Analysis of the catalytic specificity of cytochrome P450 enzymes through site-directed mutagenesis.

Analysis

of the catalytic

enzymes ERIC

through

F. JOHNSON,’

The Scripps Department

specificity

site-directed

THOMAS

KRONBACH

Key

Words:

enzyme

chrome P450

engineering

progesterone

site-directed

mutagenesis

cyto-

21-hydroxylation

SUCCESSFUL RESOLUTION AND RECONSTITUTION of cytochrome P4502 and P450 reductase by Lu and Coon and Lu et al. (2, 3), and subsequent purification of the major phenobarbital-inducible form of P450 (GYP2B4) to homogeneity by van der Hoeven and co-workers (4, 5), ushered in a productive period in which multiple forms of cytochrome P450 were isolated from both rat and rabbit by several laboratories (6). The goal of many of these early isolation procedures was to purify and characterize P450s that constitute a major portion of the total amount in hepatic microsomes as a result of an increase in their concentration by chemical induction. However, additional P450s were isolated whose existence was not inferred from studies of P450 induction. The first P450s to be isolated often differed extensively in terms of immunoreactivity, partial sequence analysis, and peptide mapping. This apparent structural heterogeneity was subsequently confirmed as the amino acid sequences of these enzymes were determined and cDNAs encoding distinct P450s were characterized. Many of these enzymes were found to exhibit less than 40% amino acid identity. As the cloning of distinct cDNAs proceeded, it also became evident that P450 diversity was much greater than had been anticipated. A recent compilation (I) lists 24 rabbit, 27 human, and 37 rat P450 sequences, and there is evidence for even greater numbers. The greatest diversity became apparent for subfamily 2C where 23 members have been identified (1) and where additional genes are evident. The sequence identity ranges from 68 to 95% or more in this subfamily.

THE

RABBIT

SUBFAMILY

In the rabbit, been reported

700

mutagenesis

and MEl-HUT

HSU

Research Institute, Division of Biochemistr La Jolla, California 92037, of Drug Metabolism, Mooswaldallee 14, 7800 Freiburg, Germany

ABSTRACT The way in which structural diversity encodes the capacity of individual P450 enzymes to metabolize multiple, structurally distinct substrates remains largely unknown. The tools of molecular biology provide a means of identifying amino acid residues among closely related P450s that are determinants of their distinct catalytic properties. Work in our laboratory has identified two substrate specificity-determining segments of the amino acid sequences of subfamily 2C P450s. A pattern has emerged from this work, and that of others, which suggests a model for the structural basis of P450 catalytic diversity.-.Johnson, E. F.; Kronbach, T.; Hsu, M.-H. Analysis of the catalytic specificity of cytochrome P450 enzymes through site-directed mutagenesis. FASEB J. 6: 700-705; 1992.

2C

more than seven subfamily 2C sequences (Table 1). However, only two of these

have pro-

P450

of ytodirorne.

USA;

and ‘Goedecke

AG,

teins, 2C5 (7) and 2C3 (8, 9), had been purified (originally designated as form 1 and form 3b or LM3b, respectively). They exhibit roughly 68% amino acid identity (10). Like other hepatic P450s, 2C3 and 2C5 metabolize a varlety of foreign compounds (xenobiotics) (7, 8). 2C5 catalyzes high rates of benzo(a)pyrene (11) and 2-acetylaminofluorene hydroxylation (12), and 2C3 catalyzes aminopyrine Ndemethylation (8), oxidation of 1-nitropyrene (13), and phenytoin 4-hydroxylation (14) at relatively high rates. In addition, they each catalyze reactions characteristic of distantly related P450s. 2C3 is the predominant steroid 63hydroxylase in rabbit liver (15), whereas this reaction is largely catalyzed in rat and human liver by 3A enzymes that display less than 40% amino acid identity with 2C3 (16). A GYP 3A 6/3-hydroxylase is also expressed in rabbits, and it is induced by rifampicin (17, 18). This mimicry is also particularly striking for 2C5, which catalyzes the formation of deoxycorticosterone from progesterone by 2 1-hydroxylation at rates similar to that of the distantly related adrenal enzyme CYP2IA (7). Thus, very different P450s can exhibit the same catalytic activities. On the other hand, genetic polymorphisms that affect single P450 genes can profoundly affect the metabolism of some substrates. The low hepatic microsomal progesterone 21-hydroxylase activity in rabbits exhibiting a loss of 2C5 expression (19) suggests that other enzymes, including closely related CYF2C P450s, contribute little to hepatic microsomal progesterone 21-hydroxylase activity in untreated animals. The other rabbit CYP2C enzymes that have been characterized exhibit between 74 and 95% amino acid identity with 2C5 (Table 1). As these proteins had not been isolated and characterized before the cDNAs were identified, heterologous expression of the enzymes from their cDNAs has been used to characterize their enzymic activity, as shown in Table 1. These results and similar observations for other species suggest that small changes in protein sequence can lead to dramatic changes in substrate metabolism, and that duplica-

‘To whom

correspondence

should

be addressed,

at: Division

of

Biochemistry/BCR-7, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10666 N. Torrey Pines Rd., La Jolla, CA 92037, USA. 2Abbreviations and conventions used in this article: The generic term P450 is used to indicate a cytochrome P450. Individual forms of P450 are designated according to the uniform system of nomenclature (1) with the exception that the common name, P45Ocam, is used for CYPCI. In many cases, the CYP or P450 prefix has been dropped, i.e., CYP2C5 or P45011C5 is designated simply as 2C5. Mutations are indicated using the one-letter abbreviation for the amino acid residue that was replaced, its position in the sequence, and the one-letter designation of the new residue in the indicated order.

flRO7j.d.tO1a/nc

,-,,

ww.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on December 23, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum

TABLE

1. Relatedness

Enzyme

between

Identity

rabbit

CYP2C

enzymes and CYP2C5

100% 95%

(10) (39)

92%

(40)

76%

(41)

CYP2C1 CYP2C2

75% 72%

(35) (35)

CYP2C3

67%

CYP2C5 CYP2C4 CYP2CI6 CYF2C14

Regiospecificity

Reference

to 11C5

67% were obtained by expression

by comparing

the cited sequence

Unpublished’ of the recombinant

l6a,

OF CYTOCHROME

(42) (31) (32) Unpublished’ Unpublished’

detectable 16cr

16cr, high Km 6/3, 16cr, low Km in COS cells. CYP2C3v software

is described

later in this article.

Percent

identity

was determined

(43).

conserved in consonance with their likely role in maintaining the overall structure of the enzyme. Epitope mapping studies for a highly specific monoclonal antibody, 1F11, developed to 2C5 (27) indicate that the seg-

AMINO ACIDS 1

100

200

300

I

I

I

I

400

487 I

%ACT

100

C5

11111

PROGESTERONE

11111111II

III

I

III

I

I

C4

To identify how the structural diversity of P450 enzymes is related to their catalytic diversity, we have focused on the steroid 21-hydroxylase activity of 2C5. Our objective was to identify amino acid residues of 2C5 that confer highly efficient 21-hydroxylase activity to other GYP2C enzymes. Initially we chose 2C4, which differs from 2C5 at 24 amino acid positions. Expression of 2C4 in COS cells indicated that, like 2C5, it catalyzes 21-hydroxylation of progesterone but the apparent Km (> 25 cM) is much greater than that of 2C5 (2 tM) (20). Chimeric cDNAs were constructed as shown in Fig. 1. It was found that a small segment of the protein containing a cluster of three amino acid differences conferred the low Km of 2C5 (2 LM) on the 2C5/2C4 hybrid (chimera G). The segment identified in these experiments aligns by sequence comparison (Fig. 2), secondary structure assignments (21-23), and computer modeling (24) to a substratecontacting loop of the soluble, bacterial enzyme P45Ocam. This loop contains three residues found in close proximity to the substrate camphor. One residue within this loop, Tyr96, forms a hydrogen bond with the substrate, thereby positioning camphor relative to the site of oxygen activation (25). As this loop exhibits high thermal motion in the crystallized enzyme, it may have sufficient flexibility to form a substrate access channel (26). This suggested that the segment determining the difference in Km for progesterone 21-hydroxylation between 2C5 and 2C4 might exhibit a similar topology and function. This surface topology would permit considerable variation in amino acid sequence among CYP2C enzymes without disrupting the overall topology of these enzymes, and at the same time this variation could alter substrate binding and/or access to the catalytic site. A comparison of CYP2C sequences indicated that they were highly variable in this region (Fig. 2), whereas adjacent segments thought to correspond to helices C and D in P45Ocam are more highly

DETERMINANTS

Unpublished’

unidentified

Not

tion of P450 genes and subsequent divergence of coding sequences could lead rapidly to the acquisition of distinct catalytic activities. As we will discuss later, single amino acid changes in genetic variants can also lead to the loss or acquisition of one of several substrate specificities displayed by a single P450. On the other hand, structurally dissimilar enzymes can catalyze the same reactions. This suggests that P450 structures can readily accommodate genetic changes in ways that affect substrate metabolism without disrupting the fundamental capacity of P450s to reduce molecular oxygen to a form reactive with a wide variety of organic chemicals. OF

(20) (20)

16cr

with that of 2C5 using GCG

MAPPING DETERMINANTS 21-HYDROXYLASE ACTIVITY

Reference

hydroxylation

21, low Km 21, high Km

(35)

CYP2C3v ‘Results

of progesterone

P450 SPECIFICITY

A

135

B C

130

D JWifii E

100

F G

.

90

III

I

123

4

SPLICE Sirts Figure 1. Structure and progesterone 21-hydroxylase activities of CYP2C5, CYP2C4, and hybrid proteins expressed in COS-1 cells. Progesterone 2l-hydroxylase activity of hybrids between 2C5 and 2C4 was determined for cultured cells incubated with 2 iM progesterone as described (20). Hybrids are represented by solid and stippled segments corresponding to each of the parent proteins

CYP2C5 (C5) or CYP2C4 (C4), respectively. Numbers at the bottom scale refer to the following splice sites in the cDNAs used for the constructions: introduced this corresponding yields a L162S tivity; 4) StyI.

1) Sty1 restriction site in 2C4, a silent mutation site into 2C5; 2) BspHI; 3) a Draill restriction site to a site in 2C5 was introduced into 2C4, which point mutation with no increased 21-hydroxylase acThe distribution of the 24 differences of amino acid

sequence is shown by vertical lines between the parent proteins. The progesterone 21-hydroxylase activity of cells transfected with chimeric genes the activity

or the parent plasmids expressed as a percentage of of CYP2C5 (@3-6 nmol deoxycorticosterone mm) is shown on the right. The initial concentra-

formed/plate/60 tion of progesterone was chosen to maximize differences between low Km and high Km enzymes. The results of two or more experiments are summarized. (Reprinted from ref 20, the Proceedings of the National Academy of Sciences, USA, Vol. 86, pp. 8262-8265, 1989.)

701


2A5) that confers coumarin 7-hydroxylase activity to 2A4, a steroid 15a-hydroxylase. A mutation at this residue is also P450c.I U ymIFSSEc,FtPREAGEAY0cIPTIIt1PPERAL*iVVQw5K. . LoxoEI.AcsuEsi.RP. . . .0 I II Ii II 11111 iii responsible for the lower coumarin hydroxylase activity of an 551510 0 00 L0EEFAGRQWPIAEkVIEGLGIAFSMKn(IIFSLNTI.INFQS6SRSI(CSV0UARCI.VEEL.RKTNALP allelic form of 2A5 found in some inbred strains of mice (29). In another study, Aoyama et al. (30) showed that the mutaS4$TITUflailS IN tion at the corresponding amino acid position in 2B1 (i.e., CYPZC 2 ii .. .1 .. I114F) was necessary in order to express a double mutant #{234}#{248} ioo ul iisii.s 130 140 iso iso (L58F, 1114F) exhibiting the altered regiospecificity for steroid hydroxylation seen for an allelic form of 2B2. Figure 2. Amino acid sequence alignment between hybrid 0 and The correspondence in aligned positions of three key P45Ocam. 96Tyr of P4SOcam, and ll3Ala, ll5Ser and ll8Lys of residues between the respective structures of 2C5, 2A4, and hybrid G, which were derived from CYP2C5, are indicated by (5). 2B1 suggests that a few key residues may exist that can readAmino acids similar in both sequences based on the evolutionary ily be altered to produce new enzyme specificities for P450s. distance as measured by Dayhoff and normalized by Gribskov (37) Moreover, each of these substitutions is rather conservative. are matched by a vertical line (I). The helical domains of P45Ocam Thus, single rather subtle changes of amino acid sequence deduced from the X-ray structure (38) are shown above the can dramatically alter the catalytic properties of P450 enP450cam sequence; gaps in the sequence are shown as dots. The zymes. frequency of substitutions among 10 subfamily 2C P450s is shown The identification of key residues reflects the number of by the histogram below the sequence of hybrid G. (Reprinted from possibilities inherent in the differences between the enzymes. ref 20, the Proceedings of the National Academy of Sciences, USA, Vol. 86, In the cases described previously, the number of sites was pp. 8262-8265, 1989.) relatively small. In this regard, it is interesting that the key residues for these three pairs of enzymes were found to vary naturally at the alignment position corresponding to alanine 113 of 2C5. This suggests that this site is a hot spot for the ment of 2C5 carried in the chimera with 2C4 (chimera G, Fig. 1), constitutes an epitope recognized by the IF11 antigeneration of P450 diversity. body. In a filter binding assay (Fig. 3), no detectable binding of the 1F11 antibody is seen with 2C4. However, binding is observed to chimera G with an apparent binding constant that is very similar to that seen for 2C5. This suggests that 20 the segment containing the three differences of amino acid sequence lies on the surface of the enzyme. The generation of single mutants in 2C4 revealed the individual contributions of the three residues derived from 2C5 to the binding of the monoclonal antibody as well as to 15 the reduction of the Km for progesterone 21-hydroxylation (27). As shown in Fig. 3, the 2C4-N118K mutant exhibits F,) tight binding with the monoclonal antibody. Weak binding

I

S

C

F

0

is also observed between the monoclonal antibody and the 2C4-TII5S mutant. No binding was observed to the V113A mutant. Thus, the capacity of the 1F11 antibody to dis-

criminate between 2C5 and 2C4 largely reflects the single amino acid difference N1I8K. It should also be noted that this region diverges among P450 2C enzymes (Fig. 2) and that this monoclonal antibody is inhibitory. Thus, this epitope is well suited for the generation of specific inhibitory antibodies that discriminate among these enzymes. Neither the N1I8K nor T115S mutants are distinct from 2C4 in their 21-hydroxylase activity (27). This is not surprising as their participation in antibody binding and hydrophillic character suggest a surface orientation for each side chain. However, the VII3A difference that does not affect antibody binding confers a lower Km (8 tiM) to 2C4 (27). The other two mutations do not individually affect the Km of the enzyme, but when present in conjunction with the VII3A mutation, as in chimera 0, they confer a Km (2 ILM) tO 2C4 indistinguishable from that of the wild-type enzyme (20).

COINCIDENCE

OF

KEY

A comparison

RESIDUES

of these results with other work suggests that amino acids that align with alanine 113 of 2C5 are key determinants of the enzymic properties of CYP2A4 and GYF2B1 (27). Lindberg and Negishi (28) have shown that the V117A mutation, which aligns with alanine 113 of 2C5, is one of three mutations (based on 11 differences between 2A4 and

702

Vol.

6

January

1992

0 *

10

E

0 C)

5

0 -11

-10

-9 [1F11

-8

-7

-6

log(M)

Figure

3. Quantification of 1F11 binding to CYP2C5, chimera G, CYP2C4-TI15S, and CYP2C4-NI18K. The binding of the 1F11 antibody to filter-bound preparations of microsomes prepared from COS cells expressing the following were assayed as described (27). C5, CYP2C5; Chi G, chimera G contains three amino acid substitutions from CYF2C5 at positions 113, 115, and 118 in CYF2C4. No binding was observed for CYP2C4 or CYP2C4-VI13A. Estimates of apparent Kd obtained from nonlinear least squares fitting are: C5, 0.6 nM; Chi G, 0.5 nM; N118K, 0.7 nM; T1I5S, >30 nM. The values of Kd for C5, Chi G, and N118K may be overestimated because the values may reflect the amount of antigen bound to the filter. (Reprinted from ref 27, The Journal of Biological Chemistry, Vol. 266, pp. 6215-6220, 1991, with permission for Biochemistry & Molecular Biology.)

The FASEB Journal

of The

American

JOHNSON

Society

ET AL.


ENZYME

ENGINEERING

To extend this approach to enzymes exhibiting a greater number of differences in amino acid sequence, we have used these results as a basis for engineering CYF2C1 to catalyze progesterone 21-hydroxylase activity. This enzyme exhibits no detectable progesterone 21-hydroxylase activity when it is expressed in COS cells (31). A chimera was constructed that placed the first 128 residues of 2C5 ahead of amino acids 129-490 of 2C1 (32). This chimera contains the segment of 2C5 that conferred a low Km to 2C4 for progesterone 21-hydroxylation (20). When expressed in COS cells, the C5/C1 chimera exhibited progesterone 21-hydroxylase activity characterized by a Km similar to that of 2C5 (32). Next, we tested whether a mutation of valine 113 of 2C1 to alanine was sufficient to confer progesterone 21-hydroxylase activity to 2C1. The resulting V113A mutant of 2C1 exhibits progesterone 21-hydroxylase activity when expressed in COS cells. Thus, this single change conferred a new catalytic activity of CYF2CI; however, the activity was only 10% or less of that seen for 2C5 (31). We therefore modified adjacent residues (Fig. 4) within the segment corresponding to the one that enhanced the apparent Km of the VI13A mutant of 2C4 (analogous to chimera G, Fig, 1). These mutations did not have a detectable effect on the activity of the V113A mutant of CYF2C1. However, substitution of the entire segment of 2C5 corresponding to the substratecontacting loop of P45Ocam discussed earlier produced a chimera, C1-hvC5 (Fig. 4) that is a highly efficient 21-hydroxylase exhibiting a Km of 1.5 1cM for progesterone (31). Thus, mutation of a single key residue conferred the catalytic activity of one enzyme to another even though these enzymes differ at 25% of their amino acid residues. In addition, a model based on the structure of P4SOcam guided us in the construction of an enzyme that substantially enhanced the catalytic efficiency of the VI13A mutant of 2C1 (31). OTHER

KEY

RESIDUES

Additional studies suggest that other segments of the mammalian P450 polypeptide chain corresponding to loops that form the substrate binding site in P45Ocam may also play a

Cl CS

*2..

:

svCS Hoot,.

.

90 110 130 SOL . . . 113 . ,Is.14 KEALVIOLGEEFSUIVFPLTA&I*GYOI VFSNICET15FSL INTUW*ClS... A GSVILEVS LA AT N

similar role in mammalian P450s. This is illustrated by two P450s found in variant forms exhibiting a selective loss of one of several catalytic capacities. Matsunaga et al. (33) described a variant of CYF2DI that catalyzes the hydroxylation of debrisoquine but exhibits a deficiency in its capacity to metabolize bufuralol, which is hydroxylated by the wildtype enzyme. The two variants exhibit differences at four amino acid positions, and one of these 1380F is responsible for the difference in bufuralol metabolism. In another example, we have described two variants of 2C3 that differ in their capacity to catalyze the 6/3hydroxylation of progesterone while exhibiting similar catalytic properties for the metabolism of other substrates (34). Expression of CYP2C3 from the cDNA cloned by Leighton et al. (35) in COS cells indicates that it encodes the 6/3hydroxylase-deficient variant of 2C3. We have recently cloned a variant of 2C3 that differs at five amino acid residues and catalyzes progesterone 613-hydroxylase activity. One of these differences, S364T, is responsible for the change in 6f3-hydroxylase activity (unpublished results). The key residues identified for 2C3 and 2D1 (33) and one of the three amino acid differences, L365M, that confers coumarin hydroxylase activity to 2A4 (28), occur within an eight amino acid segment when these sequences are aligned (21). This segment maps to a substrate contact loop in P4SOcam that is adjacent to a highly conserved region of mammalian P450s proposed to correspond to helix K of P4SOcam (21-24).

One key residue identified for CYP2A5 does not align with other key residues. The mutation of this residue, F209L, confers steroid 15a-hydroxylase activity to 2A5 (28). Current models of the mammalian P450 enzymes based on the structure of P45Ocam place this key residue at the surface of the enzyme pointing outward (24). However, additional mutagenesis studies by Iwasaki et al. (36) indicate that mutations of this residue alter the spin-state of the heme in the resting enzyme. This in turn is thought to reflect the access of H20 to the open coordination site of the heme, which leads these authors (36) to suggest that this residue is in close proximity to the substrate binding site at the interior of the protein. This region is difficult to model because it appears to contain significant amounts of inserted sequence relative to P4SOcam (21, 24). Thus, the use of alternative sequence alignments as the basis for modeling might indicate a closer relationship with P4SOcam.

A MODEL The

*2...

HELIX 5

Sussitaic

Cosiaci Loos

HELIX C

Figure 4. Partial sequence comparison of CYP2C1 and CYP2C5 in the hypervariable region and schema for the generation of the mutants and chimeras. The sequence of CYF2C1 is shown, whereas only differences found in the sequence of CYP2C5 are shown below it. The numbering above the sequence indicates the residue number. Cl-V113A was obtained by site-directed mutagenesis of the CYP2CI cDNA. C1-G was obtained by the introduction of G1I7A and R119T mutations in C1-V113A. The restriction sites used for the construction of chimera Cl-hvC5 are indicated. The Bgl II site was introduced as a silent mutation in both cDNAs by site-directed mutagenesis. Topological features of P45Ocam (model) that have been predicted to occur among the mammalian P450s (22) are shown below the schema of the constructs. (Reprinted from ref 31, Biochemistry, Vol. 30, pp. 6097-6102, 1991, with permission of the American Chemical Society.)

DETERMINANTS

OF CYTOCHROME

P450 SPECIFICITY

to

FOR

clustering two

regions

in P4SOcam vation of

P450

of most that

DIVERSITY of the key residues

correspond

of

the

primary

to date

to substrate-contacting

suggests a paradigm for P450 diversity. basic topological features evident for

maintains a basic capacity to catalyze the oxygen bond generating a powerful oxidant, segments

identified

structures

loops ConserP4SOcam

scission of the diwhereas variant

of these

enzymes

bind

and orient the substrate with respect to the heme-oxidant complex. Many of these substrate contacts are present as loop structures in P45Ocam (26), and our results are consistent with a similar function for analogous domains in mammalian P450s. These loops can readily accommodate genetic change without disruption of the topology of the enzyme. As they also function in substrate binding, substrate recognition can be altered by genetic change without disrupting other catalytic events that lead to oxygen reduction. Thus, the structures of these enzymes readily accommodate genetic change leading to the acquisition of new catalytic functions. 703


In this way, repeated gene duplication and divergence during evolution would lead to the rapid development of an extensive catalytic repertoire. This in turn can provide the range of selectivity necessary to metabolize a wide spectrum of xenobiotics. This

work

GM31001,

was

supported

by U.S.

Public

Health

Service

grant

to E.F.J.

progesterone. Biochemistry 23, 4598-4603 16. Waxman, D. J., Attisano, C., Guengerich, D. P. (1988) Human liver microsomal identification of the major microsomal hydroxylase cytochrome P-450 enzyme.

F P., and Lapenson, steroid metabolism: steroid hormone 6/3Arch. Biochem. Biophys.

263, 424-436 17. Lange, R., Larroque, C., Balny, C., and Maurel, P. (1985) Isolation and partial characterization of a rifampicin induced rabbit liver microsomal cytochrome P-450. Biochem. Biophys. Res. Cornmun. 126, 833-839 18. Schwab, 0. E., Raucy,J. L., andJohnson, E. F (1988) Modulation of rabbit and human hepatic cytochrome P450-catalyzed steroid hydroxylations by a-naphthoflavone. MoL Pharmacol. 33,

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E.

F,

M.,

Finlayson,

Hujsak,

C. M.,

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J, GuenJ. C., Sato,

U. R., and Tukey, R. H. (1989) Genetic contributions to the variation among rabbits of liver microsomal deoxycorticosterone synthesis. Arch. Biochem. Biophys. 273, 273-280

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D. R., and Strobel,

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to

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Directed evolution of cytochrome P450 enzymes for biocatalysis: exploiting the catalytic versatility of enzymes with relaxed substrate specificity.

Alteration of the substrate specificity of cytochrome P450 CYP199A2 by site-directed mutagenesis.

Analysis of human cytochrome P450 catalytic activities and expression.

Characterization of human cytochrome P450 enzymes.

Optimization of yeast-expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH-cytochrome P450 reductase and cytochrome b5.

Cytochrome P450-Mediated Phytoremediation using Transgenic Plants: A Need for Engineered Cytochrome P450 Enzymes.

Genetics, epigenetics, and regulation of drug-metabolizing cytochrome p450 enzymes.

Effects of Alismatis rhizome on rat cytochrome P450 enzymes.

Expression of mammalian cytochrome P450 enzymes using yeast-based vectors.

Effects of aescin on cytochrome P450 enzymes in rats.

Elucidation of catalytic specificities of human cytochrome P450 and glutathione S-transferase enzymes and relevance to molecular epidemiology.

An inducible NADPH-cytochrome P450 reductase from Picrorhiza kurrooa - an imperative redox partner of cytochrome P450 enzymes.

Is there more to learn about cytochrome P450 enzymes?

Hole Hopping through Tryptophan in Cytochrome P450.

Cytochrome P450 reductase, antioxidant enzymes and cellular resistance to doxorubicin.

Genetic differences in cytochrome P450 enzymes and antidepressant treatment response.

The usefulness of genotyping cytochrome P450 enzymes in the treatment of depression.

Effects of avermectin on microsomal cytochrome P450 enzymes in the liver and kidneys of pigeons.

Binding of substrates to cytochrome P450 enzymes: mathematical artifacts in the application of difference spectrophotometry.

The catalytic function of cytochrome P450 is entwined with its membrane-bound nature.

Role of residue 478 as a determinant of the substrate specificity of cytochrome P450 2B1.

Fusion to Hydrophobin HFBI Improves the Catalytic Performance of a Cytochrome P450 System.

Differential expression of cytochrome P450 enzymes from the CYP2C subfamily in the human brain.

Isolation of cytochrome P450 from hepatopancreas microsomes of the spiny lobster, Panulirus argus, and determination of catalytic activity with NADPH cytochrome P450 reductase from vertebrate liver.