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Cytochrome P450: cellular distribution and structural considerations
Cytochrome P450: cellular distribution and structural considerations Michael R. Waterman University of Texas Southwestern Medical Center, Dallas, Texas, USA The application of molecular biology to the study of cytochrome P450 has resulted in the elucidation of more than 150 primary sequences within this supeffamily of hemoproteins. Only a single tertiary structure, however, has been solved: that of the soluble bacterial cytochrome P450cam. Major questions that remain involve the topology of eukaryotic P450s in cellular membranes and whether or not amino acids that contact the substrate (camphor) in P450cam can be used as guideposts in the identification of substrate-binding sites in all other P450s. Current Opinion in Structural Biology 1992, 2:384~387
Introduction The cytochromes P450 are an extensive group of hemoprotein mixed-function oxidases found in bacteria, plants, insects, fishes, mammals and presumably all other forms of life. These enzymes play key roles in anabolic and catabolic pathways involving endogenous substrates, as well as in the metabolism of xenobiotic compounds derived from their environments. During the past decade, the primary sequences of more than 150 different forms of P450 have been elucidated, primarily by the application of the techniques of molecular biology. These sequences have been cataloged into evolutionarily defined gene families on the basis of their sequence relatedness [1o.]. P450s of all gene families have the common features of being mixed-function oxidases, thereby catalyzing reactions in which one atom of molecular oxygen is inserted into substrate and one atom is used in the generation of water, as well as demonstrating the unique absorbance at 450 nm in the reduced-CO form, which gives these b-type cytochromes their name. In spite of these common features, P450s demonstrate extraordinarily diverse substrate specificities and only a very few of the ,-~ 500 amino acids in these proteins are absolutely conserved. Thus, the diversity of the substrate specificity of these enzymes lies in the variability of their primary sequences, and an important area of investigation of these hemoproteins is focused on understanding the basis of this catalytic diversity within the context of what is thought to be a relatively conserved tertiary structure.
Compartmentation of P450 complexes With the exception of a single form of P450 found in Bacillus megaterium [2], these hydroxylases require the
assistance of other proteins for their functions; proteins which transfer electrons from NADPH or NADH to the P450 heme iron. One broad format for classifying P450s is based on the nature of these electron-transfer proteins. Bacterial P450s, with the exception of the B. megaterium form cited above, as well as P450s localized in mitochondria use an electron-transport chain which consists of a flavoprotein and an iron-sulfur protein for the transfer of reducing equivalents: NAD(P)H
--+ FAD --+ 2Fe-2S --+ FlavoIron sulfur protein protein
All bacterial P450s identified to date are soluble, whereas the mitochondrial forms are integral proteins of the inner membrane. The other class of P450s are those targeted to the endoplasmic reticulum (ER). These P450s are integral m e r e brane proteins which require another integral membrane protein, the ubiquitous flavoprotein NADPH cytochrome P450 reductase, for their function: NAD(P)H
--+ FAD, FMN --+ P450 Flavoprotein
The vast majority of mammalian P450s belong to this class. Whereas certain P450s, such as P450IA1, a polycyclic aromatic hydrocarbon hydroxylase, have a wide tissue distribution, many other forms have been and will be found to be localized to only a few cell types or even to a single cell type. It may be predicted that as many as 200 distinct forms of P450 will be found in humans, of which approximately 30 are presently known.
Abbreviation ER--endoplasmic reticulum.
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Cytochrome P450: cellular distribution and structural considerations Waterman Association of P450s with membranes One can imagine at least two reasons why eukaryotic P450s are located in membranes: their substrates are often quite lipophilic, such as cholesterol or polycyclic aromatic hydrocarbons; and, P450s require reductases for their function, and membrane association could favor this interaction. The P450s located in the ER are quite hydrophobic. Nevertheless, they contain only a single stretch of sequence which can be ascribed as membrane-spanning. This is located at the amino terminus and although not highly conserved amongst different P450s, is very hydrophobic in all cases. Careful examination of the location of specific peptide sequences within P450 in the ER membrane using anti-peptide antibodies [3] has revealed that most of the protein resides on the cytoplasmic side of the membrane. It is also clear that microsomal P450s find their way to the ER membrane via the signal recognition particle pathway [4], and that the hydrophobic amino terminus plays a key role not only in anchoring the protein to the membrane but also in the subsequent folding of the protein leading to a functional enzyme [5]. The precise location of the amino-terminal residue (luminal or cytosolic) is still a point of some controversy [6 o°] but it is clear that the amino-terminal signal anchor region of these P450s plays a major role in establishing their topology. Presumably, because of their hydrophobic nature, additional regions of the primary sequence are also involved in association of P450s with the cytoplasmic side of the membrane and may even be imbedded in the membrane, but there appear to be no other membrane-spanning sequences apart from that at the amino terminus. The location of a majority of the tertiary structure on the cytoplasmic surface of the ER membrane is also thought to be true for the flavoprotein redox partner, NADPH cytochrome P450 reductase. Although the membrane anchors of these two proteins may interact within the membrane, their func~ tional domains must interact on the cytoplasmic side of the ER. The stoichiometry of these complexes is unknown. Generally, there are more P450 molecules in ER membranes than there are P450 reductase molecules, in some tissues as many as ten times more. Thus, one reductase molecule appears to service several P450 molecules. How these complexes are arranged and whether or not more than one form of P450 exists in a single complex are unanswered questions. Mitochondrial forms of P450, on the other hand, do not have hydrophobic amino termini. Being derived from nuclear genes, these enzymes contain amino-terminal precursor sequences which are cleaved upon uptake by mitochondria to yield their mature forms. It is presumed that the targeting and uptake of these precursor P450s follows the same pathway as do other nuclear-encoded mitochondrial proteins, and it appears that the protease that cleaves the precursor sequence is similar or identical to that involved in the processing of other mitochondrial proteins [7]. The P450s are localized in the inner mitochondrial membrane. Furthermore, the flavoprotein and iron-sulfur protein required for their function are soluble proteins of the mitochondrial matrix. Accordingly, one can predict that a portion of the mitochondrial P450s, including the binding site for the iron-sulfur protein,
is located on the matrix side of the inner mitochondrial membrane. The membrane anchor(s) for these forms of P450 is unknown, however, as membrane-spanning sequences are not obvious in the primary structure. It is apparent, nonetheless, that anchors do exist as a result of the requirement for solubilization of the inner membrane before purification of mitochondrial P450s. From the above discussion, it is evident that many aspects of the cell biology of P450s are unknown. Precisely what topology they assume within cellular membranes is also not known. Because the eukaryotic P450s are membrane proteins, purification and structural analysis of even the most abundant forms have been difficult. Thus, the only high-resolution P450 structure that has been determined is that of a soluble form from Psuedomonas putida (P450cam) [8,9°o], which conver*s camphor to 5-exo hydroxycamphor. This protein possesses considerable helical character (70%) and contains a heme group which is buried within the tertiary structure. P450cam consists of 420 amino acids whereas most eukaryotic P450s contain ~ 500 amino acids. The hydrophobic amino-terminal sequence is, of course, not present in soluble bacterial forms of P450, and it is hypothesized that P450s in the ER assume a tertiary structure similar to that of P450cam, being anchored to the cytoplasmic side of the membrane by the hydrophobic membranespanning sequence [6.°]. In addition, the crucial step in their folding pathways leading to functional hydroxylases, the binding of heme, remains a mystery. At least in the case of the mitochondrial P450s, the availability of heme in the mitochondrial matrix is obvious. The microsomal forms are anchored in the ER by their amino termini, and only after complete or verg nearly complete polypeptide synthesis is the carboxy-terminal heme-binding site revealed. Thus, how heme finds its way to the cytoplasmic surface of the ER, where it binds to the apoP450 protein in a process which must be important in contributing to the final topology of the enzyme, is an important question that awaits investigation.
P450 structure-function relationships Several investigators have reported alignments of primary sequences of eukaryotic forms of P450 with those of P450cam and other bacterial forms of P450 by including gaps within the alignments, e.g. [10-12,13°',14°']. The focus of such efforts has been to locate regions of sequence homology amongst the different P450 gene families which can assist in the identification of amino acids that interact with their specific reductases or that are required for substrate binding. It is evident from these efforts that a high degree of sequence diversity exists between the different P450s, which must account for the catalytic diversity associated with this superfamily of enzymes. Inspection of alignments of eukaryotic P450s from different species and different gene families reveals no more than ten amino acids which are conserved in all forms; several of these including the heme-binding cysteine being associated with the heme pocket. It is the thiolate fifth-coordination ligand to the heme iron, which is provided by the conserved cysteine located near the carboxyl terminus, that is responsible for the
385
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Sequences and topology unique absorbance at 450 nm in the reduced-CO form. This coordination structure also provides for the unusually negative redox potential of these &type cytochromes which permits reduction of molecular oxygen. Chemical-modification studies of one mitochondrial P450, P450XIA (cholesterol side-chain cleavage cytochrome P450), have identified specific positively charged amino acids which apparently interact with negatively charged amino acids on the iron-sulfur protein adrenodoxin [15,16]. These residues lie in a positively charged region which is conserved amongst other mitochondrial P450s, and presumably contains the adrenodoxin-binding site. The location of the reductaseinteracting site in microsomal forms of cytochrome P450 has not yet been clearly established. The search for residues involved in substrate binding has included the construction of mutagenic/chimeric forms of different P450s as well as modeling of the substratebinding site or the whole protein on the basis of the known tertiary structure of the bacterial P450cam [17"]. This structure is known to 1.63 A and the specific residues that interact with the substrate camphor are well defined [8,9"]. In addition to the intrinsic interest in understanding the variability in the active site of these enzymes, which metabolize a wide variety of substrates, the possibifity of producing 'designer' forms of P450 which catalyze specific reactions of commercial interest, as well as the generation of P450 inhibitors of clinical importance, such as for aromatase cytochrome P450 [18], also serve as driving forces for such efforts. Particularly informative data on key elements of eukaryotic P450 substrate-binding regions have been obtained from the study of forms having closely related sequences, i.e. members of the same P450 subfamily. Construction of chimeric proteins between such closely related P450s upq~ expression in heterologous systems having low or undetectable background P450 activities has located specific amino acid residues which play important roles in certain enzymatic activities. For example, in the rabbit P450IIC subfamily, low-Km 21-hydroxylation of progesterone depends on the identity of residues 113 (valine), 115 (serine) and 118 (lysine) [19"]. Identification of a key residue in a different region of a different P450 has been achieved using site-directed mutagenesis based on the same principal of investigating closely related forms. A single amino acid can change the substrate specificity in this instance. Changing Phe209 to leucine is sufficient to convert the specificity of mouse coumarin hydroxylase (P450IIA5) to steroid 15a-hydroxytase [20]. This particular experiment dramatically demonstrates that a single amino acid residue can control the enzymatic specificity of a specific form of P450. The aggregate of several studies using chimeric proteins and site-directed mutagenesis indicates, as would be expected, that several different regions within the primary sequence are involved in substrate binding. Sequence alignment of these regions favors the view that the P450cam tertiary structure may be representative of eukaryotic P450s as well, at least in the region around the active site. This view is challenged, however, by the fact that residue 209 of P450IIA5, cited above as being a crucial deter-
minant of substrate specificity, is predicted to reside in the E helix near the surface of the protein, on the basis of one alignment with P450cam. Studies of additional mutations at this site show that they alter the spin-state of the heme iron, suggesting a close proximity for residue 209 to the heme group on the interior of the protein [21..]. Thus, we are left with a dilemma. Do we require alternative amino acid sequence alignments in order to better approximate the relationship of eukaryotic P450s to P450cam? Is the structure of P450cam sufficiently unusual that it can not be used to reliably model all the important features of substrate binding in eukaryotic P450s? An obvious solution to this dilemma will be the generation of additional P450 tertiary structures. We can anticipate that additional bacterial P450 tertiary structures will soon be reported [22,23] and this information will greatly assist in the evaluation of alternative sequence alignments as well as of results obtained from the investigation of mutagenic/chimeric forms of P450. However, the tertiary structure of a eukaryotic P450, which is required for final resolution of this dilemma, would seem to lie several years in the future.
Conclusions Although a great deal is known about P450 primary sequences, we know little about their tertiary structure. All structural predictions are based on the single high-resolution structure known, that of soluble bacterial P450cam, and it is not yet obvious what portions of this structure are representative of this superfamily of proteins. The inescapable need for additional tertiary structure information on other forms of P450 represents the key experimental challenge in this field. An additional challenge is the elucidation of the topology of eukaryotic P450s in cellular membranes, and the cell biology of the formation of complexes between the P450s and their necessary reductase proteins. Only upon successfully meeting these challenges will a detailed understanding of the diversity of catalytic activities found in this superfamily of mixedfunction oxidases become evident.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: . of special interest .. of outstanding interest 1. ..
NEBERTDW, NELSONDR, COON MJ, ESTABROOKRW, FEYEREISEN R, FUJII-KURIYAMAY, GONZALEZ FJ, GUENGERICH FP, GUNSALUS IC, JOHNSON ER, ET AZ.: The P450 Superfamily: Update on N e w Sequences, Gene Mapping, and R e c o m m e n d e d Nomenclature. DNA Cell Biol 1991, 10:1--4. This is the third and most recent paper in an ongoing series that cataloges the sequences of P450 from all sources into gene families and a consistent nomenclature for describing members of this gene super-
fami>. 2.
RUET'nNGERRT, WEN L-P, FUtCO AJ: Coding Nucleotide, 5' Regulatory and D e d u c e d Amino Acid Sequences of P450BM-3, a Single Peptide Cytocin'ome P450:NADPH P450 Reductase from Bacillus megaterium. J Biol Chem 1989, 264:10987-10995.
3.
DELOMoS-CHIARANDINIC, FREY AB, SABATINI DD, KREmlCH G: Determination of the Membrane Topology of the
Cytochrome P450: cellular distribution and structural considerations Waterman Phenobarbital-inducible Rat Liver Cytochrome P450 Isozyme PB-4 using Site-specific Antibodies. J Cell Biol 1987, 104:209-219. 4.
5.
SAKAGUCHIM, MIHARAK, SATO R: Signal Recognition Particle is Required for Co-translational Insertion of Cytochrome P450 into Microsomal Membranes. Proc Natl Acad Sci USA 1984, 81:3361-3364. CLARKBJ, WATERMANMI~ The Hydrophobic Amino Terminal Sequence of Bovine 17ct-Hydroxylase is Required for the Expression of a Functional Hemoprotein in COS1 Cells. J Biol Chem 1991, 266:5898-5904.
6. BLACKSB: Membrane Topology of the Mammalian P450 Cy°, tochromes. FASEB J 1992, 6:680485. A timely review of present knowledge and hypotheses of association of eukaryotic P450s with cellular membranes. 7.
8.
OMURAT, ITO A: Biosynthesis and Intracellular Sorting of Mitochondrial Forms of Cytochrome P450. Methods Enzymol 1991, 206:75-81.
POULOSTL, RAAGR: Cytochrome P450cam: Crystallography, Oxygen Activation and Electron Transfer. FASEB J 1992, 6:6744579. This article summarizes the crystal structures of several complexes of P450cam~ the product of the CYPI01 gene, and focuses on how P450s activate oxygen and control stereospecificity.
11.
12.
NELSONDR, STROBELI-{V¢':On the Membrane Topology of Vertebrate Cytochrome P450 Proteins. J Biol Chem 1988, 263:6038-6050. GOTOHO, FUJU-KuPaYAMAY: Evolution, Structure, and Gene Regulation of Cytochrome P450. In Frontiers in Biotran~ formatton. Edited by Ruckpaul K, Rein H. Berlin: AkademieVerlag; 1989; 1:196-237. KAIB VF, LOPERJC: Proteins from Eight Cytochrome P450 Families Share a Segmented Region of sequence Similarity. Proc Natl Acad Sci USA 1988, 85:7221-7225.
13. °.
OUZOUNISCA, MELVINWT: Primary and secondary Structural Patterns in Eukaryotic Cytochrome P450 Families Correspond to Structures of the Helix-rich Domain of Pseudomonas putida Cytochrome P450cam. Eur J Biochem 1991, 198:307-315. Upon sequence analysis of all P450 sequences in GenBank 56 and EMBL 15 coupled with secondary structure prediction, these authors conclude that much of the helical region of P450cam is conserved in eukaryotic P450s. This paper discusses the potential importance of the various helical segments in P450. 14. °°
Tuts J, GEREN L, MILLETr F: Fluorescein Isothiocyanate Specifically Modifies Lysine 338 of Cytochrome P450scc and Inhibits Adrenodoxin Binding. J Biol Cbem 1989, 264:16421-16425.
16.
TSUBAYOM, IWAMOTOY, HIWATASH1A, ICHIKAWAY: Inhibition of Electron Transfer from Adrenodoxin to Cytochrome P450scc by Chemical Modification with Pyridoxal 5'Phosphate: Identification of Adrenodoxin Binding Site of Cytochrome P45Oscc. Bit~.hemistry 1989, 28:6899-6907.
17. .
ZVEBEBILMJJM, WOLF R, STERNBERGMJE: A Predicted Threedimensional Structure of Human Cytochrome P450: Implications for Substrate Specificity. Protein Eng 1991, 4:271-282. This paper describes the predicted tertiary structure of human P450IAI using the crystal coordinates of P450cam and the sequence alignment of 12 representative eukaryotic P450s. 18.
POULOSTL, FINZEL BC, HOWARD AJ: High-resolution Crystal Structure of Cytochrome P450cam. J Mol Biol 1987, 195:687-700.
9. oo
10.
15.
GOTOHO: Substrate Recognition Sites in Cytochrome P450 Family 2 (CYP2) Proteins Inferred from Comparative Analysis of Amino Acid and Coding Nucleotides. J Biol Chem 1992, 267:83-90. The most recent sequence alignment, which exclusively examines the CYP2gene family, the largest of the P450 gene families. This author predicts six substrate-recognition sites within members of the CYP2gene family on the basis of sequence alignments with P450cam.
GRAHAM-LORENCE S, KAHI ~ , LORENCE MC, MENDELSON CR, SIMPSON ER: Structure-Function Relationships of Human Aromatase Cytochrome P450 Using Molecular Modeling and Site-directed Mutagenesis. J Biol Chem 1991, 266:11939-11946.
JOHNSONEF, KRONBACHT, HSU M-H: Analysis of the Catalytic Specificity of Cytochrome P450 Enzymes through Site-directed Mutagenesis. FASEB J 1992, 6:700-705. An articulate description of the use of chimeras and site-directed mutagenesis in the investigation of P450 enzymatic activities. The advantage of utilizing closely related P450s from the same subfamily is evident from this paper. 19. .
20.
LINDBERGRLP, NEGISHI M: Alteration of Mouse Cytochrome P450coh Substrate Specificity by Mutation of a Single Amino-acid Residue. Nature 1989, 339:632434.
21. o.
IWASAKIM, JUSONEN R, LINDBERGR, NEGISHI M: Alteration of High and Low Spin Equilibrium by a Single Mutation of Amino Acid 209 by Mouse Cytochromes P450. J Biol Chem 1991, 266:3380-3382. , This study illustrates, by the use of site-directed mutagenesis and spectral analysis of the mutants, the concern as to whether the tertiary structure of P450cam and a single primary sequence alignment can be used to describe all P450s. 22.
LI H, DARXWSH K, POULOS TL: Characterization of Recombinant Bacillus megaterium Cytochrome P450BM-3 and its Two Functional Domains. J Biol Chem 1991, 266:11900-11914.
23.
BODDUPALUSS, HASEMANN CA, RAVICHANDRANKG, LY J-Y, GOLDSMITH EJ, DEISENHOFERJ, PETERSON JA~ Crystallization and Preliminary X-ray Diffraction Analysis of P45Oterp and the Hemoprotein Domain of P450BM-3. Proc Natl Acad Sci USA 1992, in press.
MR Waterman, Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235, USA.
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Introduction The cytochromes P450 are an extensive group of hemoprotein mixed-function oxidases found in bacteria, plants, insects, fishes, mammals and presumably all other forms of life. These enzymes play key roles in anabolic and catabolic pathways involving endogenous substrates, as well as in the metabolism of xenobiotic compounds derived from their environments. During the past decade, the primary sequences of more than 150 different forms of P450 have been elucidated, primarily by the application of the techniques of molecular biology. These sequences have been cataloged into evolutionarily defined gene families on the basis of their sequence relatedness [1o.]. P450s of all gene families have the common features of being mixed-function oxidases, thereby catalyzing reactions in which one atom of molecular oxygen is inserted into substrate and one atom is used in the generation of water, as well as demonstrating the unique absorbance at 450 nm in the reduced-CO form, which gives these b-type cytochromes their name. In spite of these common features, P450s demonstrate extraordinarily diverse substrate specificities and only a very few of the ,-~ 500 amino acids in these proteins are absolutely conserved. Thus, the diversity of the substrate specificity of these enzymes lies in the variability of their primary sequences, and an important area of investigation of these hemoproteins is focused on understanding the basis of this catalytic diversity within the context of what is thought to be a relatively conserved tertiary structure.
Compartmentation of P450 complexes With the exception of a single form of P450 found in Bacillus megaterium [2], these hydroxylases require the
assistance of other proteins for their functions; proteins which transfer electrons from NADPH or NADH to the P450 heme iron. One broad format for classifying P450s is based on the nature of these electron-transfer proteins. Bacterial P450s, with the exception of the B. megaterium form cited above, as well as P450s localized in mitochondria use an electron-transport chain which consists of a flavoprotein and an iron-sulfur protein for the transfer of reducing equivalents: NAD(P)H
--+ FAD --+ 2Fe-2S --+ FlavoIron sulfur protein protein
All bacterial P450s identified to date are soluble, whereas the mitochondrial forms are integral proteins of the inner membrane. The other class of P450s are those targeted to the endoplasmic reticulum (ER). These P450s are integral m e r e brane proteins which require another integral membrane protein, the ubiquitous flavoprotein NADPH cytochrome P450 reductase, for their function: NAD(P)H
--+ FAD, FMN --+ P450 Flavoprotein
The vast majority of mammalian P450s belong to this class. Whereas certain P450s, such as P450IA1, a polycyclic aromatic hydrocarbon hydroxylase, have a wide tissue distribution, many other forms have been and will be found to be localized to only a few cell types or even to a single cell type. It may be predicted that as many as 200 distinct forms of P450 will be found in humans, of which approximately 30 are presently known.
Abbreviation ER--endoplasmic reticulum.
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Cytochrome P450: cellular distribution and structural considerations Waterman Association of P450s with membranes One can imagine at least two reasons why eukaryotic P450s are located in membranes: their substrates are often quite lipophilic, such as cholesterol or polycyclic aromatic hydrocarbons; and, P450s require reductases for their function, and membrane association could favor this interaction. The P450s located in the ER are quite hydrophobic. Nevertheless, they contain only a single stretch of sequence which can be ascribed as membrane-spanning. This is located at the amino terminus and although not highly conserved amongst different P450s, is very hydrophobic in all cases. Careful examination of the location of specific peptide sequences within P450 in the ER membrane using anti-peptide antibodies [3] has revealed that most of the protein resides on the cytoplasmic side of the membrane. It is also clear that microsomal P450s find their way to the ER membrane via the signal recognition particle pathway [4], and that the hydrophobic amino terminus plays a key role not only in anchoring the protein to the membrane but also in the subsequent folding of the protein leading to a functional enzyme [5]. The precise location of the amino-terminal residue (luminal or cytosolic) is still a point of some controversy [6 o°] but it is clear that the amino-terminal signal anchor region of these P450s plays a major role in establishing their topology. Presumably, because of their hydrophobic nature, additional regions of the primary sequence are also involved in association of P450s with the cytoplasmic side of the membrane and may even be imbedded in the membrane, but there appear to be no other membrane-spanning sequences apart from that at the amino terminus. The location of a majority of the tertiary structure on the cytoplasmic surface of the ER membrane is also thought to be true for the flavoprotein redox partner, NADPH cytochrome P450 reductase. Although the membrane anchors of these two proteins may interact within the membrane, their func~ tional domains must interact on the cytoplasmic side of the ER. The stoichiometry of these complexes is unknown. Generally, there are more P450 molecules in ER membranes than there are P450 reductase molecules, in some tissues as many as ten times more. Thus, one reductase molecule appears to service several P450 molecules. How these complexes are arranged and whether or not more than one form of P450 exists in a single complex are unanswered questions. Mitochondrial forms of P450, on the other hand, do not have hydrophobic amino termini. Being derived from nuclear genes, these enzymes contain amino-terminal precursor sequences which are cleaved upon uptake by mitochondria to yield their mature forms. It is presumed that the targeting and uptake of these precursor P450s follows the same pathway as do other nuclear-encoded mitochondrial proteins, and it appears that the protease that cleaves the precursor sequence is similar or identical to that involved in the processing of other mitochondrial proteins [7]. The P450s are localized in the inner mitochondrial membrane. Furthermore, the flavoprotein and iron-sulfur protein required for their function are soluble proteins of the mitochondrial matrix. Accordingly, one can predict that a portion of the mitochondrial P450s, including the binding site for the iron-sulfur protein,
is located on the matrix side of the inner mitochondrial membrane. The membrane anchor(s) for these forms of P450 is unknown, however, as membrane-spanning sequences are not obvious in the primary structure. It is apparent, nonetheless, that anchors do exist as a result of the requirement for solubilization of the inner membrane before purification of mitochondrial P450s. From the above discussion, it is evident that many aspects of the cell biology of P450s are unknown. Precisely what topology they assume within cellular membranes is also not known. Because the eukaryotic P450s are membrane proteins, purification and structural analysis of even the most abundant forms have been difficult. Thus, the only high-resolution P450 structure that has been determined is that of a soluble form from Psuedomonas putida (P450cam) [8,9°o], which conver*s camphor to 5-exo hydroxycamphor. This protein possesses considerable helical character (70%) and contains a heme group which is buried within the tertiary structure. P450cam consists of 420 amino acids whereas most eukaryotic P450s contain ~ 500 amino acids. The hydrophobic amino-terminal sequence is, of course, not present in soluble bacterial forms of P450, and it is hypothesized that P450s in the ER assume a tertiary structure similar to that of P450cam, being anchored to the cytoplasmic side of the membrane by the hydrophobic membranespanning sequence [6.°]. In addition, the crucial step in their folding pathways leading to functional hydroxylases, the binding of heme, remains a mystery. At least in the case of the mitochondrial P450s, the availability of heme in the mitochondrial matrix is obvious. The microsomal forms are anchored in the ER by their amino termini, and only after complete or verg nearly complete polypeptide synthesis is the carboxy-terminal heme-binding site revealed. Thus, how heme finds its way to the cytoplasmic surface of the ER, where it binds to the apoP450 protein in a process which must be important in contributing to the final topology of the enzyme, is an important question that awaits investigation.
P450 structure-function relationships Several investigators have reported alignments of primary sequences of eukaryotic forms of P450 with those of P450cam and other bacterial forms of P450 by including gaps within the alignments, e.g. [10-12,13°',14°']. The focus of such efforts has been to locate regions of sequence homology amongst the different P450 gene families which can assist in the identification of amino acids that interact with their specific reductases or that are required for substrate binding. It is evident from these efforts that a high degree of sequence diversity exists between the different P450s, which must account for the catalytic diversity associated with this superfamily of enzymes. Inspection of alignments of eukaryotic P450s from different species and different gene families reveals no more than ten amino acids which are conserved in all forms; several of these including the heme-binding cysteine being associated with the heme pocket. It is the thiolate fifth-coordination ligand to the heme iron, which is provided by the conserved cysteine located near the carboxyl terminus, that is responsible for the
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Sequences and topology unique absorbance at 450 nm in the reduced-CO form. This coordination structure also provides for the unusually negative redox potential of these &type cytochromes which permits reduction of molecular oxygen. Chemical-modification studies of one mitochondrial P450, P450XIA (cholesterol side-chain cleavage cytochrome P450), have identified specific positively charged amino acids which apparently interact with negatively charged amino acids on the iron-sulfur protein adrenodoxin [15,16]. These residues lie in a positively charged region which is conserved amongst other mitochondrial P450s, and presumably contains the adrenodoxin-binding site. The location of the reductaseinteracting site in microsomal forms of cytochrome P450 has not yet been clearly established. The search for residues involved in substrate binding has included the construction of mutagenic/chimeric forms of different P450s as well as modeling of the substratebinding site or the whole protein on the basis of the known tertiary structure of the bacterial P450cam [17"]. This structure is known to 1.63 A and the specific residues that interact with the substrate camphor are well defined [8,9"]. In addition to the intrinsic interest in understanding the variability in the active site of these enzymes, which metabolize a wide variety of substrates, the possibifity of producing 'designer' forms of P450 which catalyze specific reactions of commercial interest, as well as the generation of P450 inhibitors of clinical importance, such as for aromatase cytochrome P450 [18], also serve as driving forces for such efforts. Particularly informative data on key elements of eukaryotic P450 substrate-binding regions have been obtained from the study of forms having closely related sequences, i.e. members of the same P450 subfamily. Construction of chimeric proteins between such closely related P450s upq~ expression in heterologous systems having low or undetectable background P450 activities has located specific amino acid residues which play important roles in certain enzymatic activities. For example, in the rabbit P450IIC subfamily, low-Km 21-hydroxylation of progesterone depends on the identity of residues 113 (valine), 115 (serine) and 118 (lysine) [19"]. Identification of a key residue in a different region of a different P450 has been achieved using site-directed mutagenesis based on the same principal of investigating closely related forms. A single amino acid can change the substrate specificity in this instance. Changing Phe209 to leucine is sufficient to convert the specificity of mouse coumarin hydroxylase (P450IIA5) to steroid 15a-hydroxytase [20]. This particular experiment dramatically demonstrates that a single amino acid residue can control the enzymatic specificity of a specific form of P450. The aggregate of several studies using chimeric proteins and site-directed mutagenesis indicates, as would be expected, that several different regions within the primary sequence are involved in substrate binding. Sequence alignment of these regions favors the view that the P450cam tertiary structure may be representative of eukaryotic P450s as well, at least in the region around the active site. This view is challenged, however, by the fact that residue 209 of P450IIA5, cited above as being a crucial deter-
minant of substrate specificity, is predicted to reside in the E helix near the surface of the protein, on the basis of one alignment with P450cam. Studies of additional mutations at this site show that they alter the spin-state of the heme iron, suggesting a close proximity for residue 209 to the heme group on the interior of the protein [21..]. Thus, we are left with a dilemma. Do we require alternative amino acid sequence alignments in order to better approximate the relationship of eukaryotic P450s to P450cam? Is the structure of P450cam sufficiently unusual that it can not be used to reliably model all the important features of substrate binding in eukaryotic P450s? An obvious solution to this dilemma will be the generation of additional P450 tertiary structures. We can anticipate that additional bacterial P450 tertiary structures will soon be reported [22,23] and this information will greatly assist in the evaluation of alternative sequence alignments as well as of results obtained from the investigation of mutagenic/chimeric forms of P450. However, the tertiary structure of a eukaryotic P450, which is required for final resolution of this dilemma, would seem to lie several years in the future.
Conclusions Although a great deal is known about P450 primary sequences, we know little about their tertiary structure. All structural predictions are based on the single high-resolution structure known, that of soluble bacterial P450cam, and it is not yet obvious what portions of this structure are representative of this superfamily of proteins. The inescapable need for additional tertiary structure information on other forms of P450 represents the key experimental challenge in this field. An additional challenge is the elucidation of the topology of eukaryotic P450s in cellular membranes, and the cell biology of the formation of complexes between the P450s and their necessary reductase proteins. Only upon successfully meeting these challenges will a detailed understanding of the diversity of catalytic activities found in this superfamily of mixedfunction oxidases become evident.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: . of special interest .. of outstanding interest 1. ..
NEBERTDW, NELSONDR, COON MJ, ESTABROOKRW, FEYEREISEN R, FUJII-KURIYAMAY, GONZALEZ FJ, GUENGERICH FP, GUNSALUS IC, JOHNSON ER, ET AZ.: The P450 Superfamily: Update on N e w Sequences, Gene Mapping, and R e c o m m e n d e d Nomenclature. DNA Cell Biol 1991, 10:1--4. This is the third and most recent paper in an ongoing series that cataloges the sequences of P450 from all sources into gene families and a consistent nomenclature for describing members of this gene super-
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RUET'nNGERRT, WEN L-P, FUtCO AJ: Coding Nucleotide, 5' Regulatory and D e d u c e d Amino Acid Sequences of P450BM-3, a Single Peptide Cytocin'ome P450:NADPH P450 Reductase from Bacillus megaterium. J Biol Chem 1989, 264:10987-10995.
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POULOSTL, RAAGR: Cytochrome P450cam: Crystallography, Oxygen Activation and Electron Transfer. FASEB J 1992, 6:6744579. This article summarizes the crystal structures of several complexes of P450cam~ the product of the CYPI01 gene, and focuses on how P450s activate oxygen and control stereospecificity.
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NELSONDR, STROBELI-{V¢':On the Membrane Topology of Vertebrate Cytochrome P450 Proteins. J Biol Chem 1988, 263:6038-6050. GOTOHO, FUJU-KuPaYAMAY: Evolution, Structure, and Gene Regulation of Cytochrome P450. In Frontiers in Biotran~ formatton. Edited by Ruckpaul K, Rein H. Berlin: AkademieVerlag; 1989; 1:196-237. KAIB VF, LOPERJC: Proteins from Eight Cytochrome P450 Families Share a Segmented Region of sequence Similarity. Proc Natl Acad Sci USA 1988, 85:7221-7225.
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OUZOUNISCA, MELVINWT: Primary and secondary Structural Patterns in Eukaryotic Cytochrome P450 Families Correspond to Structures of the Helix-rich Domain of Pseudomonas putida Cytochrome P450cam. Eur J Biochem 1991, 198:307-315. Upon sequence analysis of all P450 sequences in GenBank 56 and EMBL 15 coupled with secondary structure prediction, these authors conclude that much of the helical region of P450cam is conserved in eukaryotic P450s. This paper discusses the potential importance of the various helical segments in P450. 14. °°
Tuts J, GEREN L, MILLETr F: Fluorescein Isothiocyanate Specifically Modifies Lysine 338 of Cytochrome P450scc and Inhibits Adrenodoxin Binding. J Biol Cbem 1989, 264:16421-16425.
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TSUBAYOM, IWAMOTOY, HIWATASH1A, ICHIKAWAY: Inhibition of Electron Transfer from Adrenodoxin to Cytochrome P450scc by Chemical Modification with Pyridoxal 5'Phosphate: Identification of Adrenodoxin Binding Site of Cytochrome P45Oscc. Bit~.hemistry 1989, 28:6899-6907.
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ZVEBEBILMJJM, WOLF R, STERNBERGMJE: A Predicted Threedimensional Structure of Human Cytochrome P450: Implications for Substrate Specificity. Protein Eng 1991, 4:271-282. This paper describes the predicted tertiary structure of human P450IAI using the crystal coordinates of P450cam and the sequence alignment of 12 representative eukaryotic P450s. 18.
POULOSTL, FINZEL BC, HOWARD AJ: High-resolution Crystal Structure of Cytochrome P450cam. J Mol Biol 1987, 195:687-700.
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GOTOHO: Substrate Recognition Sites in Cytochrome P450 Family 2 (CYP2) Proteins Inferred from Comparative Analysis of Amino Acid and Coding Nucleotides. J Biol Chem 1992, 267:83-90. The most recent sequence alignment, which exclusively examines the CYP2gene family, the largest of the P450 gene families. This author predicts six substrate-recognition sites within members of the CYP2gene family on the basis of sequence alignments with P450cam.
GRAHAM-LORENCE S, KAHI ~ , LORENCE MC, MENDELSON CR, SIMPSON ER: Structure-Function Relationships of Human Aromatase Cytochrome P450 Using Molecular Modeling and Site-directed Mutagenesis. J Biol Chem 1991, 266:11939-11946.
JOHNSONEF, KRONBACHT, HSU M-H: Analysis of the Catalytic Specificity of Cytochrome P450 Enzymes through Site-directed Mutagenesis. FASEB J 1992, 6:700-705. An articulate description of the use of chimeras and site-directed mutagenesis in the investigation of P450 enzymatic activities. The advantage of utilizing closely related P450s from the same subfamily is evident from this paper. 19. .
20.
LINDBERGRLP, NEGISHI M: Alteration of Mouse Cytochrome P450coh Substrate Specificity by Mutation of a Single Amino-acid Residue. Nature 1989, 339:632434.
21. o.
IWASAKIM, JUSONEN R, LINDBERGR, NEGISHI M: Alteration of High and Low Spin Equilibrium by a Single Mutation of Amino Acid 209 by Mouse Cytochromes P450. J Biol Chem 1991, 266:3380-3382. , This study illustrates, by the use of site-directed mutagenesis and spectral analysis of the mutants, the concern as to whether the tertiary structure of P450cam and a single primary sequence alignment can be used to describe all P450s. 22.
LI H, DARXWSH K, POULOS TL: Characterization of Recombinant Bacillus megaterium Cytochrome P450BM-3 and its Two Functional Domains. J Biol Chem 1991, 266:11900-11914.
23.
BODDUPALUSS, HASEMANN CA, RAVICHANDRANKG, LY J-Y, GOLDSMITH EJ, DEISENHOFERJ, PETERSON JA~ Crystallization and Preliminary X-ray Diffraction Analysis of P45Oterp and the Hemoprotein Domain of P450BM-3. Proc Natl Acad Sci USA 1992, in press.
MR Waterman, Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235, USA.
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