: 23847-23850Abstract Full Text Full Text PDF PubMed Scopus (995) Google Scholar). The induction is mediated by a ligand-activated transcription factor, the aryl hydrocarbon receptor, which is a basic-loop-helix PAS domain protein that binds to enhancer elements flanking the CYP1A1, CYP1A2, and CYP1B1 genes and stimulates transcription. The oxidation of PAHs is generally protective. However, some P450-catalyzed reactions can transform these relatively inert compounds into genotoxic metabolites that can initiate mutagenesis and cancer. Human P450 1A2 is notable among family 1 enzymes for the capacity to N-oxidize arylamines, the major metabolic process in the bioactivation of arylamines to potent mutagenic or carcinogenic compounds (2Kim D. Guengerich F.P. Annu. Rev. Pharmacol. Toxicol. 2005; 45: 27-49Crossref PubMed Scopus (215) Google Scholar). α-Naphthoflavone (ANF), a prototype flavonoid, is known to competitively inhibit P450s of family 1, albeit at different concentrations, and has been used to discriminate between P450 family 1 enzymes (3Shimada T. Yamazaki H. Foroozesh M. Hopkins N.E. Alworth W.L. Guengerich F.P. Chem. Res. Toxicol. 1998; 11: 1048-1056Crossref PubMed Scopus (204) Google Scholar). Flavonoids have gained recent interest in view of their potential therapeutic and prophylactic effects on P450-mediated chemical carcinogenesis (4Hodek P. Trefil P. Stiborova M. Chem. Biol. Interact. 2002; 139: 1-21Crossref PubMed Scopus (537) Google Scholar). CYP1A2 is the principal family 1 enzyme expressed in human liver, and CYP1A2 contributes significantly to the hepatic metabolism of drugs, as recently reviewed (5Agundez J.A. Curr. Drug Metab. 2004; 5: 211-224Crossref PubMed Scopus (262) Google Scholar). Among liver P450 drug-metabolizing enzymes, P450 1A2 plays a predominant role in the metabolic clearance of caffeine and melatonin as well as of marketed drugs such as flutamide, lidocaine, olanzapine, tacrine, theophylline, triamterene, and zolmitriptan. Large inter-individual differences in CYP1A2 expression levels and catalytic activity contribute to significant differences between individuals in drug clearance. The basis for this variation is only partially understood. This report provides the first structural characterization of human CYP1A2 and is the first structure of a microsomal P450 from family 1. The structure reveals an enzyme that is highly adapted for the oxidation of relatively large, planar molecules such as (heterocyclic) arylamines and PAHs and provides a basis for understanding the unique roles this enzyme plays in the biotransformation of xenobiotics. CYP1A2 exhibits less than 40% amino acid sequence identity when compared with other structurally characterized mammalian microsomal P450s 2A6 (6Yano J.K. Hsu M.H. Griffin K.J. Stout C.D. Johnson E.F. Nat. Struct. Mol. Biol. 2005; 12: 822-823Crossref PubMed Scopus (290) Google Scholar), 2B4 (7Scott E.E. White M.A. He Y.A. Johnson E.F. Stout C.D. Halpert J.R. J. Biol. Chem. 2004; 279: 27294-27301Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar), 2C5 (8Williams P.A. Cosme J. Sridhar V. Johnson E.F. McRee D.E. Mol. Cell. 2000; 5: 121-132Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar), 2C8 (9Schoch G.A. Yano J.K. Wester M.R. Griffin K.J. Stout C.D. Johnson E.F. J. Biol. Chem. 2004; 279: 9497-9503Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar), 2C9 (10Williams P.A. Cosme J. Ward A. Angove H.C. Matak V.D. Jhoti H. Nature. 2003; 424: 464-468Crossref PubMed Scopus (794) Google Scholar, 11Wester M.R. Yano J.K. Schoch G.A. Yang C. Griffin K.J. Stout C.D. Johnson E.F. J. Biol. Chem. 2004; 279: 35630-35637Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar), 2D6 (12Rowland P. Blaney F.E. Smyth M.G. Jones J.J. Leydon V.R. Oxbrow A.K. Lewis C.J. Tennant M.G. Modi S. Eggleston D.S. Chenery R.J. Bridges A.M. J. Biol. Chem. 2006; 281: 7614-7622Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar), and 3A4 (13Williams P.A. Cosme J. Vinkovic D.M. Ward A. Angove H.C. Day P.J. Vonrhein C. Tickle I.J. Jhoti H. Science. 2004; 305: 683-686Crossref PubMed Scopus (743) Google Scholar, 14Yano J.K. Wester M.R. Schoch G.A. Griffin K.J. Stout C.D. Johnson E.F. J. Biol. Chem. 2004; 279: 38091-38094Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar). The P450 1A2 active site cavity complements the sizes, shapes, and chemical properties of the substrate binding sites determined for other human drug-metabolizing P450s, contributing to the overall capacity of P450s to oxidize a vast array of structurally dissimilar substrates. Expression and Purification of hP450 1A2—Plasmid vectors were constructed to express modified forms of human P450 1A2 in Escherichia coli. The modifications were designed to facilitate crystallization by removing the N-terminal transmembrane helical domain to reduce aggregation, increase solubility, and eliminate a flexible appendage that is not part of the catalytic domain. In the expression construct, the native sequence upstream of the polyproline motif at residue 42 was modified to correspond to a modified, truncated N-terminal sequence successfully employed for crystallization of P450s 2C5/3LVdH (8Williams P.A. Cosme J. Sridhar V. Johnson E.F. McRee D.E. Mol. Cell. 2000; 5: 121-132Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 15Wester M.R. Johnson E.F. Marques-Soares C. Dijols S. Dansette P.M. Mansuy D. Stout C.D. Biochemistry. 2003; 42: 9335-9345Crossref PubMed Scopus (190) Google Scholar), 2C8dH (9Schoch G.A. Yano J.K. Wester M.R. Griffin K.J. Stout C.D. Johnson E.F. J. Biol. Chem. 2004; 279: 9497-9503Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar), 2B4dH E.E. He Y.A. Wester M.R. White M.A. Halpert J.R. Johnson E.F. Stout C.D. S. A. 2003; PubMed Scopus Google Scholar), and (6Yano J.K. Hsu M.H. Griffin K.J. Stout C.D. Johnson E.F. Nat. Struct. Mol. Biol. 2005; 12: 822-823Crossref PubMed Scopus (290) Google Scholar). This was using a that a the modified N-terminal sequence and sequence the native protein at The a a and of the from a template the human CYP1A2 C. S. A. PubMed Scopus Google by of using the was and with the for into the of alternative was also that the transmembrane helical domain different of the native the motif at the of the catalytic domain. the used in this with were into the expression which been with the and The of the by for and of the used in the of the and are in the of expression for the predominant was with a CYP1A2 expression with an expression for the and to further the and of the were based on and for the and of the by and in the of the M.R. Stout C.D. Johnson E.F. 2002; PubMed Scopus Google Scholar). used for and crystallization of the protein to the modified human P450 1A2 P. T. Guengerich F.P. PubMed Scopus Google Scholar). The of the protein was by for the of the enzyme the enzyme. Protein modified CYP1A2 were by The were at a well of in a Protein were in a of protein to The to the by only the in the the well The protein of a protein with to and and for the from a protein at a of in ANF, and of the were by a protein in acid ANF, and with of and in was to resolution was on a using at the to the was in a and in Data and employed PubMed Scopus Google Scholar), and Biol. PubMed Scopus Google Scholar). in with R.J. Biol. 2005; PubMed Scopus Google using the structure of as a CYP1A2 molecules were in the and with was with the M. PubMed Scopus Google and with of the and by using W.L. P. J. M. R.J. T. Biol. 1998; PubMed Scopus Google with the of This of an CYP1A2 for of the native protein with the of an on and are in 1. the that the not to and P450 1A2 between in the resolution between in the resolution of between in the resolution between in the resolution between in the resolution is the of an and is the of that range used in or and are the and structure to the and between in the resolution is the of an and is the of that or and are the and structure to the and Biol. PubMed Scopus Google in a The in the and in the were at on that were in The and Biol. PubMed Scopus Google were used to process the for with the CYP1A2 for the as a in using R.J. Biol. 2005; PubMed Scopus Google Scholar). in generally to resolution and have of P450 1A2 in complex with in the on a of of these to a resolution of is in 1. resolution to 1.95 at the on a to was used to and the The CYP1A2 of the native protein and the that not in the determined for the at the and at the of the truncated and not in the of molecules were and the inhibitor was in the distal active site above the heme prosthetic as by at Data and are in 1 with the of the protein Protein and was modified to the N-terminal transmembrane domain to and to reduce as for the crystallization of other microsomal P450s (6Yano J.K. Hsu M.H. Griffin K.J. Stout C.D. Johnson E.F. Nat. Struct. Mol. Biol. 2005; 12: 822-823Crossref PubMed Scopus (290) Google Scholar, G.A. Yano J.K. Wester M.R. Griffin K.J. Stout C.D. Johnson E.F. J. Biol. Chem. 2004; 279: 9497-9503Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 15Wester M.R. Johnson E.F. Marques-Soares C. Dijols S. Dansette P.M. Mansuy D. Stout C.D. Biochemistry. 2003; 42: 9335-9345Crossref PubMed Scopus (190) Google Scholar, E.E. He Y.A. Wester M.R. White M.A. Halpert J.R. Johnson E.F. Stout C.D. S. A. 2003; PubMed Scopus Google Scholar). Expression of CYP1A2 as a protein for crystallization was in In the construct, a sequence that has been successfully employed for the crystallization of other human P450 enzymes was for the native N-terminal sequence upstream of the motif at the of the catalytic domain M.R. Stout C.D. Johnson E.F. 2002; PubMed Scopus Google Scholar). The alternative employed a of the CYP1A2 sequence for which including the transmembrane were that to three different This to in the to the N-terminal sequence of the construct, in a and a differences in the catalytic domain were not evident for from to or for the and for the for the of of the three not Aå. The was to a resolution of 1.95 for a in and of the native protein Data and are in 1. Human P450 1A2 structure of P450 1A2 exhibits the and and which are generally for the P450 some of which are also in other P450 are present and by or compared with mammalian P450s of known the most are the of the protein the heme binding site and the surface that is to binding sites for the cytochrome P450 and cytochrome Johnson E.F. and Biochemistry. 2005; P. Scopus Google Scholar). The most between known P450 are the that the distal of the substrate binding the and and the microsomal P450 enzymes generally have a between and which exhibits a that part of a surface on the of the protein to the transmembrane domain. This surface forms part of the surface of the catalytic domain of P450s (8Williams P.A. Cosme J. Sridhar V. Johnson E.F. McRee D.E. Mol. Cell. 2000; 5: 121-132Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, C. Biochemistry. 2006; 45: PubMed Scopus Google Scholar). and are generally in this for other mammalian P450 structures. In the P450 1A2 and are helical than a is This and surface are not generally in P450 structures. P450 1A2 less than 40% amino acid sequence identity with P450 3A4 or structurally characterized family 2 as based on the sequence the P450 1A2 structure from of families 2 and 3 in the and of structure sequence of P450s 1A2, and 3A4 is in is present between and as well as an at the the and a which forms a that into the of the P450 1A2 structure is the of as the distal surface of the active site cavity P450 family 2 (6Yano J.K. Hsu M.H. Griffin K.J. Stout C.D. Johnson E.F. Nat. Struct. Mol. Biol. 2005; 12: 822-823Crossref PubMed Scopus (290) Google Scholar, E.E. White M.A. He Y.A. Johnson E.F. Stout C.D. Halpert J.R. J. Biol. Chem. 2004; 279: 27294-27301Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, P.A. Cosme J. Sridhar V. Johnson E.F. McRee D.E. Mol. Cell. 2000; 5: 121-132Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, G.A. Yano J.K. Wester M.R. Griffin K.J. Stout C.D. Johnson E.F. J. Biol. Chem. 2004; 279: 9497-9503Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, P.A. Cosme J. Ward A. Angove H.C. Matak V.D. Jhoti H. Nature. 2003; 424: 464-468Crossref PubMed Scopus (794) Google Scholar, 11Wester M.R. Yano J.K. Schoch G.A. Yang C. Griffin K.J. Stout C.D. Johnson E.F. J. Biol. Chem. 2004; 279: 35630-35637Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, P. Blaney F.E. Smyth M.G. Jones J.J. Leydon V.R. Oxbrow A.K. Lewis C.J. Tennant M.G. Modi S. Eggleston D.S. Chenery R.J. Bridges A.M. J. Biol. Chem. 2006; 281: 7614-7622Abstract Full Text Full Text PDF PubMed Scopus (406) Google an for this in the structure of P450 3A4 (13Williams P.A. Cosme J. Vinkovic D.M. Ward A. Angove H.C. Day P.J. Vonrhein C. Tickle I.J. Jhoti H. Science. 2004; 305: 683-686Crossref PubMed Scopus (743) Google Scholar, 14Yano J.K. Wester M.R. Schoch G.A. Griffin K.J. Stout C.D. Johnson E.F. J. Biol. Chem. 2004; 279: 38091-38094Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar), a structure and above the active site cavity In the P450 1A2 the is at and helical in the of to molecules the to between and and and The of the of the in the of the the active site in the cavity above the distal surface of the heme prosthetic group. In the P450 1A2 structure of the the active site is closed evident or substrate The of the cavity was to which is than that of P450 2A6 The compact, closed active site cavity of P450 1A2 and P450 2A6 with the more active site architectures by P450 3A4 and P450 with of and Human P450 1A2 in with protein was and in the of The structure and of are in The of in the active site cavity was well by for a that not The of the is clearly by the of the and further by for the and the which were by the of the at the the to the This that binds in a which the to the heme view of the binding of in the human P450 1A2 active the of and the heme by and of the amino acid the active site cavity in with of the protein as a The inhibitor and the heme prosthetic are in with and and are and is as a Large is to a inhibitor of human P450 reactions with an of (3Shimada T. Yamazaki H. Foroozesh M. Hopkins N.E. Alworth W.L. Guengerich F.P. Chem. Res. Toxicol. 1998; 11: 1048-1056Crossref PubMed Scopus (204) Google Scholar, 2003; PubMed Scopus Google Scholar). structural are to contribute to the binding of P450 1A2 for the and of the substrate binding cavity to and from the is to contribute to a for In and aromatic between and and contribute to a binding the of an the of provides an binding The to to the of as well as to the of on This is the only present in the active and that the active site cavity with the protein surface are The from the heme to of is Aå. This at the of the range of for substrate binding in other P450 and is for the more of the on the aromatic S. J. Chem. 2003; PubMed Scopus Google Scholar). The capacity of human enzymes to oxidize has been D. Guengerich F.P. Chem. Res. Toxicol. PubMed Scopus Google Scholar). was readily by to and CYP1A2 oxidation of was for to The for in the P450 1A2 structure that the site of CYP1A2 oxidation at the other of the to the site of oxidation. The of oxidation the binding of in a for oxidation and a of the These are to contribute to the binding of P450 1A2 by substrate binding cavity is and is by on and that a relatively planar substrate binding on as the heme prosthetic group. a the of the substrate binding cavity a relatively of the in a for the the and the The of a from the of on the of to this the other of the the of on a substrate binding The of as the substrate binding cavity contributes to the cavity only is the of the to a active site the in also for the with The of the active site cavity is further by a between the of on with the of on at the of the and in an of molecules and including and these a binding cavity that with planar compounds such as and CYP1A2 substrates such as tacrine, olanzapine, arylamines, and In this the structure of the P450 2A6 active site is most with that of P450 1A2, which is in planar compounds (6Yano J.K. Hsu M.H. Griffin K.J. Stout C.D. Johnson E.F. Nat. Struct. Mol. Biol. 2005; 12: 822-823Crossref PubMed Scopus (290) Google Scholar), the differences in active site well with the substrate P450 1A2 is in that the enzyme has a which the of a bound as the to the site of the heme The relatively substrate binding cavity is to reduce the of to the cavity in a binding The of in the structure of P450 1A2 in complex with further to this that the of has on the P. T. Guengerich F.P. PubMed Scopus Google Scholar). acid for and have been to CYP1A2 enzymes that a The and Guengerich F.P. Biochemistry. 2000; PubMed Scopus Google have the potential to reduce between that the compact, closed active site to that the binding of to the heme In these three a catalytic of an on active site for of and which a catalytic activity in the on the catalytic of in substrate site J. Biol. Chem. Full Text PDF PubMed Google on the substrate used in the activity The of A. Guengerich F.P. Biochemistry. PubMed Scopus Google can based on the in the active site cavity and to prominent role in binding a wide range of substrates. the generally the catalytic of the the catalytic for the of by the is to that of the CYP1A2 the exhibits a for A. Guengerich F.P. Biochemistry. PubMed Scopus Google Scholar). This is to the of the on substrate binding as well as the catalytic of the enzyme. The the importance of at this for the and of the P450 J. Biol. Chem. Full Text PDF PubMed Google Scholar). was that the the of the Guengerich F.P. Biochemistry. 2000; PubMed Scopus Google Scholar). with the role of in that the substrate binding cavity to CYP1A2 at this that are relatively for most substrates. Human P450 family 1 enzymes for which the of substrates and is of great the planar active site topology in the P450 1A2 which is well adapted for the oxidation of relatively aromatic compounds, is to among the family 1 the the active site cavity in the P450 1A2 structure are and an are among human family 1 enzymes in the enzyme active site in the of for the of CYP1A2 a for the and CYP1A2 J. M. D. 2004; 424: PubMed Scopus Google Scholar). In the between and and of is only and which demonstrates the at the of the CYP1A2 active site cavity and the of CYP1A2 for in that are to have more on P450 family 1 active site are the of and in the is that the of in the P450 1A2 structure is not a of P450 family 1 The plays a role in the of by in with molecules that the by the of helical and by in the between and Human and CYP1B1 an at the of which as well as between and The of a in in a less active site The in the P450 1A2 structure is characterized by the of 3 polar and into the active site in to the more of the of amino the are unique to on Lewis and PubMed Scopus Google that and in to of The P450 1A2 structure that at for this is to The of to to CYP1A2 substrates such as caffeine contribute to substrate binding and positioning by a prominent role in that is to in the distal of the active site cavity binding of substrates. The importance of in substrate binding was also in the which significantly activity for and the enzyme J. M. D. 2004; 424: PubMed Scopus Google Scholar). In activity for of the CYP1A2 by to the The of with a on substrate binding and at the of the P450 1A2 active site also in the for N-hydroxylation of arylamines. on the aromatic and for ANF, such as and can in the P450 1A2 structure in a for in which the to the site of metabolism is for a with the of the active site the and active site the to an in to the and the potential for of the distal part of the active site cavity can identified as of human CYP1A2 The less P450 active site in which have less of an on substrate binding and is with the substrates that are more and than CYP1A2 substrates and the differences in catalytic for substrates with these human P450 1A2 structure exhibits a relatively planar substrate binding cavity that is highly adapted for the and of substrates that are by the enzyme. The structure in the of P450 1A2 for substrates that are of great importance in drug metabolism as well as in procarcinogen activation. The topology of the active site is characterized by on and that substrate binding on of the The unique active site demonstrates how P450 family 1 enzymes have evolved to catalyze efficiently polycyclic aromatic hydrocarbon oxidation and structural properties that a substrate binding site that has not been in P450 family 2 and 3 structures. The recent increase in the of available human P450 provides a understanding of how the diversity in the active site is and forms the basis for the capacity of these enzymes to oxidize an wide range of structurally distinct substrates. In this the first structure of a human microsomal P450 from family 1, in the basis of drug by human P450s and a for P450 1A2 for is for the the human CYP1A2 and Wester for and on the of the were at the a by on of the of of The is by the of of and and by the for and of the of with