Expression of Catalytically Active Human Cytochrome P450scc inEscherichia coliand Mutagenesis of Isoleucine-462

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 353, No. 1, May 1, pp. 109 –115, 1998 Article No. BB980621

Expression of Catalytically Active Human Cytochrome P450scc in Escherichia coli and Mutagenesis of Isoleucine-4621 Stephen T. Woods, Jade Sadleir, Tristan Downs, Thrasivoulos Triantopoulos, Madeleine J. Headlam, and Robert C. Tuckey2 Department of Biochemistry, The University of Western Australia, Nedlands, Australia

Received November 24, 1997, and in revised form January 27, 1998

Cytochrome P450scc (P450scc) catalyzes the first step in steroid hormone synthesis, the conversion of cholesterol to pregnenolone. Human P450scc has been poorly studied due to the difficulty of purifying reasonable quantities of enzyme from human tissue. To provide a more convenient source of the human enzyme and to enable structure–function studies to be done using site-directed mutagenesis, we expressed the mature form of human P450scc in Escherichia coli. The expression system enabled us to produce larger quantities of active cytochrome than have previously been isolated from placental mitochondria. The expressed P450scc was purified to near homogeneity and shown to have catalytic properties comparable to the enzyme purified from the human placenta. The mature form of human adrenodoxin was also expressed in E. coli and supported cholesterol side chain cleavage activity with the same Vmax as that observed using bovine adrenodoxin but with a higher Km. Mutation of Ile-462 to Leu in human P450scc caused a decrease in the catalytic rate constant (kcat) with cholesterol as substrate, increased the Km for 22R-hydroxycholesterol, but did not affect the kinetic constants for 20ahydroxycholesterol. This suggests that Ile-462 lies close to the side chain binding site and that the side chains of cholesterol, 22R-hydroxycholesterol, and 20a-hydroxycholesterol occupy slightly different positions in the active site. © 1998 Academic Press Key Words: P450scc; adrenodoxin; pregnenolone.

1

This work was supported by the Australian Research Council (Grant A09600225). 2 To whom correspondence should be addressed. 0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

Cytochrome P450scc (P450scc)3 catalyzes the three hydroxylation reactions required to convert cholesterol to pregnenolone, the first step in steroid hormone biosynthesis. Electrons for the hydroxylation reactions are supplied by NADPH via the electron transfer proteins adrenodoxin reductase and adrenodoxin. Bovine and human cytochrome P450scc genes (CYP11A1) encode 520 and 521 amino acid proteins, respectively (1, 2). The N-terminal 39 amino acids of bovine P450scc have been shown to constitute a presequence, which targets the enzyme to the inner mitochondrial membrane and is cleaved to give the 481-amino-acid mature cytochrome (1, 3). The human and bovine forms of cytochrome P450scc have been characterized and have similar catalytic properties (4, 5). The enzymes have an amino acid sequence homology of 72% (2). Only a single gene encoding the human enzyme has been found, suggesting that steroidogenic tissues within a species contain the same isoform of cytochrome P450scc (2). The expression of active bovine cytochrome P450scc in E. coli was first reported by Wada et al. (6). Site-directed mutagenesis studies on the expressed bovine enzyme confirmed the predicted role of two lysine residues (377 and 381 of precursor protein4) in the binding of adrenodoxin (7). Substitutions of these conserved lysine residues for neutral or positive residues dramatically increased the Kd for adrenodoxin binding. Bacterial expression and site-directed mutagenesis of human adrenodoxin have similarly been used to identify the acidic resi3 Abbreviations used: P450scc, cholesterol side-chain cleavage cytochrome P450, the product of the CYP11A gene; PCR, polymerase chain reaction; kcat, the catalytic rate constant or turnover number; DTT, dithiothreitol. 4 Amino acid numbering refers to the mature form of P450scc unless the precursor form is specified.

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dues on adrenodoxin that interact with the positively charged lysines on the P450scc (8, 9). Mutagenesis has also been used to investigate the roles of the tyrosine residues at positions 93 and 94 of bovine P450scc (10). Mutations at Tyr-94 decreased the binding of cholesterol, 22R-hydroxycholesterol, and 25-hydroxycholesterol, while mutations at Tyr-93 affected only the binding of cholesterol. An important inference to be drawn from these results is that the hydroxylated substrates have a different orientation in the active site than cholesterol, which prevents interaction with Tyr-93. Ile-395 of cytochrome P450cam is a hydrophobic residue located in the camphor binding pocket (11) and is conserved in cytochrome P450scc (residue 461 for the bovine enzyme). The hydrogen bonding partner for this residue (Thr-185 in P450cam) is also conserved in P450scc (residue 224 for the bovine enzyme). In a modelled structure of bovine P450scc, based on the crystal structure of P450cam, Ile-461 is near the heme group, close to the proposed cholesterol binding site (12). It is conserved in all the species of cytochrome P450scc for which sequences are currently available. To test whether this Ile is involved in cholesterol binding, we have expressed human P450scc in Escherichia coli and used sitedirected mutagenesis to change this residue (Ile-462 in human P450scc) to Leu and to Thr. We have also expressed functional human adrenodoxin in E. coli. EXPERIMENTAL PROCEDURES Materials. The expression vector pTrc99A (13) and phenyl Sepharose CL-4B were from Pharmacia Biotech. The human placental cDNA library was from Clontech. Restriction endonucleases and other modifying enzymes were from Promega or Boehringer Mannheim. Reagents for bacterial growth media were from Difco. 20a,22R-Dihydroxycholesterol was prepared enzymatically from 22R-hydroxycholesterol (14). All other chemicals were purchased from Sigma or BDH. Bacterial expression of P450scc. Full-length cDNA encoding human cytochrome P450scc was isolated from a human placental library in lgt11. Initially the library was screened with antiserum to bovine P450scc (15–17). The resulting 50 plaques producing a positive signal were screened by hybridization using the synthetic oligonucleotide 59-ATGTTCCACACCAGCCTCCCCATG-39, end labeled with [32P]phosphate (17). From this a full-length cDNA was isolated. PCR was used to remove the precursor sequence from the P450scc cDNA to enable bacterial expression of the mature protein. A synthetic oligonucleotide containing EcoRI and NcoI sites and the initiator codon ATG within the NcoI site (underlined) was used as a 59-primer and an oligonucleotide containing a KpnI site (underlined) was used as the 39-primer. The sequences of the primers were as follows: 5’ primer, 5’-GGGAATTCCATGGCAAGTACAAGAAGTCCTCGCCCCTTCAATGAG-3’ 3’ primer, 5’-GCAGGTACCCCTTGGGCCCCACCCCTGGGCCT-3’ To create the NcoI site, the codon following the ATG was changed to encode alanine, a preferred second codon for E. coli, rather than

isoleucine in the native form. Silent mutations were made in the following six codons to enhance the AT richness of this region, which has been shown to be important in obtaining maximal yields of cytochromes P450 expressed in bacteria (7). The PCR product was cloned into the pTrc99A vector using the NcoI and KpnI restriction sites created. The correct orientation of the insert was confirmed by restriction mapping and sequencing confirmed that it encoded the expected amino acid sequence (2). Escherichia coli (JM109) was transformed with the above plasmid using the CaCl2 method (18). The transformed cells were grown on minimal media plates containing thiamine (1 mg/ml) and ampicillin (100 mg/ml). Individual colonies were removed and grown overnight in 50 ml Luria–Bertani broth. One milliliter of this culture was used to inoculate 750 ml of double-strength Luria–Bertani broth (pH 6.9), which was then incubated until the absorbance at 600 nm was 0.75. Isopropyl-b-D-thioglactopyranoside and d-aminolevulinic acid were then added to a final concentration of 1 mM each and the cultures incubated for a further 24 h at 28°C. The E. coli was harvested and resuspended in 10 mM Tris-HC1 (pH 7.8) containing 0.75 M sucrose. After treatment with lysozyme (100 mg/ml), cells were collected by centrifugation and resuspended in 100 mM potassium phosphate (pH 7.4), 0.1 mM EDTA, and 0.1 mM DTT. Cells were then sonicated on ice using a Branson sonicator with four 1-min pulses with 30-s intervals, at a power setting of 7. The sonicated E. coli were centrifuged at 106,900g for 60 min and the pellet (membrane fraction) resuspended in 100 mM potassium phosphate (pH 7.4), 20% glycerol, 0.1 mM EDTA, and 0.1 mM DTT. The protein concentration of the resuspended membranes was determined using the Lowry protein assay (19). The suspension was diluted with the same buffer to give a protein concentration of 8 mg/ml. Cytochrome P450scc was extracted by adding cholate and Emulgen 911 to final concentrations of 0.5 and 0.2%, respectively, stirring for 1 h at 0°C, then centrifugation at 106,900g for 60 min to give supernatant containing the P450scc. The expression of functional P450scc was optimized with respect to isopropyl-b-D-thioglactopyranoside and d-aminolevulinic acid concentrations, type of growth media, and media pH, as well as for the temperature and time of bacterial culture following induction of P450scc expression. Expression of human adrenodoxin in E. coli. Full-length cDNA encoding human adrenodoxin was isolated from a human placental cDNA library in lgt11 in a similar manner to that described above for the P450scc cDNA. Initially the library was screened with antiadrenodoxin antiserum (15). A full-length clone was identified by screening the resulting positives with the synthetic oligonucleotide 59-CCCGCCTCTTTGGAGTCTCCG-39. PCR was used to remove the precursor sequence from the adrenodoxin cDNA to enable bacterial expression of the mature protein. The primers used to do this were: 5’ primer, 5’-GGGAATTCCATGGCATCATCAGAAGATAAAATAACAGTC-3’ 3’ primer, 5’-GTTCTAGTTCAGGAGGTCTTGCC-3’. The 59-primer introduced an EcoRI and a NcoI site (underlined) containing the methionine initiator codon, encoded alanine as the second amino acid rather than serine (the first amino acid of the mature enzyme), and contained silent mutations to enhance the AT richness of this region. The PCR product was ligated into pGEM-T and sequenced to confirm that the sequence was correct (20). The plasmid was then digested with NcoI and SalI to enable cloning of the cDNA into these sites within pTrc99A. JM109 cells transformed with pTrc99A containing the cDNA for mature adrenodoxin were grown as described above for P450scc expression except that d-aminolevulinic acid was excluded from the culture medium. Purification of recombinant P450scc. The procedure was based on that used for purification of P450scc from placental mitochondria

EXPRESSION OF HUMAN P450scc AND MUTAGENESIS OF ILE-462 with the following modifications (4). The detergent extract from a 2-liter culture was diluted 10-fold with 0.55 M NaCl, 0.1 mM EDTA, 0.1 mM DTT, and 20% glycerol to reduce the detergent concentration. The diluted extract was applied to a column of phenyl Sepharose CL-4B equilibrated with 10 mM potassium phosphate (pH 6.8), 0.5 M NaCl, 0.1 mM DTT, 0.1 mM EDTA, 0.05% cholate, 0.02% Emulgen 911, and 20% glycerol. The gel was washed with 5 column volumes of the same buffer and the P450scc eluted by raising the detergent concentrations to 0.125% cholate and 0.05% Emulgen 911. The eluent was concentrated to 15 ml, dialyzed overnight against 10 mM potassium phosphate (pH 6.8), 0.1 mM DTT, 0.1 mM EDTA, and 20% glycerol, then passed through a column of Amberlite XAD-2 equilibrated with the same buffer, to remove remaining Emulgen 911. Emulgen 911 was then added to a final concentration of 0.05% and the sample further chromatographed on DEAE-Sephacel followed by a second phenyl Sepharose column, as described before (4). Detergent was then removed by passing the solution through a column of Amberlite XAD-2 as above and concentrated. P450scc from this step was used in most activity assays and was stored at 280°C. In an attempt to further purify the P450scc, it was applied to an adrenodoxin–Sepharose affinity column (prepared from cyanogen bromide-activated Sepharose 4B and human adrenodoxin) equilibrated with 10 mM potassium phosphate (pH 6.8), 0.1 mM DTT, 0.1 mM EDTA, and 20% glycerol. The P450scc bound efficiently to the gel and was eluted with the same buffer containing 0.1 M KCl. The recovery of P450scc from the gel was 36%. Following overnight dialysis against 100 mM potassium phosphate (pH 7.4), 0.1 mM DTT, 0.1 mM EDTA, and 20% glycerol no visible absorbance remained, indicating complete loss of the heme group. Purification of adrenodoxin and adrenodoxin reductase. Bovine adrenodoxin and adrenodoxin reductase were purified from adrenal glands as described previously (21, 22). Expressed adrenodoxin was purified by a similar procedure to that used for the bovine enzyme with some modifications. Bacterial membranes from 2 liters of culture were sedimented as described above in the P450scc purification. The brown supernatant was applied to a DE52-cellulose column (2.5 3 6 cm) equilibrated with 75 mM potassium phosphate (pH 7.4). The column was washed with 10 mM potassium phosphate (pH 7.4) and 0.15 M KCl. The adrenodoxin was eluted by increasing the KCl concentration in the buffer to 0.25 M. Fractions were analyzed by absorption spectrophotometry and those containing adrenodoxin were diluted with 10 mM potassium phosphate (pH 7.4) to adjust the KCl concentration to 0.1 M and applied to a second DEAE-cellulose column (1.5 3 7 cm). The column was washed as above and the adrenodoxin eluted with a gradient of 0.15– 0.25 M KCl. Fractions containing adrenodoxin were concentrated to 4 ml using an Amicon YM 10 membrane and then applied to a Sephadex G100 column (1.5 3 60 cm) equilibrated with 50 mM Hepes (pH 7.4). The adrenodoxin fractions were pooled, concentrated as before, and stored at 270°C. Catalytic activity of recombinant P450scc. Activity of cytochrome P450scc was measured in a reconstituted system containing 0.3% Tween 20, as described previously (4). Pregnenolone was measured by radioimmunoassay (5). Generation of site-directed mutants of P450scc. Mutant human cytochromes P450scc were constructed using the Altered Sites in vitro mutagenesis system (Promega). Wild-type human P450scc cDNA was excised from pTrc99A with NcoI and BamHI and subcloned into the NcoI–BamHI site of pALTER-Ex1. Single-stranded DNA was made by helper phage R408 infection of JM109 E. coli containing this construct. Oligonucleotide primers (with mutations underlined) were as follows: I462L, 59-ACATTCAACCTTCTTCTGATGCCT-39; I462T, 59-ACATTCAACCTGACTCTGATGCCTG-39. The I462L primer also introduced a MboII restriction site while the I462T primer introduced a HinfI site, from silent mutations. The oligonucleotides were annealed to the single-stranded DNA for second strand DNA synthesis. Second strand DNA synthesis also incor-

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porated a primer to repair the inactivated ampicillin resistance gene, and was carried out using T4 DNA polymerase and T4 DNA ligase. Following transformation, colonies with mutated plasmid were selected for by recovery of ampicillin resistance. Mutants were further identified by restriction analysis and confirmed by DNA sequencing. Mutant P450scc cDNA was subcloned into the NcoI–BamHI site of the expression vector pTrc99A and the recombinant plasmid used to transform JM109 cells. Expression of the P450scc mutants was done under the conditions described above for wild-type enzyme. Other methods. Reduced-CO minus reduced difference spectra of membrane extracts were measured with a Varian DMS 100 spectrophotometer using an extinction coefficient of 91 mM21 cm21 for the absorbance difference between 450 and 490 nm (23). SDS–polyacrylamide electrophoresis and Western blotting were performed as described before (22, 24). DNA sequencing was carried out using the Prism Dye-Deoxy Terminator kit (Perkin–Elmer). Protein sequencing was carried out on human P450scc following transfer from an SDS–polyacrylamide gel to PVDF membrane, by automated N-terminal Edman degradation using an Applied Biosystems Model 476A protein sequencer.

RESULTS

Identification of the presequence cleavage point of the human P450scc precursor protein. It has been established that the bovine P450scc precursor contains a 39-amino-acid presequence which is cleaved upon mitochondrial uptake (1, 3). Similar processing of the human P450scc has been assumed, but not directly shown. Since this information is critical to expressing the mature enzyme in E. coli, we determined the point of precursor cleavage by analyzing the N-terminal amino acid sequence of mature human P450scc isolated from placental mitochondria. The sequence, ISTRXPRPFNEI, confirmed that a 39-amino-acid presequence is removed with cleavage occurring between Gly-39 and Ile-40 of the precursor form, as for the bovine cytochrome. Expression and purification of human P450scc. The level of expression of human P450scc was insufficient to permit accurate measurement of its concentration via CO-reduced minus reduced difference spectra in whole cells so measurement was made following extraction of bacterial membranes with cholate and Emulgen 911. Expression was variable even under the optimized conditions (see Experimental Procedures). The level of expression (10 –35 nmol/liter culture) was lower than that reported for the bovine enzyme (6, 7, 10) but sufficient to make this a more convenient source of the enzyme than the human placenta, with 1 liter of culture yielding as much P450scc as up to three placentae (4). Expressed cytochrome P450scc was purified by a procedure based on the purification of the cytochrome from placental mitochondria (4). The changes made to the procedure involved omitting the ammonium sulfate fractionation step, which gave low yields of active enzyme (placental or expressed), and decreasing the detergent concentration to get stronger binding of P450scc to the phenyl Sepharose. Following

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FIG. 1. Analysis of the purity of expressed P450scc. Samples were run on two 12.5% SDS–polyacrylamide gels. One gel was stained with Coomassie blue R250 (A) and the other used for a Western transfer to a nitrocellulose membrane which was subsequently probed with anti-P450scc antiserum (B). Lane 1, P450scc following the second phenyl Sepharose step; lane 2, P450scc after chromatography on adrenodoxin Sepharose. The arrow indicates the position of the P450scc band.

the purification, analysis by SDS–polyacrylamide gel electrophoresis revealed several smaller contaminating bands present with the P450scc (Fig. 1). Further purification of the P450scc using an adrenodoxin affinity column was attempted. This gave an increase in purity but the loss of activity of the enzyme, with all of the cytochrome losing its heme group. Western blot analysis showed that some of the contaminating bands reacted with anti-P450scc antiserum, suggesting that they were P450scc degradation products (Fig. 1). This was confirmed by extracting the P450scc from the gel, boiling it with SDS–sample buffer, and running it on another gel, where the major contaminating band was again observed. Therefore it is likely that the generation of multiple bands arises from lability of the protein during sample preparation. Absolute and CO-reduced minus reduced difference spectra of the purified expressed P450scc (from the second phenyl Sepharose

step) were the same as those for P450scc purified from the human placenta. Expression and purification of human adrenodoxin. High-level expression of the mature form of human adrenodoxin was achieved with 2.7 mmol of pure protein being purified from 2 liters of bacterial culture. The adrenodoxin was purified by a procedure analogous to that used for bovine adrenodoxin. The final product ran as a single band on an SDS–polyacrylamide gel stained with Coomassie blue and displayed a 414- to 276-nm absorbance ratio of 0.78. The visible absorbance spectrum of the expressed adrenodoxin was indistinguishable from that reported for adrenodoxin purified from the human placenta (25). Catalytic properties of expressed proteins. Purified expressed cytochrome P450scc gave a turnover number (kcat) between 15 and 25 mol pregnenolone/min/mol P450scc with cholesterol as substrate, overlapping the range reported for the enzyme purified from the human placenta (4, 5). Analysis of catalytic activity with the reaction intermediates 22R-hydroxycholesterol and 20a,22R-dihydroxycholesterol, as well as with 20a-hydroxycholesterol, gave comparable Km and relative kcat values to those reported for the placental enzyme (Table I). This indicates that the expressed P450scc is a suitable source of human enzyme for catalytic studies. For both sources of human P450scc, relative kcat values indicate that the first hydroxylation in the 22R-position occurs more slowly than the subsequent hydroxylations. Also, for both sources of enzyme, the hydroxycholesterol substrates gave Km values an order of magnitude lower than that observed for cholesterol, indicating tighter binding to the active site (4, 5). The Vmax value observed for expressed human P450scc was the same whether bovine or expressed human adrenodoxin was used as electron donor. The Km measured for expressed human adrenodoxin (1.4 mM) was double that observed with bovine adreno-

TABLE I

Comparison between Catalytic Properties of Expressed and Placental Cytochromes P-450scc Placental P-450scca

Expressed P-450sccb

Substrate

Kmc (mM)

Relative kcatd

Km (mM)

Relative kcat

Cholesterol 22R-Hydroxycholesterol 20a,22R-Dihydroxycholesterol 20a-Hydroxycholesterol

63 8 10 5

1.0 2.5 5.5 0.39

71 8 3.2 15

1.0 3.5 9.3 0.85

a

Data for the placental enzyme are from Tuckey and Cameron (4). Expressed P450scc was purified to near homogeneity as described under Experimental Procedures. Data are from two P450scc preparations except for 20a,22R-dihydroxycholesterol where a single preparation was used. c Catalytic activity was measured in 0.3% Tween 20 using bovine adrenodoxin and adrenodoxin reductase. Km and kcat values were determined from Lineweaver–Burk plots. d Relative kcat refers to the ratio of the kcat for each substrate relative to the kcat for cholesterol. b

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FIG. 2. Comparison between bovine and expressed human adrenodoxin. The rate of pregnenolone synthesis from cholesterol (100 mM) by expressed human P450scc (0.02 mM) was measured using bovine adrenodoxin reductase (0.5 mM) and either bovine or expressed human adrenodoxin . The concentration of free adrenodoxin was calculated as described by Hanukoglu and Jefcoate (26). This primarily accounts for the binding of some of the adrenodoxin to adrenodoxin reductase and is important when the adrenodoxin concentration is low.

doxin (Fig. 2). The Km for expressed adrenodoxin measured using P450scc purified from the human placenta was 1.5 mM, similar to the value obtained using expressed P450scc. Mutagenesis of Ile-462 of cytochrome P450scc. Mutations were made to human cytochrome P450scc at position 462 to test the role of Ile at this position in cholesterol binding. The mutation I462T resulted in no detectable expression of holo-enzyme as judged by COdifference spectroscopy of the detergent extract of bacterial membranes. The lack of expression of active protein may have been due to the mutation causing disruption of the hydrogen bond predicted to occur

between the carbonyl backbone group of the isoleucine and the hydroxyl group of Thr-224 (12). The more conservative mutation, I462L, gave expression of active cytochrome at a level of approximately 20% that of the native enzyme. The catalytic properties of the P450scc with the I462L mutation were analyzed following partial purification of the enzyme using phenyl Sepharose chromatography. The mutant enzyme displayed a kcat for cholesterol approximately half that of the native enzyme (Table II). In contrast, no effects were seen on kcat with the I462L mutation when 22Rhydroxycholesterol or 20a-hydroxycholesterol were used as substrates. The I462L mutation caused a fourfold increase in the Km for 22R-hydroxycholesterol without affecting the Km for cholesterol or 20a-hydroxycholesterol. The data suggest that Ile-462 of human P450scc is positioned in close proximity to carbon 22 of the cholesterol side chain. DISCUSSION

We have successfully expressed human P450scc in E. coli. The level of expression is sufficient to facilitate the study of human P450scc, with 1 liter of bacterial culture typically providing as much P450scc as that extracted from between one and three placentas (4). The level of expression of the human enzyme was variable (10 –35 nmol/liter) and lower than that reported for the bovine enzyme employing a similar expression strategy (6, 7, 10). The lower level of expression of human P450scc may be due to its greater lability. For example, the exclusion of glycerol from the buffers used for purification, the use of cholate in the absence of Emulgen 911, or chromatography on adrenodoxin Sepharose result in the loss of the heme group from the human but not the bovine enzyme (4). The catalytic properties of human and bovine cytochromes P450scc are very similar although small differences have been noted (5). Greater differences exist between their chromato-

TABLE II

Comparison between Wild Type and the I462L Mutant Forms of Cytochrome P450scc Wild typea

Substrate Cholesterol 22R-Hydroxycholesterol 20a-Hydroxycholesterol

Kmb (mM) 156 6 67c 66 2 86 2

I462L mutant kcat (min21)

Km (mM)

kcat (min21)

22 6 7 44 6 19 15 6 7

160 6 68 25 6 6 12 6 7

96 2 46 6 15 11 6 4

a Wild type (expressed) and mutant enzymes were partially purified by chromatography of the detergent extract of bacterial membranes, on phenyl Sepharose. b Electron transfer to cytochrome P450scc was supported by bovine adrenodoxin reductase (0.5 mM) and expressed human adrenodoxin (15 mM). Km and kcat values for wild type and mutant enzymes were determined from Lineweaver–Burk plots. c Data are mean 6 SD of a triplicate series of determinations. Data for the wild-type P450scc are combined from two preparations of enzyme while data for the I462l mutant are from a single preparation.

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graphic properties (probably reflecting surface amino acids) with different purification protocols being required for the two proteins (4, 27). Another possible explanation for the lower level of expression of human P450scc compared with bovine P450scc could be the difference in codon usage between the human and bovine genes. The plasmid encoding the mature form of the human enzyme (but not the bovine) contains three regions in which there are four consecutive codons which are rarely used in E. coli. Stretches like these are believed to cause ribosomes to stall, causing premature termination and/or less frequent initiation of translation (28). Since the ribosome covers a region of at least nine codons, numerous rare codons within this distance are proposed to be effective in slowing down the movement of the ribosome, as well as that of the following ribosomes on the same mRNA (29). The expressed human P450scc was purified to near homogeneity, as judged by SDS–polyacrylamide gel electrophoresis. However, the final purification step utilizing adrenodoxin Sepharose resulted in the complete loss of visible absorbance, indicating the loss of the heme prosthetic group. Most of the contaminating bands present immediately prior to and following adrenodoxin–Sepharose affinity chromatography reacted with anti-P450scc antiserum, which suggests that they are breakdown products of P450scc. It is likely they were produced during sample preparation for electrophoresis. Thus the lability of human P450scc not only involves the loss of heme, but also is associated with the breakage of peptide bonds. Human P450scc purified from the placenta similarly showed lability, with degradation being observed in samples stored at 280°C for several years. The expressed human P450scc has very similar catalytic properties to human P450scc purified from the human placenta. The purification scheme used for the expressed enzyme is comparable to that for the placental enzyme. Minor differences represent an improvement to the purification protocol, rather than changes made specifically to accommodate different chromatographic properties of the expressed protein. In contrast to P450scc, high-level expression of human adrenodoxin was achieved. The expressed protein was functional in electron transport to human P450scc and gave the same Vmax as for side-chain cleavage supported by bovine adrenodoxin but a higher Km. Previously, low expression of mature human adrenodoxin fused to the mitochondrial leader sequence of cytochrome c oxidase subunit Va was reported for Saccharomyces cerevisiae (30). Human adrenodoxin has also been expressed as a fusion protein in E. coli, with subsequent cleavage required to release the mature enzyme (8). We have shown that a threefold higher level of expression of mature adrenodoxin can be achieved without the need to produce fusion protein.

Like us, Xia et al. (31) directly expressed mature human adrenodoxin in E. coli and reported expression of holoprotein at 25–50% of the level that we obtained. They were able to improve their yield of holoprotein more than threefold by in vitro reconstitution of the iron–sulfur cluster into expressed apoprotein. Little is known concerning residues in P450scc involved in holding substrate in the active site. There is good evidence from catalytic and binding studies that there is a residue which hydrogen bonds to the 3bhydroxyl group of cholesterol and one or more others which hydrogen bond to the 20a-hydroxyl and 22Rhydroxyl groups of the reaction intermediates (5, 32– 35). All other interactions between amino acid residues and cholesterol or the reaction intermediates are likely to be hydrophobic. Pikuleva et al. (10) have used sitedirected mutagenesis of Tyr-93 and Tyr-94 of bovine P450scc to implicate these residues in cholesterol binding. In the present study we have found that changing Ile-462 of human P450scc to Leu caused the kcat for cholesterol to decrease, without affecting the kcat for 20a-hydroxycholesterol or 22R-hydroxycholesterol. It also caused a fourfold increase in the Km for 22Rhydroxycholesterol but did not affect the Km measured with cholesterol or 20a-hydroxycholesterol as substrates. The weakening of the binding of 22R-hydroxycholesterol, indicated by the increased Km, may be caused by the mutation interfering with the hydrogen bonding of this intermediate to the active site. The data suggest that Ile-462 is near or in the active site, close to the site of binding of the cholesterol side chain. We have previously presented evidence that the cholesterol side chain occupies a different orientation in the active site of human P450scc then that of derivatives carrying C24 –C26 hydroxyl or keto groups (5). These analogues of cholesterol bind tightly to the active site but are only slowly converted to pregnenolone. Pikuleva et al. (10) have also provided evidence that the cholesterol side chain occupies a slightly different position in the active site compared to that of hydroxylated analogues. Mutation of Tyr-93 of the bovine enzyme suggested that this residue interacts with cholesterol but not 22Rhydroxycholesterol or 25-hydroxycholesterol. Mutation of Tyr-94 indicated that this tyrosine interacts with both cholesterol and 25-hydroxycholesterol but not with 22R-hydroxycholesterol. The present study shows that Ile-462 interacts differently with cholesterol and 22R-hydroxycholesterol. This can also be explained by 22R-hydroxycholesterol occupying a slightly different position in the active site than cholesterol. The results further suggest that there is a difference in the positioning of 22R-hydroxycholesterol and 20a-hydroxycholesterol in the active site, compatible with our previous report that 22R-hydroxycholesterol is converted to pregnenolone by hu-

EXPRESSION OF HUMAN P450scc AND MUTAGENESIS OF ILE-462

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