©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification by in Vitro Mutagenesis of the Interaction of Two Segments of C2MstC1, a Chimera of Cytochromes P450 2C2 and P450 2C1 (*)

(Received for publication, October 3, 1994; and in revised form, November 18, 1994)

Manjunath K. Ramarao (§) Petra Straub (¶) Byron Kemper (**)

From the Department of Physiology and Biophysics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A hybrid cytochrome P450, C2MstC1, with 306 N-terminal amino acids derived from cytochrome P450 2C2 sequence and 184 C-terminal amino acids from cytochrome P450 2C1 acquires a novel progesterone 21-hydroxylase activity which is absent in the parent enzymes. Extension of the cytochrome P450 2C2 sequence to residue 382 reduced progesterone hydroxylase activity to 5% of that of C2MstC1, while further extension to residue 411 or 462 increased activity back to about 30 or 40%, respectively. In the chimera with cytochrome P450 2C2 sequence to residue 382, substitution of cytochrome P450 2C1 amino acids at positions 368, 369, and 374 increased progesterone hydroxylase activity to a level equivalent to that of C2MstC1. In the chimera with cytochrome P450 2C2 sequence extending to residue 411, substitutions of P450 2C1 amino acids at positions 386 and 388, in addition those at 368, 369, and 374, were required to obtain activities equivalent to that of C2MstC1, which suggests an interaction between these two regions. The lauric acid hydroxylase activities of all chimeras and mutant cytochromes P450 differed by 2-fold or less, demonstrating that the changes in progesterone hydroxylase activity reflected altered interactions with the substrate. Alignment of cytochrome P450 2C1 sequence with cytochromes P450, P450, and P450 predicts that residues 368/369 and 386/388 are in adjacent antiparallel strands of the same beta-sheet, in agreement with the experimental data suggesting an interaction between these two regions.


INTRODUCTION

Cytochromes P450 (P450), (^1)constitute a superfamily of proteins involved in oxidative metabolism of various endogenous substances such as steroids, fatty acids, biogenic amines, prostaglandins, and leukotrienes as well as exogenous substances including drugs, plant metabolites, and a variety of pollutants(1) . The P450 superfamily consists of at least 221 genes and 12 putative pseudogenes present in species from bacteria to man with the 51 P450s described for the rat being the largest number described for a single species(2) . The large number of P450s accounts in part for the wide variety of substrates metabolized by P450s, but individual P450s also may metabolize molecules with quite different structures. Among the P450 subfamilies, subfamily C with 28 mammalian members has the greatest diversity.

In rabbit, eight subfamily 2C genes have been reported(3) . These P450s metabolize a variety of xenobiotics but most also metabolize endogenous steroid molecules(3) . P450 2C3 is the predominant progesterone 16alpha-hydroxylase in rabbit liver, but P450 2C14 and P450 2C16 also are progesterone 16alpha-hydroxylases. A variant of P450 2C3, 2C3v, has progesterone 6beta in addition to 16alpha-hydroxylase activity. P450 2C5 is the major progesterone 21-hydroxylase in liver and the closely related P450 2C4 has low progesterone 21-hydroxylase activity. In contrast, hydroxylation of steroids by P450 2C1 and P450 2C2 either cannot be detected or occurs at a very low rate(3, 4) . Substrates of these two P450s include the fatty acids, lauric acid and arachidonic acid. Both catalyze the (-1)-hydroxylation of lauric acid, and 11,12- and 14,15-epoxygenation of arachidonic acid(4, 5, 6) . P450 2C2, but not P450 2C1, also catalyzes the 19-hydroxylation of arachidonic acid. Interestingly, a chimeric P450 consisting of the N-terminal 1-306 amino acids of P450 2C2 and C-terminal 307-490 amino acids of P450 2C1 acquired progesterone C21-hydroxylase activity, while the (-1)-lauric acid hydroxylase activity of P450 2C2 was retained(7) .

We have shown that the amino acids in the N-terminal portion of P450 2C2 are important for the progesterone hydroxylase activity of the chimera and for the lauric acid hydroxylase activity of both the chimera and P450 2C2(5, 7, 8) . These amino acids are within SRS-1, one of six such sites predicted from the alignment of P450 2C sequences with P450, for which the three-dimensional structure is known(9) . The remaining SRSs are scattered along the entire P450 molecule. In the absence of a three-dimensional structure for the mammalian P450 2C proteins, the validity of such alignments with P450 can be tested by analysis of chimeric and mutated P450s. The acquisition of progesterone hydroxylase activity when the C-terminal 184 amino acids of P450 2C1 were substituted for those of P450 2C2 indicates that regions in the C-terminal portion of the P450 also are critical for recognition of steroid substrates. In order to define the C-terminal regions responsible for recognition of the progesterone substrate, several chimeric genes encoding hybrid proteins containing N-terminal P450 2C2 sequence and successively shorter substitutions of P450 2C1 C-terminal sequence have been constructed. Specific critical amino acids have been identified by site-directed mutagenesis. The results indicate that three C-terminal regions are important for progesterone hydroxylase activity and that two of these regions interact with each other. These experimental data are consistent with the relative positions of these regions predicted by alignment with the amino acid sequences of bacterial P450s for which three-dimensional structures have been determined.


EXPERIMENTAL PROCEDURES

Materials

The following oligonucleotides, with mutated residues underlined, were synthesized on an Applied Biosystem Model 380A DNA synthesizer at the Biotechnology Center of the University of Illinois at Urbana-Champaign: O368/369, 5`-CGTGCCCTTGGGGATGAGATAGTTTCTGAACTTAAGGTTACAGATTGTTGT/CACGGGGCAC-3`; O369/371, 5`-CGTGCCCTTGGGGATGAGATAGTTTCTGAACTTAAGGTTACAGG/ATTGTTGC/TATGGG-3`; O374/378, 5`-CGTGCCCTTGGGGATGAGATAGC/TTTCTGAACTTAAG/CGTTACA-3`; and O386/388, 5`- TATCTCATCCCCAAGGGCACAGC/ATGTAA/CTAACA-3`. All PCR reagents and the enzyme Amplitaq were purchased from Perkin Elmer Cetus. The pCMV5 expression vector was obtained from Dr. Stinski (University of Iowa). COS-1 cells were obtained from the American Type Cell Collection. Cell culture media and reagents were obtained from Life Technologies, Inc. DEAE-dextran was purchased from Pharmacia LKB Biotechnology Inc., and calf serum, chloroquine, and dimethyl sulfoxide were from Sigma. [1,2,6,7-^3H]Progesterone and [1-^14C]lauric acid were purchased from Amersham Corp. TranS-label was purchased from ICN Biomedicals.

Construction of Chimeras of P450 2C2 and P450 2C1

Vectors based on pCMV5 for expression of P450 2C1, P450 2C2, and C2MstC1 have been described(5) . C2MstC1 contains 306 N-terminal amino acids from P450 2C2 and 184 C-terminal amino acids from P450 2C1. Additional chimeras were constructed with shorter P450 2C1 C-terminal sequences by exploiting common restrictions sites, StyI at amino acid residue 382, BalI at residue 411, and HincII at residue 462 to produce pCMV5-C2StyC1, pCMV5-C2BalC1, and pCMV5-C2HincC1, respectively. To construct pCMV5-C2StyC1, the BclI-BamHI fragments encoding P450 2C1 and P450 2C2 were isolated from pCMV5-2C1 and pCMV-2C2, respectively, and digested with StyI. The BamHI-BclI fragment from pCMV-2C2 containing the pCMV5 vector, the BclI-StyI fragment encoding P450 2C2 sequence, and the StyI-BamHI fragment encoding P450 2C1 sequence were ligated together in a single step to produce pCMV5-C2StyC1. To construct pCMV5-C2BalC1, KpnI-BamHI fragments containing P450 2C1 or P450 2C2 cDNA sequence were digested with BalI. KpnI/BamHI-digested pCMV5 DNA, the KpnI-BalI fragment encoding P450 2C2, and the BalI-BamHI fragment encoding P450 2C1 were ligated in a single step to produce pCMV-C2BalC1. pCMV5-C2HincC1 was constructed in the same way except that the KpnI-BamHI fragments encoding the cDNAs were digested with HincII.

Site-specific Mutagenesis by PCR

For mutations at residues 368, 369, 371, 374, and 378, the MstII end of the MstII-BamHI fragment from pCMV5-C2StyC1 was filled in with the Klenow fragment of DNA polymerase I. This fragment was then inserted into pTZ18R digested with SmaI and BamHI, which results in regeneration of the MstII site. This template was used in PCR reactions with the T7 promoter oligonucleotide (Promega Corp.) in combination with one of the mutating primers, O368/369, O369/371, or O374/378. The mutating primers extended from the StyI site to the positions to be mutated. PCR conditions were as described (10) with 250 ng of each primer for 30 cycles at 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 2 min. The resulting amplified fragment was digested with MstII and StyI and ligated with MstII/BamHI-digested pCMV5-2C2 DNA and the StyI-BamHI fragment from pCMV5-2C1 to introduce the mutations in C2StyC1. A triple mutant of C2StyC1 containing H368R, T369A, and L374V is referred to as C2tmStyC1. Some of these mutations were introduced into pCMV5-2C2, pCMV5-C2BalC1, and pCMV5-C2HincC1, by substituting the MstII-StyI PCR fragments, with the mutations, for the corresponding fragment of each of these plasmids. The triple mutants of H368R, T369A, and L374V in these plasmids are referred to as C2tm, C2tmBalC1, and C2tmHincC1.

For mutations at residues 386 and 388, pCMV5-C2tmBalC1 was used as a template in a PCR reaction. The primer at one end hybridized to the pCMV5 sequence just 3` of the C2tmBalC1 insert and had the sequence 5`-CCACCCGGGGATCC-3`. The other mutating primer, O386/388, extended from the StyI site to the region to be mutated. The PCR fragment was digested with StyI and BamHI and ligated with MstII/BamHI-digested pCMV5-C2tm DNA and the MstII-StyI fragment of pCMV5-C2tm containing the mutations at 368, 369, and 374, to introduce the additional mutations at 386 and 388 into C2tmBalC1. This penta-mutant is referred to as C2pmBalC1. The mutations at 386 and 388 were then introduced into C2tm and C2tmHincC1 by substituting the StyI-BamHI fragments containing the mutations and are referred to as C2pm and C2pmHincC1, respectively. Mutated sequences were confirmed by sequencing using the Sanger's dideoxynucleotide chain termination method with Sequenase^R version 2 (U. S. Biochemical Corp.) according to the manufacturer's protocol. Mutants in pCMV-C2StyC1 and pCMV5-C2tmBalC1 were sequenced using the primers 5`-GTACAGCATAGAAGTCAG-3` and 5`-AAGTGGCCAGGGTC-3`, respectively.

Cell Culture and Assay of Progesterone and Lauric Acid Hydroxylation

COS-1 cells were cultured and transfected with expression plasmid DNA as described (5) except that the cells were grown in 10% calf serum rather than 10% fetal bovine serum. To assay for hydroxylase activity, 48 h after transfection, lysates of the COS-1 cells were prepared (5) and divided into two equal portions for assay of progesterone and lauric acid hydroxylation. Metabolism of lauric acid and separation of metabolites by HPLC was as described (5) except that the reaction time was 15 min. Progesterone 21-hydroxylase activity was determined under the same conditions except that 20 µM cold progesterone and 4 µCi of [1,2,6,7-^3H]progesterone was added in place of lauric acid. The tubes containing the reaction mixture were incubated at 37 °C for 15 min, and the reaction was stopped by the addition of 1/10 (v/v) of 1 M HCl. Progesterone and its metabolites were extracted with chloroform and separated by thin layer chromatography as described previously(7) . For quantitation of the radiolabeled products, radioactive spots were scraped from the TLC plates, and radioactivity was assayed by scintillation counting. The activity of the hybrid construct C2MstC1 was arbitrarily assigned a value of 100%, and the activities of the other constructs were calculated relative to this value.

Immunoprecipitation of Expressed Proteins

Forty-eight h after transfection, cells were incubated for 4 h with 50 µCi/ml of TranS-label in Met and Cys free minimal essential medium before lysis. Radioactive proteins were immunoprecipitated from cell lysates and analyzed by SDS-PAGE as described previously(5) .


RESULTS

Progesterone 21-Hydroxylase Activity of Chimeras of P450 2C2 and P450 2C1

A chimeric P450 with the N-terminal 1-306 amino acids of P450 2C2 and the C-terminal 307-490 amino acids of P450 2C1, referred to as C2MstC1, acquired a novel progesterone 21-hydroxylase activity, distinct from either of the parents(7) . To initially identify the specific P450 2C1 C-terminal sequence required for progesterone 21-hydroxylase activity, additional chimeric P450s were constructed with successively shorter C-terminal sequences from P450 2C1 (Fig. 1). Each of the chimera was assayed for activity in whole cell extracts of transfected COS-1 cells, and progesterone metabolites were separated by thin layer chromatography. An autoradiogram of a typical thin layer chromatogram is shown in Fig. 2A, and the results of seven experiments are summarized in Fig. 3, top panel. Mock-transfected cells or cells expressing P450 2C1 and P450 2C2 had no detectable progesterone 21-hydroxylase activity. Relative to C2MstC1, the activity of the C2StyC1 chimera was reduced to about 5%, suggesting that the P450 2C1 coding sequence between the MstII and StyI sites was critical for maximal progesterone hydroxylase activity. Paradoxically, chimeras with even less P450 2C1 C-terminal sequence, C2BalC1 and C2HincC1, recovered 30-40% of progesterone 21-hydroxylase activity relative to C2MstC1, 6- to 8-fold higher activity than C2StyC1.


Figure 1: Schematic representation of the cDNAs of the parental, chimeric, and the mutant P450s that were analyzed. The P450 2C1 cDNA is represented by solid black, and the P450 2C2 cDNA is represented by cross-striped rectangles. The numbering at the top, represents the amino acid positions of the coding region of the protein. The six SRSs(9) , are represented by a horizontal line at the appropriate amino acid positions and are labeled as SRS1-SRS6. Splice sites represent the restriction enzyme sites that were used to make the chimeric P450s. The protein encoded by each of the cDNAs is labeled on the right-hand side of the diagram. The chimeras are designated by the N-terminal sequence, the restriction site at which the switch was made, and the C-terminal sequence in that order. In the case of C2StyC1 mutants, the sequence between amino acid residues 368 and 378 is shown, and, in the case of other mutants, the sequence between the amino acid positions 368 and 388 is shown. The P450 2C2 residues were mutated to P450 2C1 residues, by PCR, as described under ``Experimental Procedures.'' The P450 2C2 sequences are shown at the top, and the mutant amino acids at the corresponding positions are shown below.




Figure 2: Analysis of progesterone metabolites produced by the cell lysates of COS-1 cells. A mock-transfected culture, used as a negative control, is labeled as MOCK. The cell lysates were obtained and assayed for progesterone hydroxylase activity as described under ``Experimental Procedures.'' The positions of 21-hydroxyprogesterone and progesterone are indicated. Individual lanes are labeled by the corresponding proteins which catalyzed the reaction. A, a representative autoradiogram of a TLC of progesterone metabolites produced after transfection with the P450 2C1, P450 2C2, and their chimeric P450s. B, a representative autoradiogram of the progesterone metabolites, produced by the cell lysates of the COS-1 cells, transfected with the C2StyC1 mutants. As a control, activities of C2MstC1 and C2StyC1 are also included. The single and the double mutants are designated as wild type amino acid, the position, and the mutant amino acid in that order. A triple mutant (tm), of C2StyC1 with H368R, T369A, and L374V, is referred to as C2tmStyC1.




Figure 3: Progesterone and lauric acid hydroxylase activity in COS-1 cells transfected with the P450s 2C1, 2C2, their chimeras, and the C2StyC1 mutants. Values plotted represent means and standard errors for seven independent transfections. Top panel, progesterone hydroxylase activity was determined as described in Fig. 2. The position of the 21-hydroxylated product was determined by co-migration with the unlabeled 21-hydroxyprogesterone. The bands containing the product were scraped, and radioactivity was assayed by scintillation counting. Values for the individual hydroxylase activities were normalized against that of C2MstC1. Middle panel, (-1)-lauric acid hydroxylation. A second aliquot of the cell lysates from COS-1 cells, which had been used for the progesterone hydroxylase assay, was assayed for lauric acid hydroxylase activity and analyzed by HPLC as described under ``Experimental Procedures.'' The means of seven independent transfections for each of the proteins are plotted with the standard error indicated. Values for the individual hydroxylase activities were normalized against activity of the wild type P450 2C2. Bottom panel, ratio of the means of the progesterone 21-hydroxylase activity and the lauric acid hydroxylase activity.



These changes in progesterone hydroxylase activity potentially could be due to changes in the levels of expression of functional enzymes for each chimera in the COS-1 cells. To assess the effect of mutations on the stability of the proteins, transfected COS-1 cells were labeled for 4 h with TranS-label, and the expressed proteins were immunoprecipitated(5) . A weak band co-migrated with P450 for the mock-transfected cells, and a second band, of unknown origin, migrated slightly faster than P450 2C2 and served as an useful internal control for labeling and precipitation (Fig. 4). Since the half-life of the newly expressed P450 in COS-1 cells has been shown to be less than 1 h, labeling the cells for 4 h provides a reasonable measure of the steady state levels of the protein(5) . No differences greater than 2-fold in the amount of radioactive proteins immunoprecipitated for P450 2C1, P450 2C2, the chimeric proteins, and all the other mutants analyzed in this study were detected in five independent experiments. The results demonstrate that the differences in progesterone activity are not the result of altered stability or expression of the P450 proteins.


Figure 4: Immunoprecipitation of the P450s expressed in COS-1 cells. Expression of immunoreactive P450 2C1, 2C2, their chimeras, and mutants in COS-1 cells was analyzed. Forty-eight h after transfection, COS-1 cells were labeled for 4 h with 60 µCi/ml Tran[S]-label. After lysis, P450s were immunoprecipitated using a polyclonal antiserum raised against P450 2C3 (12) . Immunoprecipitated proteins were analyzed by SDS-PAGE and fluorography. P450 protein bands are marked by an arrow at the left. Individual lanes are labeled by the corresponding parental, chimeric, or the mutant P450 expressed, and the sample from mock-transfected cells is labeled MOCK.



It is possible that either differences in the fraction of total immunoprecipitable protein that folds into a functional P450 or general effects of the mutations on the catalytic activity rather than specific effects on interactions of the substrate with P450 might cause the changes in progesterone activity. The lauric acid hydroxylase activity of each of the chimeric proteins, therefore, was analyzed in duplicate aliquots of the whole cell extract. Results from seven experiments are summarized in Fig. 3, middle panel. Mock-transfected cells do not show any detectable lauric acid hydroxylase activity. P450 2C1 is a weak (-1)-lauric acid hydroxylase with activity about 10% of that of P450 2C2 under these conditions. The activities of the chimeric P450s ranged from 75% to 150% relative to that of P450 2C2 indicating that the mutations did not have substantial effects on the expression of functional enzyme or general catalytic activity. These changes are similar to those reported for a chimera in which the last 28 amino acids of P450 2C2 were replaced with those of P450 2C14(11) .

In Fig. 3, bottom panel, the progesterone hydroxylase activities of the chimeric proteins are normalized to those of the lauric acid hydroxylase activity by calculating the ratio of the two activities. The relative activities for the chimeras were similar for either the progesterone activity (Fig. 3, top panel) or normalized activity (Fig. 3, bottom panel). These results indicate that the P450 2C1 coding sequence between the MstII and StyI sites is important for interaction of progesterone with the P450. Further, P450 2C1 coding sequence between the StyI site and the BalI site interfered with these interactions since the C2BalC1 and C2HincC1 chimeras had similar activities severalfold greater than C2StyC1. Finally, the P450 2C1 sequence C-terminal of the HincII site, encoding 28 amino acids, was critical for progesterone interactions since the C2HincC1 chimera was a progesterone 21-hydroxylase, while P450 2C2 was not.

Mutations between Residues 306 and 382 (MstII-StyI Sites)

To determine which amino acid residues were responsible for the loss of activity of C2StyC1 compared to C2MstC1, amino acids present in P450 2C1 were substituted for those of P450 2C2 in C2StyC1. The region between amino acids 306-382 in P450 2C1 and P450 2C2 differs only at five positions, 368, 369, 371, 374, and 378. P450 2C1 amino acids were substituted at each of these positions and in various combinations (see C2StyC1 mutants in Fig. 1). A representative separation of the progesterone metabolites for each mutant is shown in Fig. 2B. Progesterone and lauric acid hydroxylase activities and the ratio of progesterone to lauric acid hydroxylase activities from seven experiments are summarized in Fig. 3(C2StyC1 mutants). Lauric acid hydroxylase activities of the mutants differed little ranging from 50% to 90% of that of C2MstC1. Progesterone 21-hydroxylase activities of the mutants relative to each other, therefore, were very similar for both the actual (Fig. 3, top panel) and normalized (Fig. 3, bottom panel) values. Single substitutions of H368R, T369A, and L374V resulted in mutants with substantially increased activity by 5- to 7- fold compared to C2StyC1, but still only 20%-30% of the activity of C2MstC1. Substitutions at two positions, I371T and N378S, did not increase progesterone hydroxylase activity. These results indicated that single mutations could not restore the activity of C2StyC1 to that of C2MstC1; therefore, a combination of the individual mutations was tested. Double mutants containing one mutation that had increased activity and one that had not, T369A/I371T and L374V/N378S, had activities similar to the single mutants with increased activity. In contrast, a double mutant of H368R and T369A and a triple mutant of these two substitutions plus L374V had activities equivalent to that of C2MstC1. These results indicate that the P450 2C1 amino acids Arg-368, Ala-369, and Val-374 together are the major contributors to the progesterone 21-hydroxylase activity between the residues 306 and 382.

These three substitutions, which increase progesterone hydroxylase activity of C2StyC1 by 20-fold, were also introduced into P450 2C2, C2BalC1, and C2HincC1 to test whether they could similarly increase activity in these enzymes. No progesterone hydroxylase activity was detected when a single substitution was made in P450 2C2 at position 369, and barely detectable levels were observed for substitutions at 368 and 369 or at 368, 369, and 374 ( Fig. 5and Fig. 6). This result shows that in addition to these three P450 2C1 amino acids, there is still a requirement for the C-terminal P450 2C1 sequence for progesterone recognition. To identify the C-terminal P450 2C1 sequence required for the progesterone 21-hydroxylase activity, the three substitutions were introduced into C2BalC1 and C2HincC1 to produce C2tmBalC1 and C2tmHincC1, respectively. In these mutants, the three mutations are combined with the P450 2C2 sequence between the StyI and BalI sites and the P450 2C1 sequence from the HincII site to the termination codon, both of which increase progesterone hydroxylase activity based on the studies with the chimeras. Activities equivalent to C2MstC1 or greater were, therefore, anticipated. The results, however, showed that introduction of the three mutations into C2BalC1 and C2HincC1 had little or no effect on the activity of these two chimeras in contrast to the effects of these substitutions in C2StyC1 ( Fig. 5and Fig. 6). These results indicate that there is a requirement for the P450 2C1 sequence from the StyI to BalI sites encoding amino acids 382-411 for the increase in progesterone hydroxylation mediated by the three mutations introduced between the MstII and StyI sites encoding amino acids 306-382. Similar progesterone 21-hydroxylase activities of C2tmBalC1 and C2tmHincC1 eliminate a specific role for the P450 2C1 sequence between BalI and HincII for progesterone hydroxylation. These results suggest that amino acids at 368, 369, or 374 might interact with amino acids between residues 382 and 411.


Figure 5: A typical autoradiogram of the TLC showing the separated products of progesterone metabolism, catalyzed by the mutants of P450 2C2, C2StyC1, C2BalC1, and C2HincC1, expressed in COS-1 cells. The assays were done as described in the legend for Fig. 2. The ``tm'' refers to the triple mutation, involving H368R, T369A, and L374V. Activities of C2MstC1 and P450 2C2 are shown as controls. The positions of C21-hydroxyprogesterone and progesterone are indicated.




Figure 6: Summary of progesterone and lauric acid hydroxylase activities of mutations of P450 2C2, C2StyC1, C2BalC1, and C2HincC1. Values plotted represent means and standard errors for seven independent transfections. Both the progesterone and lauric acid hydroxylase activity assays were done on the same cell lysates. Analysis of progesterone hydroxylation (top panel), lauric acid hydroxylation (middle panel), and the ratio of the means of progesterone hydroxylation to lauric hydroxylation (bottom panel) were carried out as described in the legend to Fig. 3.



Mutation of Amino Acids between Residues 382 and 411

There are six amino acid differences between P450 2C1 and P450 2C2 in the 30 amino acids between residues 382 and 411. In order to identify which P450 2C1 residues might be required for the increase in progesterone hydroxylase activity resulting from substitutions at 368, 369, and 374, additional substitutions of P450 2C1 for P450 2C2 amino acids were introduced into C2tmBalC1. The single mutants, D386A and L388I, and a double mutant at both positions were made. Introduction of Ala at position 386 of C2tmBalC1 resulted in a 10-20% increase in the activity, whereas Ile at position 388 did not significantly alter the activity of C2tmBalC1 ( Fig. 7and Fig. 8). However, the activity of the double mutant of C2tmBalC1, termed C2pmBalC1, was increased to a level similar to that of C2MstC1 and C2tmStyC1. This result demonstrated that the increase in progesterone hydroxylase activity due to substitution of P450 2C1 amino acids at residues 368, 369, and 374 requires the additional P450 2C1 residues at positions 386 and 388 and is consistent with an interaction between the amino acids in these two regions.


Figure 7: A typical autoradiogram showing the separated products of progesterone metabolism of COS-1 cells expressing P450 mutants at positions 386 and 388. The activity assays were done on the cell lysates as described in the legend for Fig. 2A. ``tm'' refers to the triple mutant indicated in the legend for Fig. 2B, and ``pm'' refers to the penta mutant, involving H368R, T369A, L374V, D386A, and L388I. When these five mutations are in P450 2C2, C2BalC1, and C2HincC1, the constructs are referred to as C2pm, C2pmBalC1, and C2pmHincC1, respectively. The mobilities of 21-hydroxyprogesterone and progesterone are indicated.




Figure 8: Summary of progesterone and lauric acid hydroxylase activities of P450s with mutations at 386 and 388. The values plotted here represent the means and standard errors of four independent transfections. Both the progesterone and lauric acid hydroxylase activities were performed on the same cell lysates of COS-1 cells transfected with the indicated constructs. Analysis of progesterone hydroxylation (top panel), lauric acid hydroxylation (middle panel), and the ratio of the means of progesterone hydroxylation to lauric hydroxylation (bottom panel) were carried out as described in the legend to Fig. 3.



These five substitutions were introduced into P450 2C2 and C2HincC1, producing C2pm and C2pmHincC1, respectively, to determine if they were sufficient to explain the progesterone 21-hydroxylase activity of C2MstC1. The introduction of the five mutations conferred detectable progesterone hydroxylase activity to P450 2C2 but only about 10% that of C2MstC1 ( Fig. 7and Fig. 8). This indicates that there is still a requirement for additional C-terminal amino acids of P450 2C1 in addition to the 5 residues identified, for maximum progesterone 21-hydroxylase activity. The introduction of the five mutations into C2HincC1 increased activity to levels comparable with those of C2MstC1 and C2pmStyC1 and about 10-fold higher than that of C2pm. This result indicates that the additional C-terminal P450 2C1 sequence required is in the C-terminal 28 residues encoded by sequence from the HincII site to the termination codon.


DISCUSSION

We have exploited the novel progesterone hydroxylase activity in a chimera with N-terminal P450 2C2 sequence and C-terminal P450 2C1 sequence to identify regions and amino acids of P450 2C1 that confer this activity. Previously, position 113 of P450 2C1 had been shown to be important for progesterone 21-hydroxylase activity(12) . In addition, amino acids near the C terminus were implicated in steroid substrate recognition by the observation of a novel testosterone 16beta-hydroxylase activity if the 28 C-terminal amino acids of P450 2C2 were replaced with those of P450 2C14(11) , but not with those of P450 2E1 or P450 2B5(13) . In this study, the C-terminal 28 amino acids and two additional regions in the C-terminal half of P450 2C1 were shown to contribute to progesterone 21-hydroxylase activity. Remarkably, the activity of lauric acid hydroxylation in all of the chimeras and mutations tested differed by only 2-fold while progesterone hydroxylation varied by 20-fold or more. The relatively constant activity for lauric hydroxylation indicates that the variations in progesterone hydroxylase activity are not due to changes in either the expression levels of functional P450 in the COS-1 cells or the inherent activity of the enzyme and can be ascribed to altered interactions of the substrate with the enzyme.

The experimental data indicate that the regions containing amino acids 368-374 and amino acids 386-388 may interact with each other. If P450 2C1 sequence was present in both regions as in C2MstC1 or C2tmStyC1, maximal activity was observed, but if P450 2C2 sequence replaced the P450 2C1 sequence in either the first region, C2StyC1, or the second region, C2tmBalC1, activity was reduced. Further, substitution of P450 2C1 amino acids at 368, 369, and 374 into C2StyC1 increased progesterone activity 20-fold. However, the same substitutions in C2BalC1 or C2HincC1 resulted in no increase in activity. Therefore, in order for the P450 2C1 substitutions at 368, 369, and 374 to increase progesterone 21-hydroxylase activity, P450 2C1 sequence had to be present in the region encoded by sequence between the StyI and BalI sites which indicates that amino acids in these two regions interact with each other. In the StyI/BalI region, substitutions of P450 2C1 amino acids at 386 and 388 in combination with those at 368, 369, and 374 resulted in maximal progesterone hydroxylase activity, suggesting that these 2 residues were near 368, 369, or 374 in P450 2C1.

The experimental results indicating an interaction between the 368-374 and 386-388 regions are reinforced by predicted positions of these amino acids in P450 2C1 based on alignments with bacterial P450s for which the three-dimensional structure is known. Alignment of rabbit P450 2C proteins with P450 as proposed by Gotoh (9) and the alignment of P450 and P450 with P450 based on the three-dimensional structure(14, 15) are shown in Fig. 9. In this alignment, residues 368 and 369 of P450 2C1 correspond to P450 residues Arg-299 and Ile-300 which are in one strand of beta-sheet 3 and line the substrate binding pocket. P450 2C1 residues, 386 and 388, correspond to Glu-317 and Leu-319, respectively, which are in the second strand of the same beta3 sheet. The side chains of Glu-317 and Leu-319 also protrude into the substrate binding pocket. This alignment suggests, therefore, that these 4 residues are near the substrate binding pocket and that Ala-386 and Ile-388 can interact with Arg-368 and Ala-369 or nearby residues through hydrogen bonding, influencing the conformation of the beta3 sheet and the shape of the substrate binding pocket. The effect of changes at 374 which aligns at the end of the beta3 sheet strand are not as easily rationalized. Presumably amino acid changes at this position might indirectly affect the beta-strand lining the substrate binding pocket. Based on the structure of P450, Poulos and his co-workers (16) have suggested that in order to accommodate larger substrates yet maintain the required reaction trajectory and interactions with the iron-bound oxygen atom, the beta3 sheet is one of the segments that may undergo topological adjustments. Our results suggesting an interaction between amino acids on separate strands of the beta3 sheet which affects interactions with a steroid more than with a fatty acid substrate are consistent with this proposal.


Figure 9: Comparison of the sequences of rabbit P450 2C sequences with those of the bacterial P450s, P450, P450, and P450. The P450 2C sequences are aligned with P450 as described by (9) , and the P450 and P450 are aligned with P based on the three-dimensional structure as described(14, 15) . Residues corresponding to positions 368, 369, 374, 386, and 388 of P450 2C1, which contribute to progesterone hydroxylase activity, are shaded. Brackets at the bottom indicate which of the bacterial amino acids are present in two antiparallel strands of the same beta-sheet.



Alignment of P450 2C1 and P450 2C2 amino acid sequences with P450 is also consistent with interactions between the amino acids at 368/369 and 386/388. Residues 363 to 370 of P450 2C1 and P450 2C2 correspond to residues 329 to 335 of P450 which forms the beta-strand 1-4, and that between 384 and 390 corresponds to residues 350 to 356 in the adjacent antiparallel beta-strand 1-3. In P450, these two beta strands line the substrate binding pocket. Similar results were obtained when the amino acid sequences of P450 2C1 and P450 2C2 were aligned with that of P450. The region between amino acids 366 and 371 of P450 2C1 and P450 2C2 corresponds to the beta-strand 1-4 of P450 (from residues 317 to 322), and that between residues 384 and 390 corresponds to the adjacent antiparallel beta-strand 1-3 (from residues 335 to 341). These two strands also line the substrate binding pocket although the substrate-bound models of P450 have implicated only one amino acid, Phe-317 (corresponding to Val-366 of P450 2C1), in substrate binding. The predicted positions of the residues 368/369 and 386/388 in P450 2C1 on the basis of alignment with all the P450s for which the three-dimensional structure is known is consistent with the experimental results that residues in these two regions interact and affect interactions of the substrate with the enzyme.

Gotoh (9) proposed 6 SRSs scattered along the P450 molecule which might be involved in the P450 substrate recognition. Of the five amino acids we have identified in the C-terminal portion of P450 2C1 that contribute to progesterone hydroxylase activity, only 368 and 369 are within an SRS (SRS-5, residues 359 to 369) and would have been predicted to affect substrate interactions. Mutations within SRS-5 have been shown to affect the activity of other P450s. A S364(365)T change is responsible for the difference in progesterone 6beta-hydroxylase activity in two variants of P450 2C3(17) . The equivalent residue in P450 2C1 as aligned by Gotoh and Fujii-Kuriyama (18) is shown in parentheses. An Ala substitution for Val-363(362) conferred androgen 15alpha-hydroxylase activity to P450 2B1, and an Ala substitution for Val-367(366) conferred androgen 6beta-hydroxylase activity to this enzyme(19, 20) . An I380(369)F mutation in P450 2D1 affected bufuralol but not debrisoquine metabolism (21) and a M366(361)L change was one of three changes required for the alteration in substrate specificity of P450 2A4 to that of 2A5(22) . The effects of mutations at 368 and 369 are, therefore, consistent with observations in other P450s and provide additional support for the assignment of this region as an SRS. In contrast, the other mutations contributing to progesterone hydroxylase activity at positions 374, 386, and 388 are not within SRSs. It seems most likely that these residues exert their effects by interactions indirectly altering the geometry of the substrate pocket. This possibility is consistent with the proposed interactions of residues 386 and 388 with 368 and 369.

Since all the rabbit P450 2C enzymes, except P450 2C1 and P450 2C2, are steroid hydroxylases, it is possible that mutations of ``steroid-determining'' amino acids in the N- and C-terminal regions of P450 2C1 and P450 2C2, respectively, may explain the lack of progesterone 21-hydroxylase activity in the parent proteins, which is recovered in the C2MstC1 hybrid. Comparison of P450 2C1 and P450 2C2 sequences with P450 2C5 should be most instructive since P450 2C5, like C2MstC1, is a progesterone 21-hydroxylase. There is, however, no consistent similarity between the amino acids in P450 2C5 or the other P450 2C steroid hydroxylases and the amino acids at five positions in P450 2C1 that contribute to the steroid hydroxylase activity (Fig. 9). At position 368, all the steroid hydroxylases have His as in P450 2C2 rather than Arg as in P450 2C1. Most of the steroid hydroxylases have the P450 2C1 amino acid at 369 and the P450 2C2 amino acid at 386. The contribution of specific amino acids at these positions to steroid hydroxylase activity, therefore, is not simple but is context-dependent.

Previous studies in which C-terminal 28 amino acid substitutions conferred steroid hydroxylase activity led to the suggestion that the 28 C-terminal amino acids prevented or were not permissive for steroid substrates in P450 2C2(11) . The observation that 5 substitutions outside of the 28 C-terminal positions can also confer progesterone hydroxylase activity demonstrates the this C-terminal sequence does not completely block steroid substrate interactions. The substitutions in both the residue 368 to 388 region and the C-terminal region independently confer steroid hydroxylase activity and are additive in combination.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM35897 (to B. K.) and a grant from the Deutsche Forschungsgemeinschaft (to P. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Harvard Medical School, Dept. of Neurobiology, 220 Longwood Ave., Boston, MA 02115.

Present address: Gastroenterologie und Hepatologie, Zentrum Innere Medizin, Medizinische Hochschule Hannover, D-30625 Hannover, Germany.

**
To whom correspondence and reprint requests should be addressed: Dept. of Physiology and Biophysics, University of Illinois, 524 Burrill Hall, 407 S. Goodwin Ave., Urbana, IL 61801. Tel: 217-333-1146; Fax: 217-333-1133.

(^1)
The abbreviations used are: P450, cytochrome P450; HPLC, high pressure liquid chromatography, PAGE, polyacrylamide gel electrophoresis; SRS, substrate recognition site; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Thomas Kronbach for initially reporting to us his observation that C2MstC1 acquired a novel progesterone 21-hydroxylase activity and Eric F. Johnson for supplying P450 antisera and for helpful discussions.


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