A Rational Approach to Re-engineer Cytochrome P450 2B1 Regioselectivity Based on the Crystal Structure of Cytochrome P450 2C5*

Santosh KumarDagger, Emily E. Scott, Hong Liu, and James R. Halpert

From the Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555-1031

Received for publication, December 9, 2002, and in revised form, February 27, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The regioselectivity for progesterone hydroxylation by cytochrome P450 2B1 was re-engineered based on the x-ray crystal structure of cytochrome P450 2C5. 2B1 is a high Km progesterone 16alpha -hydroxylase, whereas 2C5 is a low Km progesterone 21-hydroxylase. Initially, nine individual 2B1 active-site residues were changed to the corresponding 2C5 residues, and the mutants were purified from an Escherichia coli expression system and assayed for progesterone hydroxylation. At 150 µM progesterone, I114A, F297G, and V363L showed 5-15% of the 21-hydroxylase activity of 2C5, whereas F206V showed high activity for an unknown product and a 13-fold decrease in Km. Therefore, a quadruple mutant, I114A/F206V/F297G/V363L (Q), was constructed that showed 60% of 2C5 progesterone 21-hydroxylase activity and 57% regioselectivity. Based on their 2C5-like testosterone hydroxylation profiles, S294D and I477F alone and in combination were added to the quadruple mutant. All three mutants showed enhanced regioselectivity (70%) for progesterone 21-hydroxylation, whereas only Q/I477F had a higher kcat. Finally, the remaining three single mutants, V103I, V367L, and G478V, were added to Q/I477F and Q/S294D/I477F, yielding seven additional multiple mutants. Among these, Q/V103I/S294D/I477F showed the highest kcat (3-fold higher than that of 2C5) and 80% regioselectivity for progesterone 21-hydroxylation. Docking of progesterone into a three-dimensional model of this mutant indicated that 21-hydroxylation is favored. In conclusion, a systematic approach to convert P450 regioselectivity across subfamilies suggests that active-site residues are mainly responsible for regioselectivity differences between 2B1 and 2C5 and validates the reliability of 2B1 models based on the crystal structure of 2C5.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Until the recent elucidation of the rabbit cytochrome P450 2C5 x-ray crystal structure (1), chimeragenesis, site-directed mutagenesis, and homology modeling based on bacterial structures have been the primary tools available to identify key residues responsible for the substrate specificities of mammalian P450 enzymes (2). Most of these residues belong to the substrate recognition sites (SRSs)1 proposed by Gotoh (3) based on analogy with the crystal structure of bacterial P450 101 and have direct counterparts in the active site of P450 2C5. However, most P450 structures reveal that the heme group is buried deep within the protein matrix, indicating that residues outside of the active site may also be required to guide the substrate into the heme pocket by recognizing substrates at the protein surface and/or comprising part of a substrate access channel (4-6).

The role of SRS and non-SRS residues in differential substrate specificity and stereo- and regioselectivity within P450 subfamilies has been studied thoroughly, especially in the case of P450 2A, 2B, and 2C enzymes. In most cases, the functions of the enzymes could be interconverted by making multiple reciprocal substitutions at SRS residues (7-10). However, in a number of other studies, non-SRS residues were shown to play a crucial role in determining substrate specificities (11-17). These non-SRS residues were predicted to be part of a substrate access channel near a region between the F and G helices, as seen in bacterial P450 101, or between the B-C loop and N terminus of the I helix, as seen in bacterial P450 51 (18-20). The mammalian P450 2C5 structure supports the existence of both substrate access channels (1).

Structure-function studies across P450 subfamilies have been largely neglected until very recently (21, 22). Renewed interest in this area was sparked by the considerable effort in a number of laboratories to use the P450 2C5 structure to model other mammalian P450 enzymes and to predict their substrate specificities and stereo- and regioselectivity (2, 23-25). An implicit assumption in all such models based on a single template is that the backbones of the enzymes are essentially invariant and that active-site differences alone are responsible for specificity differences. In this study, we sought to confer the progesterone hydroxylation specificity of 2C5 on 2B1. 2B1 is a high Km progesterone 16alpha -hydroxylase, whereas 2C5 is a low Km progesterone 21-hydroxylase (26). 2B1 was used as a model enzyme because of extensive previous site-directed mutagenesis studies that have verified experimentally all 13 active-site residues inferred from the 2C5 structure (2, 21, 27-32). Through a systematic approach of site-directed mutagenesis, a 2B1 enzyme (V103I/I114A/F206V/S294D/F297G/V363L/I477F) was constructed that showed a 3-fold higher kcat compared with 2C5 and 80% regioselectivity for progesterone 21-hydroxylation. The results suggest a dominant role of active-site side chains in determining regioselectivity differences across these two P450 subfamilies and extend previous evidence for the reliability of 2B models based on the 2C5 structure (24).

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Oligonucleotide primers were obtained from the University of Texas Medical Branch Molecular Biology Core Laboratory. [4-14C]Progesterone and [4-14C]testosterone were obtained from PerkinElmer Life Sciences and Amersham Biosciences), respectively. All other chemicals were purchased from sources previously described (33) or from standard suppliers. Rat NADPH-cytochrome P450 reductase and cytochrome b5 were prepared as described (34).

Site-directed Mutagenesis-- The truncated version of 2B1 that served as the background for all mutations described in this study, 2B1dH, was generated using overlap extension PCR as described (33). Mutants were constructed either by overlap extension PCR or by subcloning using pKK2B1dH as a template. The primers and templates used are shown in Fig. 1. Primary forward and reverse primers were as previously described (33). To confirm the desired mutation and to verify the absence of unintended mutations, all constructs were sequenced at the University of Texas Medical Branch Protein Chemistry Laboratory.


View larger version (72K):
[in this window]
[in a new window]
 
Fig. 1.   Primers for the construction of 2B1dH mutants. The codons changed to make the desired mutation are shown in boldface, and nucleotide(s) changed to remove a restriction site are underlined. Multiple mutants constructed by subcloning are also shown with the restriction sites used.

Expression and Purification of P450 2B1dH Mutants-- 2B1dH and its mutants were expressed in Escherichia coli TOPP3 (Stratagene) and purified as described (33). In brief, bacteria were grown in Terrific Broth salts for ~2.5 h at 37 °C before induction using isopropyl-beta -D-thiogalactopyranoside and supplementation with the heme precursor delta -aminolevulinic acid. After 72 h at 30 °C, the cells were harvested by centrifugation, and protein was extracted from lysed membranes using potassium phosphate buffer (pH 7.4). Protein extract was loaded onto a Ni2+ affinity column, which was washed with 20 volumes of buffer, and then eluted with 200 mM imidazole. Cytochrome P450 was quantitated using the reduced CO difference spectrum (35). Specific content was determined using the Pierce BCA kit with bovine serum albumin as a standard. The specific content for 2B1dH was 18 nmol of P450/mg of protein, whereas the specific content for most of the mutants was between 8 and 16. However, the I114A/F206V/S294D/F297G/V363L, I114A/F206V/S294D/F297G/V363L/I477F, and V103I/I114A/F206V/S294D/F297G/V363L/I477F multiple mutants had specific contents of 4, 5, and 2 nmol, respectively. Low specific content in these mutants is accounted for by the presence of significant amounts of P420.

Enzymatic Assays-- Progesterone and testosterone hydroxylation assays were carried out essentially as described (33, 36) using a 1:4:2 molar ratio of P450/cytochrome P450 reductase/cytochrome b5 in the absence of lipid. 16alpha - and 15alpha -hydroxyprogesterone were not resolved. However, the progesterone 16alpha -hydroxylase activity of 2B1 has been measured previously using two-dimensional chromatography (21). Km and kcat values were determined by regression analysis using Sigma Plot (Jandel Scientific, San Rafael, CA).

Computer Modeling-- A molecular model of P450 2B1 was constructed using the InsightII software package (Homology, Discover_3, Biopolymer, Builder, and Docking from Molecular Simulations Inc., San Diego, CA) and P450 2C5 as the template as described previously (24). For the 2B1 mutant (V103I/I114A/F206V/S294D/F297G/V363L/I477F), the coordinates of the corresponding residues were changed in the 2B1 three-dimensional model by Biopolymer, and the resulting 2B1 mutant was minimized.

The structure of progesterone was constructed using the Builder module. The parameters for heme and ferryl oxygen were those described by Paulsen and Ornstein (37, 38). During the docking calculations, the system energy minimization and molecular dynamics simulations were carried out with the Discover_3 program using the consistent valence force field with a non-bond cutoff of 10 Å to a maximum gradient of 5 kcal mol-1 Å-1. Progesterone was automatically docked into the three-dimensional models of 2B1 and V103I/I114A/F206V/S294D/F297G/V363L/I477F in a reactive binding orientation with the Docking module of InsightII, leading to 16alpha - or 21-hydroxylation. Because the initial oxidation step involves hydrogen abstraction, the C-16 or C-21 atom was placed 3.7 Å from ferryl oxygen, with the 16alpha -hydrogen or one of the hydrogen atoms bonded to C-21 directed toward ferryl oxygen (C-H-ferryl oxygen angle of 180°) to promote hydrogen bond formation. During the subsequent energy minimization process, the substrate molecule, along with the side chains of protein residues within 5 Å of the substrate, was allowed to move. The non-bond interaction energies were evaluated with the Docking module of InsightII, and the lowest energy orientation obtained after molecular mechanics minimization of 2B1 and its mutant is shown in Fig. 6.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

2B1dH Single Mutants I114A, F297G, and V363L Show Significant Progesterone 21-Hydroxylase Activity-- 2B1dH single mutants were constructed at the nine active-site positions where 2B1 and 2C5 differ and tested for oxidation of progesterone. Consistent with previous findings, 2B1dH had negligible progesterone 21-hydroxylase activity, but significant progesterone 16alpha -hydroxylase activity (>2 min-1) at 150 µM progesterone (Fig. 2), which was the highest substrate concentration obtainable under our conditions. However, the 2B1dH single mutants I114A, F297G, and V363L had 5-15% of 2C5dH progesterone 21-hydroxylase activity. With regard to progesterone 16alpha - and 15alpha -hydroxylation, the I114A mutant showed enhanced activity, whereas F297G and V363L showed decreased activity (Fig. 2). Although V363L demonstrated lower activity for progesterone 21-hydroxylation compared with I114A, it showed high regioselectivity for this reaction (>70%). The F206V substitution yielded relatively high activity (6 min-1) and >95% regioselectivity for an unknown product (Fig. 2). The progesterone hydroxylation profile of V103I was similar to that of 2B1dH, whereas S294D, V367L, I477F, and G478V showed negligible progesterone hydroxylase activity.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 2.   Progesterone hydroxylation profiles of 2C5dH, 2B1dH, and 2B1dH single mutants. The progesterone hydroxylase activities determined are indicated. The activity was measured at 150 µM substrate as described under "Experimental Procedures." The bars represent the means obtained from two independent determinations. 16alpha - and 15alpha -hydroxyprogesterone do not separate under the conditions used. The structure of progesterone is shown in the inset, with the 16alpha - and 21-positions indicated.

The Km for Progesterone Is Decreased by ~13-Fold upon F206V Substitution-- As shown in Table I, the Km for progesterone of 2B1dH was determined to be 200 µM. Mutants I114A and F297G showed a 3-fold decrease in Km, whereas V363L showed a 2-fold decrease. Interestingly, a F206V substitution decreased the Km by ~13-fold. The kcat values for progesterone 21-hydroxylation by I114A, F297G, and V363L were in the range of 1-2 min-1 versus 16 min-1 for 2C5dH (Table I).


                              
View this table:
[in this window]
[in a new window]
 
Table I
Steady-state kinetics of 2B1dH single mutants for progesterone 16alpha +15alpha - and 21-hydroxylase and unknown activities
Results are the means ± S.D. of three independent experiments.

I114A/F206V/F297G/V363L Shows Enhanced kcat and Regioselectivity for Progesterone 21-Hydroxylation-- A number of multiple mutants were constructed by combining I114A, F297G, and V363L to test the additive effect on progesterone 21-hydroxylase activity. In addition, F206V was also added to assess whether this mutation decreases the Km. The progesterone hydroxylation profiles of the multiple mutants are presented in Fig. 3. As expected, I114A/F297G (Fig. 3, A bars) showed enhanced progesterone 21-hydroxylase activity compared with either single mutant. Addition of V363L to I114A/F297G increased the regioselectivity for progesterone 21-hydroxylation, although activity was suppressed (Fig. 3, B bars versus A bars). Addition of F206V to I114A and I114A/V363L caused high activity for unknown products while suppressing progesterone 21- and 16alpha /15alpha -hydroxylase activities (Fig. 3, C and E bars). However, addition of F206V to I114/F297G did not suppress progesterone 21- and 16alpha /15alpha -hydroxylase activities (Fig. 3, D bars). Interestingly, addition of F206V to I114A/F297G/V363L enhanced activity for progesterone 21-hydroxylation and suppressed progesterone 16alpha - and 15alpha -hydroxylase activity, but still allowed significant production of unknown products (Fig. 3, F bars). Unlike 2B1dH, which produced only 16alpha -hydroxyprogesterone, and 2C5dH, which produced only 21-hydroxyprogesterone, the quadruple mutant I114A/F206V/F297G/V363L (Q) produced two major and two minor unknown products (Fig. 4). 21-Hydroxyprogesterone was determined to compose 57% of all the products in the quadruple mutant. I114A/F206V, I114A/F206V/F297F, and I114A/F206V/V363L had a decreased Km for progesterone versus those mutants without Val206. The Km for progesterone was unaltered in I114A/F297G and I114A/F297G/V363L compared with the individual single mutants (Table II), further suggesting the crucial role of F206V in enhancing the affinity for the substrate. The kcat for the quadruple mutant was 60% of that for 2C5dH, and the Km was decreased by 3-4-fold compared with 2B1dH (Tables I and II). The 10-fold increase in kcat for progesterone 21-hydroxylation with no change in Km upon addition of F206V to I114A/F297G/V363L is striking and is different from other multiple mutants that include F206V.


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3.   Progesterone hydroxylation profiles of 2B1dH multiple mutants. A bars, I114A/F297G; B bars, I114A/F297G/V363L; C bars, I114A/F206V; D bars, I114A/F206V/F297G; E bars, I114A/F206V/V363L; F bars, I114A/F206V/F297G/V363L (quadruple mutant). The progesterone hydroxylase activities determined are indicated. The activity was measured at 150 µM substrate as described under "Experimental Procedures." The bars represent the means obtained from two independent determinations. 16alpha - and 15alpha -hydroxyprogesterone were isolated together.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 4.   Thin-layer chromatography of progesterone metabolites produced by 2B1dH, 2C5dH, and the 2B1dH quadruple mutant. The quadruple mutant is 2B1dH I114A/F206V/F297G/V363L. Unknown 3 is identical to the unknown obtained with F206V in Fig. 2. 16alpha - and 15alpha -hydroxyprogesterone co-migrate and were isolated together.


                              
View this table:
[in this window]
[in a new window]
 
Table II
Steady-state kinetics of 2B1dH multiple mutants for progesterone 21-hydroxylase and unknown activities
Results are the means ± S.D. of three independent experiments.

The Testosterone Hydroxylation Profiles of S294D and I477F Are Similar to That of 2C5dH-- Testosterone hydroxylation profiles were determined for 2B1dH single mutants and compared with those of 2B1dH and 2C5dH. As reported above, 2B1dH produced equal amounts of 16alpha - and 16beta -hydroxytestosterone, whereas 2C5dH demonstrated mainly testosterone 16beta -hydroxylase activity (Fig. 5). Of the nine single mutants tested, S294D, V363L, and I477F had decreased testosterone 16alpha -hydroxylase activity and enhanced 16beta -hydroxylase activity (similar to 2C5dH). The Km of 2C5dH for testosterone (Table III) was similar to the reported Km of 2B1dH (39). However, the S294D and I477F single mutants demonstrated a 2-fold lower Km for testosterone. Interestingly, F206V showed decreased 16alpha - and 16beta -testosterone hydroxylase activities and a new testosterone 6alpha -hydroxylase activity. This profile of testosterone hydroxylation is similar to the progesterone hydroxylation profile in that F206V primarily showed activity for a new product (Figs. 2 and 5). On the other hand, V103I showed unaltered testosterone hydroxylation, and F297G, V367L, and G478V showed decreased testosterone 16alpha - and 16beta -hydroxylase activities. The similar testosterone hydroxylation profiles and affinities for substrate of 2C5dH, S294D, and I477F suggested that addition of S294D and/or I477F to the 2B1dH quadruple mutant might enhance progesterone 21-hydroxylase activity and/or regioselectivity.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 5.   Testosterone hydroxylation profiles of 2B1dH, 2C5dH, and 2B1dH single mutants. The testosterone hydroxylase activities determined are indicated. The activity was measured at 200 µM substrate as described under "Experimental Procedures." The bars represent the means obtained from two independent determinations.


                              
View this table:
[in this window]
[in a new window]
 
Table III
Steady-state kinetics and regioselectivity for testosterone 16beta -hydroxylase activity of 2B1dH, 2C5dH, and 2B1dH single mutants
Results are the means ± S.D. of two independent experiments.

Q/I477F and Q/S294D/I477F Show Enhanced Progesterone 21-Hydroxylase Activity and Regioselectivity-- Q/S294D, Q/I477F, and Q/S294D/I477F were examined for progesterone hydroxylation using 150 µM substrate (Table IV). Progesterone 21-hydroxylase activity was largely unaffected, whereas regioselectivity for Q/S294D, Q/S294D/I477F, and Q/I477F was increased to 73, 69, and 67%, respectively, compared with 57% for the quadruple mutant. Steady-state kinetic parameters were also measured for these multiple mutants (Table IV). The kcat for Q/S294D and Q/S294D/I477F was similar to that for the quadruple mutant, but was increased for Q/I477F (similar to 2C5dH). However, the Km was either unaffected in the case of Q/S294D and Q/S294D/I477F or increased in the case of Q/I477F. These observations suggested that addition of S294D to the quadruple mutant increased regioselectivity for progesterone 21-hydroxylation, whereas I477F enhanced the activity as well as the regioselectivity. The kcat and regioselectivity for progesterone 21-hydroxylation were much improved over those for the quadruple mutant and close to those for 2C5dH. To test further improvement of progesterone 21-hydroxylase activity and regioselectivity, the remaining three mutants (V103I, V367L, and G478V) were added to Q/I477F and Q/S294D/ I477F.


                              
View this table:
[in this window]
[in a new window]
 
Table IV
Progesterone hydroxylation at 150 µM substrate and steady-state kinetics and regioselectivity for progesterone 21-hydroxylase activity

Addition of V103I to Q/I477F and Q/S294D/I477F Further Enhances kcat and Regioselectivity for Progesterone 21-Hydroxylation-- Q/V103I/I477F and Q/V103I/S294D/I477F showed ~80% regioselectivity for progesterone 21-hydroxylation (Table V). The kcat for progesterone 21-hydroxylation by Q/V103I/I477F and Q/V103I/S294D/I477F was 1.5- and 3-fold higher than that for 2C5dH, respectively. The Km for progesterone was largely unchanged from that of the mutants lacking V103I (Table V versus Table IV). Addition of S294D to Q/I477F and Q/V103I/I477F yielded high P420, as described under "Experimental Procedures," whereas addition of S294D/V367L to Q/V103I/I477F and Q/V103I/I477F/G478V yielded only P420 (data not shown). As expected based on the V367L and G478V single mutants, Q/V103I/V367L/I477F, Q/V103I/I477F/G478V, and Q/V103I/S294D/I477F/G478V showed much lower activity and regioselectivity for progesterone 21-hydroxylation compared with Q/V103I/I477F and Q/V103I/S294D/I477F (Table V). The Km for substrate was not substantially affected compared with Q/V103I/I477F and Q/V103I/S294D/I477F.


                              
View this table:
[in this window]
[in a new window]
 
Table V
Progesterone hydroxylation at 150 µM substrate and steady-state kinetics and regioselectivity for progesterone 21-hydroxylase activity
Q/V103I/S294D/V367L/I477F and Q/V103I/S294D/V367L/I477F/G478V do not express.

Docking of Progesterone into the Active Site of 2B1dH and Q/V103I/S294D/I477F Models-- To explain the changes in regioselectivity observed for progesterone in 2B1 Q/V103I/S294D/I477F, a molecular model was constructed. Fig. 6 (A and B) shows progesterone docked into the active site of the wild-type 2B1 and 2B1 Q/V103I/S294D/I477F models, respectively. The substrate fit well in the wild-type 2B1 active site, with no van der Waals overlaps when docked in an orientation that leads to formation of 16alpha -hydroxyprogesterone (Fig. 6A). However, the substrate did not fit in a 21-OH orientation. The estimated angle (C-H-ferryl oxygen) and distance (between C-16 and ferryl oxygen) in 2B1 are 153.0° and 4.43 Å, respectively. Active-site residues Ile114, Phe206, Phe297, Val363, and Ile477 are within 5 Å of the substrate. In contrast, progesterone fit well in the 2B1 Q/V103I/S294D/I477F active site when docked in a 21-OH orientation, but not in a 16alpha -OH orientation (Fig. 6B). The estimated angle (C-H-ferryl oxygen) and distance (between C-21 and ferryl oxygen) are 160.6° and 3.69 Å, respectively. These are close to the angle and distance required for hydrogen bond formation (180° and 3.7 Å, respectively) and are similar to those for 2C5 (Ref. 1 and data not shown). Active-site residues Ile103, Ala114, Val206, Asp294, Gly297, Leu363, and Phe477 are within 5 Å of the substrate, with Ala114 and Phe477 lying closest at 3 Å. Our modeling results are consistent with the biochemical data that 2B1 favors 16alpha -hydroxylation, whereas 2B1 Q/V103I/S294D/I477F favors the formation of 21-hydroxyprogesterone. Consistent with the experimental data, modeling of progesterone in 2B1 mutants that included V367L and/or G478V along with Q/V103I/S294D/I477F showed poor fits (data not shown). This suggests that V367L and G478V mutations, either individually or in combination with others, change the active-site structure in a way that is unfavorable for progesterone 21-hydroxylation.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 6.   Docking of progesterone into the active site of P450 2B1 models. A, substrate was docked in 2B1dH in an orientation leading to formation of 16alpha -hydroxyprogesterone; B, substrate was docked into Q/V103I/S294D/I477F in an orientation leading to 21-hydroxyprogesterone. The heme (red sticks), progesterone (brown space-filling representation), and active-site residues (purple sticks) are shown. The carbon atoms at positions 16 and 21 of progesterone are shown in blue and green, respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The recent elucidation of the x-ray crystal structure of the first mammalian P450 (rabbit 2C5) has sparked intense interest in understanding the structural basis of P450 function to facilitate drug discovery/design and engineering of novel biocatalysts (1). This breakthrough has led to advanced homology models of drug-metabolizing cytochromes P450, especially the enzymes from the P450 2 subfamily, relative to those constructed based on bacterial enzymes (25). The P450 2C5 crystal structure has also provided an enhanced framework for identifying active-site residues and investigating their role in differential substrate specificities and stereo- and regioselectivity across subfamilies (2, 22). By extensive site-directed mutagenesis studies and more recently by analogy with 2C5, 13 2B1 active-site residues have been identified, nine of which differ from those of 2C5 (2). In the present study, 2B1 residues have been replaced systematically by the corresponding active-site residues of 2C5 to confer a novel progesterone hydroxylase activity (progesterone 21-hydroxylation).

The major finding is that simultaneous substitution of seven 2B1 active-site residues (positions 103, 114, 206, 294, 297, 363, and 477) with the corresponding 2C5 residues resulted in a 3-fold higher kcat for progesterone 21-hydroxylation compared with 2C5 with 80% regioselectivity. However, the Km for the substrate remained an order of magnitude higher than that for 2C5. Consistent with the experimental data, substrate docking in the active site of a model of the multiple mutant showed that the formation of 21-hydroxyprogesterone is favored, unlike wild-type 2B1, which favors 16alpha -hydroxyprogesterone. To produce a 2B1 multiple mutant with the desired phenotype, a three-tiered approach was used. First, 2B1 single mutants were made at all nine non-identical active-site positions, and three mutants that showed progesterone 21-hydroxylation (I114A, F297G, and V363L) and a fourth with a decreased Km (F206V) were combined to yield a quadruple mutant. Second, additional substitutions (S294D and I477F) were added based on a 2C5-like testosterone hydroxylation profile. Finally, V103I, which had no effect on progesterone or testosterone hydroxylation profiles on its own, was added to Q/S294D/ I477F.

New insights can be gained by comparing and contrasting the present study with our recent investigation of active-site determinants of specificity differences between P450 2B6 and 2E1 (22). In that case, a single point mutation at the alignment position corresponding to residue 477 in 2B enzymes conferred on 2E1 significant activity for the 2B6-selective substrate 7-ethoxy-4-trifluoromethylcoumarin and abolished activity for the 2E1-selective substrate p-nitrophenol. However, none of six 2B6 single mutants gained activity for p-nitrophenol. The two major advances in this investigation were the focus on regioselectivity differences for a common substrate, progesterone, and the generation of multiple 2B mutants, many of which included substitutions that, on their own, did not enhance the activity of interest. Based on the results, it appears that, in addition to direct interactions of active-site residues with substrate, residue-residue interactions and/or an influence of active-site backbone residues on orientation of the substrate may also be important (4, 24). Residue-residue interactions have been implicated in determining stereo- and regioselectivity for androstenedione hydroxylation and differential inhibition by 4-phenylimidazole in 2B4 and 2B5 (7, 24). For example, mutagenesis experiments and molecular modeling suggest that the side chains of residues 114 and 294 in 2B4 and 2B5 move in concert to influence the 4-phenylimidazole binding orientation (24). In the present case, there were increases in progesterone 21-hydroxylase activity of 10-, 2-, and 3-fold upon addition of F206V to I114A/F297G/V363L, I477F to I114A/F206V/F297G/V363L, and V103I to Q/I477F, respectively, even though F206V, I477F, and V103I alone showed negligible progesterone 21-hydroxylase activity. These observations may reflect an additional interaction between Phe477 and/or Phe206 and progesterone that leads to tighter packing in the active site and an increased frequency of productive collisions (Fig. 6). Ile103 is also closer to progesterone in Q/V103I/S294D/I477F compared with 2B1.

The almost complete conversion of stereoselectivity for testosterone 16-hydroxylation by 2B1 S294D, V363L, and I477F to that of 2C5 aided in making multiple mutants with higher progesterone 21-hydroxylase activity and regioselectivity. However, it should be recognized that the 2C5-like testosterone hydroxylation profiles of S294D, V367L, and I477F resulted from abolished testosterone 16alpha -hydroxylase activity and enhanced 16beta -hydroxylase activity, rather than acquisition of a novel activity as with progesterone 21-hydroxylation by I114A, F297G, and V363L. There are numerous prior examples of stereoselective loss of activity upon single amino acid substitutions in bacterial and mammalian P450 enzymes (28, 40, 41). On the other hand, F206V exhibited a novel activity with both progesterone and testosterone along with greatly suppressed original activities. Residue 206 in 2B1 has previously been shown to be critical in converting steroid 16- to 15alpha -hydroxylation (21), and the analogous residue in 2a4 and 2a5 is a major determinant of differences in substrate specificity (40). The F205V substitution in 2C5 results in almost a complete loss of progesterone 21-hydroxylase activity and gain of new activity, suggesting that this residue is critical in determining P450 regioselectivity (26). However, the structural basis for these observations remains unclear.

In the absence of deleterious steric interactions with 2B1, the more hydrophobic progesterone should be characterized by a lower Km compared with testosterone, as observed with 2C5. However, wild-type 2B1 exhibited a 7-fold higher Km for progesterone than for testosterone. This suggests that the larger 17beta -acetyl group in progesterone, as opposed to the hydroxyl group in testosterone, clashes with one or more residues in the 2B1 active site. Furthermore, a F206V substitution in 2B1, either individually or in combination with I114A, I114A/F297G, or I114A/V363L, decreased the Km for progesterone by 6-13-fold, suggesting a major role of Val206 in determining affinity. Other multiple mutants that included F206V exhibited an increased Km, which may be due to the occurrence of additional unfavorable interactions. The Km is generally affected by parameters such as size, shape, and hydrophobicity of the substrate and of active-site residues (42). A similar Phe-to-Val substitution at residue 226 in P450 1A2 (analogous to residue 206 in 2B1) has also been shown to have a strong bearing on substrate affinity (43). An excellent correlation has been observed between Km values and side chain size at residue 209 in P450 2a5 (analogous to residue 206 in 2B1), in which the Km values decrease as the side chains become larger regardless of the hydrophobicity (44). The larger side chain of Phe206 in 2B1 may interact sterically with the additional acetyl group in progesterone compared with testosterone (Fig. 6) (1). The F206V substitution may provide more room for entry in a particular orientation leading to increased affinity for substrate.

In summary, our results demonstrate that active-site residues are mainly responsible for determining differences in regioselectivity for progesterone hydroxylation between 2B1 and 2C5. A synergistic effect on progesterone 21-hydroxylation activity and regioselectivity by certain multiple substitutions suggests a role of residue-residue interactions in determining active-site topology and substrate orientation. This report suggests the feasibility of rational redesign of mammalian P450 specificity based on analogy with P450 2C5, as previously performed for bacterial P450 enzymes of known three-dimensional structure (45). This approach provides an excellent complement to directed evolution by random mutagenesis, which tends to mainly pinpoint non-active-site residues (46, 47).

    ACKNOWLEDGEMENTS

We thank Drs. Tammy L. Domanski, Kishore K. Khan, and Dmitri R. Davydov and You Qun He for invaluable help while pursuing this work and preparing the manuscript. We also thank Dr. Eric F. Johnson for providing a P450 2C5dH protein sample.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant ES03619 and Center Grant ES06676.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1031. Tel.: 409-772-9677; Fax: 409-772-9642; E-mail: sakumar@utmb.edu.

Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M212515200

    ABBREVIATIONS

The abbreviation used is: SRSs, substrate recognition sites.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Williams, P. A., Cosme, J., Sridhar, V., Johnson, E. F., and McRee, D. E. (2000) Mol. Cell 5, 121-131[Medline] [Order article via Infotrieve]
2. Domanski, T. L., and Halpert, J. R. (2001) Curr. Drug Metab. 2, 117-137[Medline] [Order article via Infotrieve]
3. Gotoh, O. (1992) J. Biol. Chem. 267, 83-90[Abstract/Free Full Text]
4. Haseman, C. A., Kurumbail, R. G., Boddupalli, S. S., Peterson, J. A., and Deisenhofer, J. (1995) Structure 3, 41-62[Medline] [Order article via Infotrieve]
5. Graham-Lorence, S., and Peterson, J. A. (1996) FASEB J. 10, 206-214[Free Full Text]
6. Waller, C. L., Evans, M. V., and McKinney, J. D. (1996) Drug Metab. Dispos. 2, 203-210
7. He, Y. Q., Szklarz, G. D., and Halpert, J. R. (1996) Arch. Biochem. Biophys. 335, 152-160[CrossRef][Medline] [Order article via Infotrieve]
8. Strobel, S. M., and Halpert, J. R. (1997) Biochemistry 36, 11697-11706[CrossRef][Medline] [Order article via Infotrieve]
9. Negishi, M., Uno, T., Honkakoski, P., Sueyoshi, T., Darden, T., and Pedersen, L. P. (1996) Biochimie (Paris) 78, 685-694
10. Goldstein, J. A., and de Morais, S. M. (1994) Pharmacogenetics 4, 285-299[Medline] [Order article via Infotrieve]
11. Hoch, U., Falck, J. R., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 26952-26958[Abstract/Free Full Text]
12. Domanski, T. L., Schultz, K. M., Roussel, F., Stevens, J. C., and Halpert, J. R. (1999) J. Pharmacol. Exp. Ther. 290, 1141-1147[Abstract/Free Full Text]
13. He, Y. Q., Harlow, G. R., Szklarz, G. D., and Halpert, J. R. (1998) Arch. Biochem. Biophys. 350, 333-339[CrossRef][Medline] [Order article via Infotrieve]
14. Ibeanu, G. C., Ghanayem, B. I., Linko, P., Li, L., Pederson, L. G., and Goldstein, J. A. (1996) J. Biol. Chem. 271, 12496-12501[Abstract/Free Full Text]
15. Pikuleva, I., Puchkaev, A., and Bjorkhem, I. (2001) Biochemistry 40, 7621-7629[Medline] [Order article via Infotrieve]
16. Nakayama, K., Puchkaev, A., and Pikuleva, I. (2001) J. Biol. Chem. 276, 31459-31465[Abstract/Free Full Text]
17. Tsao, C. C., Wester, M. R., Ghanayem, B., Coulter, S. J., Chanas, B., Johnson, E. F., and Goldstein, J. A. (2001) Biochemistry 40, 1937-1944[CrossRef][Medline] [Order article via Infotrieve]
18. Poulos, T. L., Cupp-Vickery, J., and Li, H. (1995) in Cytochrome P450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P. R., ed) , pp. 125-150, Plenum Press, New York
19. Poulos, T. L., Finzel, B. C., Gunsalus, I. C., Wagner, G. C., and Kraut, J. (1985) J. Biol. Chem. 260, 16122-16130[Abstract/Free Full Text]
20. Podust, L. M., Poulos, T. L., and Waterman, M. R. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3068-3073[Abstract/Free Full Text]
21. Luo, Z., He, Y. A., and Halpert, J. R. (1994) Arch. Biochem. Biophys. 309, 52-57[CrossRef][Medline] [Order article via Infotrieve]
22. Spatzenegger, M., Liu, H., Wang, Q., Debarber, A., Koop, D. R., and Halpert, J. R. (2003) J. Pharmacol. Exp. Ther. 304, 477-487[Abstract/Free Full Text]
23. Afzelius, L., Zamora, I., Ridderström, M., Andersson, T. B., Karlén, A., and Masimirembwa, C. M. (2001) Mol. Pharmacol. 59, 909-919[Abstract/Free Full Text]
24. Spatzenegger, M., Wang, Q., He, Y. Q., Wester, M. R., Johnson, E. F., and Halpert, J. R. (2001) Mol. Pharmacol. 59, 475-484[Abstract/Free Full Text]
25. Lewis, D. F. V. (2002) J. Inorg. Biochem. 91, 502-514[CrossRef][Medline] [Order article via Infotrieve]
26. Cosme, J., and Johnson, E. F. (2000) J. Biol. Chem. 275, 2545-2553[Abstract/Free Full Text]
27. Kedzie, K. M., Balfour, C. A., Escobar, G. Y., Grimm, S. W., He, Y. A., Pepperl, D. J., Regan, J. W., Stevens, J. C., and Halpert, J. R. (1991) J. Biol. Chem. 266, 22515-22521[Abstract/Free Full Text]
28. Halpert, J. R., and He, Y. A. (1993) J. Biol. Chem. 268, 4453-4457[Abstract/Free Full Text]
29. He, Y. A., Luo, Z., Klekotka, P. A., Burnett, V. L., and Halpert, J. R. (1994) Biochemistry 33, 4419-4424[Medline] [Order article via Infotrieve]
30. Szklarz, G. D., He, Y. A., and Halpert, J. R. (1995) Biochemistry 34, 14312-14322[Medline] [Order article via Infotrieve]
31. Kobayashi, Y., Strobel, S. M., Hopkins, N. E., Alworth, W. L., and Halpert, J. R. (1998) Drug Metab. Dispos. 26, 1026-1030[Abstract/Free Full Text]
32. Domanski, T. L., He, Y. Q., Scott, E. E., Wang, Q., and Halpert, J. R. (2001) Arch. Biochem. Biophys. 394, 21-28[CrossRef][Medline] [Order article via Infotrieve]
33. Scott, E. E., Spatzenegger, M., and Halpert, J. R. (2001) Arch. Biochem. Biophys. 395, 57-68[CrossRef][Medline] [Order article via Infotrieve]
34. Harlow, G. R., He, Y. A., and Halpert, J. R (1997) Biochim. Biophys. Acta 1338, 259-266[Medline] [Order article via Infotrieve]
35. Omura, T., and Sato, R. (1964) J. Biol. Chem. 239, 2379-2387[Free Full Text]
36. Ciaccio, P. J., and Halpert, J. R. (1989) Arch. Biochem. Biophys. 271, 284-299[Medline] [Order article via Infotrieve]
37. Paulsen, M. D., and Ornstein, R. L. (1991) Proteins 11, 184-204[Medline] [Order article via Infotrieve]
38. Paulsen, M. D., and Ornstein, R. L. (1992) J. Comput. Aided Mol. Des. 6, 449-460[Medline] [Order article via Infotrieve]
39. Scott, E. E., He, Y. Q., and Halpert, J. R. (2002) Chem. Res. Toxicol. 15, 1407-1413[CrossRef][Medline] [Order article via Infotrieve]
40. Lindberg, R. L., and Negishi, M. (1989) Nature 339, 632-634[CrossRef][Medline] [Order article via Infotrieve]
41. Uno, T., Mitchell, E., Aida, K., Lambert, M. H., Darden, T. A., Pedersen, L. G., and Negishi, M. (1997) Biochemistry 36, 3193-3198[CrossRef][Medline] [Order article via Infotrieve]
42. Negishi, M., Uno, T., Darden, T. A., Sueyoshi, T., and Pedersen, L. G. (1996) FASEB J. 10, 683-689[Abstract/Free Full Text]
43. Parikh, A., Josephy, P. D., and Guengerich, F. P. (1999) Biochemistry 38, 5283-5289[CrossRef][Medline] [Order article via Infotrieve]
44. Juvonen, R. O., Iwasaki, M., and Negishi, M. (1991) J. Biol. Chem. 266, 16431-16435[Abstract/Free Full Text]
45. Miles, C. S., Ost, T. W. B., Noble, M. A., Munro, A. W., and Chapman, S. K. (2000) Biochim. Biophys. Acta 1543, 383-407[Medline] [Order article via Infotrieve]
46. Farinas, E. T., Bulter, T., and Arnold, F. H. (2001) Curr. Opin. Biotechnol. 12, 545-551[CrossRef][Medline] [Order article via Infotrieve]
47. Cirino, P. C., and Arnold, F. H. (2002) Curr. Opin. Chem. Biol. 6, 130-135[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.