Molecular Basis for a Functionally Unique Cytochrome P450IIB1 Variant

Vol. 266, No . 33, Issue of November 25, pp . 22515-22521, 1991 Printed in U.S.A.

, OF BIOLOGICAL CHEMISTRY THEJOURNAL (c

1991 bv The American Society for Biochemistry and Molecular Biology, Inc.

Molecular Basis for a Functionally Unique CytochromeP450IIB1 Variant* (Received for publication, June 24, 1991)

Karen M. KedzieS, Celia A. Balfour, GinaY. Escobar, ScottW. Grimm, You-ai He, David J. Pepperl, John W. Regang, Jeffrey C. Stevensll, and JamesR. Halpert)) From the Department of Pharmacology and Toxicology, Collegeof Pharmacy, University of Arizona, Tucson, Arizona 85721

tiple forms of the enzyme (1).A question of great interest is Liver microsomes from phenobarbital-treated rats of four inbred strains expressing distinct allelic var- thestructuralbasis for the specificity of eachindividual iants of cytochrome P450IIB1 were analyzed.The cytochrome P450 form.At present,nothree-dimensional Wistar Munich (WM) strain exhibited 5- to 10-fold structure has been reported for a mammalian cytochrome (a P450, and theregions of the molecule directly responsible for lowerandrostenedione16B-hydroxylaseactivity specific P450IIB1 marker) than the Lewis, Wistar substrate binding and catalysis are not well defined. Recent Kyoto, and Wistar Furth strains. The androstenedione studies with site-directed mutants and hybrid enzymes have 16@-hydroxylaseinthe WM liver microsomes was begun to identify such regions (2-8), although the ability to refractory inactivation to by N-(Z-p-nitrophen- relate the information from one P450 family to another is ethyl)chlorofluoroacetamide, a selective P450IIB1 limited. Models of mammalian P450s’ (9, 10) may also permit inactivator in the other three strains. Purified P450IIB1-WM was insensitive to the inactivator and the identification of functionally important regions of the molecule, but much of the work is still speculative. exhibited &fold lower androstenedione 16b-hydroxOne very useful method for establishing structure-function ylase, testosterone 16-hydroxylase, and 7-ethoxycourelationships among cytochromes P450 is through comparison marin deethylase activities but the same benzphetamine demethylase activity and slightly higher andro- of allelic variants. These are encodedby the same genetic stenedione 16a-hydroxylase activity thana P450IIB1 locus in different strains or individuals of the same species purified from outbred Sprague-Dawley rats, which but ap-containone or more amino acid substitutions. Allelic variants with different catalytic propertiesprovide an excelpears to correspond to the form in Lewis rats. The stereoselectivity of androstenedione 16-hydroxylation lent means for pinpointing individual amino acid residues of = 1.4) is potential functional importance.For example, two allelic varcatalyzed by P4501IBl-WM (16@-OH:16a-OH thus distinct from that (16@-OH:16a-OH= 12-15) of iants of cytochrome P450IID1 were identified that differ at other P45011B1 preparations described. A cDNA en- only four positions in the amino acid sequence yet exhibit a coding P450IIB1-WM was cloned and sequenced, re- 10-fold differencein monooxygenase activity towardbufuralol vealing a single amino acid substitution (Gly-478 + (11).Subsequent studies led to the identification of a single Ala) compared with the published sequence (Fujii-Ku-residue as largely responsible for the catalytic difference (4). riyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., One of thebeststudiedexamples of allelic variants of and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U. cytochromes P450 involves the IIB subfamily in rats. Based S. A. 79, 2793-2797). Heterologousexpression of on two-dimensional gel electrophoresis, four P45011B12 and P450IIB1 and P450IIB1-WM confirmed the striking two P450IIB2 variants have been found in outbred animals differenceinandrostenedionemetaboliteprofiles, strongly implicating the involvement of Ala-478 in The abbreviations used are: P450, cytochrome P450; PB, phenodefining the distinctive catalytic properties of barbital; LB, Luria broth; SDS, sodium dodecyl sulfate; IgG, immuP450IIBl-WM. noglobulin G; androstenedione, androst-4-ene-3,17-dione; PCR, polymerase chain reaction; WM, Wistar Munich; LEW, Lewis; WKY, Wistar Kyoto; WF, Wistar Furth; SD, Sprague-Dawley; HPLC, highperformance liquid chromatography; HEPES, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid. The broad substrate specificity of the cytochrome P450 Nomenclature: the P450IIB gene subfamily is composed of cytosuperfamily is largely attributable to the expression of mul- chromes P450 from different species which have been grouped together on the basis of amino acid sequence identity, in accordance * This research was supported in partby Grant ES03619 from the with the recently suggested nomenclature of Nebert et al. (12). The National Institutes of Health. The costs of publication of this article protein referred to as P450IIBl in this study has thededuced amino were defrayed in part by the payment of page charges. This article acid sequence defined by Fujii-Kuriyama et al. (13) (Accession Nummust thereforebe hereby marked “advertisement” in accordance with ber 500719 in GenBank).As shown in this report, the protein referred 18 U.S.C. Section 1734 solely to indicate this fact. to as P450IIB1-WM differs from by IIBl one aminoacid substitution. $ Recipient of National Research Service Award F32 ES05502. T o In accordance with the definitions set forth by Nebert et al. (12), whom correspondence and reprint requests should be addressed Dept. IIB1-WM is considered an allelic variant of IIB1. P450IIB1-3 is a of Pharmacology and Toxicology, College of Pharmacy, University of variant cDNA (3), which encodes a protein with Val as residue 282, Arizona, Tucson, AZ 85721. Tel.: 602-626-4489; Fax: 602-626-4063. instead of the Glu in IIB1. P450IIB1-WM corresponds to electropho§ Supported by thePharmaceuticalManufacturers Association retic variant 4 of Rampersaud and Walz (14). This protein appears Foundationand by theHawaii Affiliate of the American Heart not tohave been purified previously nor has the protein corresponding Association. to P450IIBl-WF (variant 7). In contrast, proteins corresponding to ll Current address: Drug Metabolism and Disposition, Lilly Re- P450IIB1-LEW(variant3)andP450IIBl-WKY(variant 6) have search Laboratories, Eli Lilly and Company, Indianapolis, IN46285. been described previously and called P-450b-H and P-450b-LE, re(1 Recipient of ResearchCareer DevelopmentAward ES00151 spectively (15, 16). It is not known which of the variants is encoded (1985-1990). by the cDNA of Fujii-Kuriyama et al. (13).

22515

22516

A Functionally Unique

(15), and inbred strains expressing individual variants have beenidentified(14). Two of theIIBlvariantshave been purified and shown to exhibit electrophoretic but no appreciable catalytic differences (16, 17). More recently, cDNAs encoding two allelic variants of P450IIB1 (3, 18) and a IIB2 variant (3) have been cloned and sequenced. Analysis of the sequence of the IIB2 variant prompted the construction of P450IIBl/IIB2 hybrids, ultimately leading to the identification of amino acid residues 58 and 114 in P4501IB1 as important determinantsof the stereospecificity and regioselectivity of steroid hydroxylation by the enzyme (3). To date, this is the only report of a role for individual cytochrome P450IIB residues in regulating substrate specificity. In order to identify amino acid residues of potential functional importance inP450IIB1, we analyzed liver microsomes from phenobarbital-treated ratsof four inbred strains, previ(14). ously identified as expressing distinct P450IIB1 variants These studies revealed unique functional properties of P4501IB1 in Wistar Munich rats (P450IIBl-WM), a finding confirmed in reconstituted systems. We have isolated a cDNA encoding P450IIBl-WM and expressed it and P450IIB1 in heterologoussystems. This has led to the identification of amino acid residue 478 in P4501IB1 as an important determinant of substrate specificity.

P45OIIBl Variant

Sprague-Dawley rats, NADPH-cytochrome P450 reductase, and rabbit anti-P450IIB1 were available from previous studies (21-23). Media and Growth Conditions-Yeast S. cerevisiae 334 (Mata, pep4-3, prbll-1122, urd-52, leu2-3, 112, regl-501, gall) was obtained from R. A. Sclafani (University of Colorado Heath Science Center, Denver,CO)(24). 334 was grown ineitherYPD(yeastextractpeptone-dextrose) or synthetic minimal medium with dextrose supplemented with uracil and leucine (25). Transformed 334 strains were grown insyntheticminimal medium withdextrosesupplemented with uracil and were induced with 2% galactose (24). All yeast cultures were grown a t 28 "C. Intact yeast were transformed using lithium acetate (26). The E. coli strain utilized for all plasmid isolations and genetic constructions was DH5a (27, 28). DH5a was cultivated in LB (29); for those strains containinga plasmid, LB was supplemented with 50 pg/ml ampicillin. All bacterial cultures were grown a t 37 "C. DH5a was transformed by the CaCI, method (29). COS cellswere grown in Dulhecco's modified Eagle's medium,10% fetal bovine serum,with 100 units/ml penicillin and 100 rg/ml streptomycin a t 37 "C under 5% C 0 2 . They were transfected using DEAE-dextran (30). Cells were exposed to plasmid DNA in phosphate-buffered saline-DEAE dextran for 30 min and then treated with 100 r M chloroquine for 2.5 h. After exposure to 10% dimethyl sulfoxide for 2.5 min, the reagents were removed and replaced with medium containing 5% fetal bovine serum. The medium was changed a t 24- and48-hpost-transfect.ion. Cells were assayed 72 h after transfection. Plasmids and cDNAs-The E. coli plasmid usedfor all genetic constructs, suhcloning, and DNA sequencingwas Bluescript I1 KS-. The yeast expression vector was YEp51 (31),obtained from C. DieckEXPERIMENTALPROCEDURES man (University of Arizona, Tucson, AZ). The COS cell expression Materials-Restriction endonucleases, DNA modification en- vector was pBC12BI (30). The cDNA encoding P4501IBl-WM was zymes, and growth media for COS cells were purchased from GIBCO- generated by PCR for this study. Apartial cDNAencoding P450IIB1Bethesda Research Laboratories. The Gene Clean Kit was obtained 3 was obtained from F. G. Gonzalez (National Institutes of Health, from BiolOl (La Jolla, CA). The Fast Track RNA Isolation Kit. was Bethesda,MD)(3). All restrictionfragments were isolatedfrom purchased from In Vitrogen (San Diego, CA). PCR reagents were agarose gels and purified with the use of the Gene Clean kit. All obtained from Perkin-Elmer Cetus Instruments. Primer oligo(dT)clo, ligations were performed by standard methods. DNA sequencing was was obtained from Boehringer Mannheim. Primersfor PCR and site- carried out on double-stranded DNA hy the dideoxynucleotide chaindirected mutagenesis were obtained from the University of Arizona termination method (32) with a Sequenase kit. Site-directed mutaMacromolecular StructureFacility(Tucson, AZ). The Sequenase genesis wascarried outby the methodof Kunkel(33) using the MutaDNA Sequencing Kit was purchased from United States Biochemical Gene Phagemid in vitro Mutagenesis kit. Microsome Preparation-Microsomesfrom rat livers andyeast Corp. Growth media for Escherichia coli and Saccharomyces cerevisiae cells were prepared as described previously (34)." COS microsomes were purchased from Difco. Bluescript I1 KS- was purchased from Stratagene(San Diego, CA). TheMuta-GenePhagemidin vitro were prepared from trypsin-treated cells. The cells from each plate Mutagenesis kit and all electrophoresis reagents were obtained from were collected in 2 ml of phosphate-bufferedsaline, pelletedby centrifugation,andresuspendedin 2 ml of COS cell sonication Bio-Rad. Glassheads, nitro blue tetrazolium,5-bromo-4-chloro-3indolyl phosphate, goat anti-rabbit IgG conjugated to alkaline phos- solution (250 mM sucrose, 1 mM EDTA). Cells were broken by sonication for 15 s with a microtip. Debris was removed by centrifuphatase, 16a-OH androstenedione, chloramphenicol, dilauroyl-~-3phosphatidyl chlorine,Lubrol PX, and NADPH were purchased from gation a t 10,000 X g for 5 min. Microsomes were collected by centrifSigma. 6D-OH androstenedione was purchased from Steraloids (Wil- ugation a t 150,000 X g for 20 min. The pelleted microsomes were ton,NH). 4-['4CC]Androst-4-ene-3,17-dione and 4-["CC]testosterone washed once and then resuspended in 1 ml of COS cell microsome resuspension buffer (50 mM K H ~ P O I , p H7.4, 0.2 mM EDTA, 20% were purchased from Du Pont-New England Nuclear. Thin layer chromatography (TLC) plates (silica gel, 250 pm, Si 250 PA (19C)) glycerol) and stored a t -70 "C. Steroid Hydroxylase Assay-The assays of androstenedione hywere purchased from Baker Chemicals (Phillipsburg, NJ). DEAESephacel, Sepharose 4B, Sepbadex G-25, and Sephadex G-100 were droxylase activity (22):' and testosterone hydroxylase activity(36) obtained from Pharmacia LKBBiotechnology Inc. Cyanogen bromide were performedas described. Metabolites were resolved onTLC was purchased from Eastman Kodak. Sodium deoxycholate, sodium plates by two cycles of chromatography in ethy1acetate:chloroform (2:1, v/v) for androstenedione or dich1oromethane:acetone (4:1, v/v) cholate, and HEPES were purchasedfrom Calbiochem. All other reagents andsupplies not listed were obtained from standard sources. for testosterone andvisualized by autoradiography. Metabolite bands Treatment of Animals-Male rats (200-250 g) of the Wistar Mun- were identified by the use of unlabeled standards and quantitatedby ich (WM), Lewis (LEW), and Wistar Furth (WF) strains were ob- scintillation counting. Other Monooxygenase Assays-The formation of formaldehyde tained from Harlan-Sprague-Dawley (Indianapolis, IN) and of the Wistar Kyoto (WKY) strain from Charles River (Wilmington, MA). from benzphetamine was measured by the methodof Nash (37), after Animals were pretreated for 5 days with 0.1% (w/v) PB in drinking termination of the reaction with1.0 ml of ice-cold 10% trichloroacetic water. Animals were killed by cervical dislocation and livers removed acid (21). 7-Ethoxycoumarin deethylase activity was determined fluorimetrically using an excitation wavelength of 366 nmandan immediately. Portions of individual livers were used to prepare miof454 nm. The deethylationreaction was crosomes or RNA, with the remaining tissue frozen in liquid nitrogen emissionwavelength stopped by the addition of 2 N HCI and metabolites extracted with and stored at -70 "C. RNA Preparation-RNAfrom WMrat liverwas prepared as chloroform and back-extracted with 30mM sodium borate (38). Immunochemical Methods-SDS-polyacrylamide gels (7.5%) were described (19). mRNA was isolated with the use of the Fast Track run as described (39). Proteins were electrophoretically transferred Kit according tothemanufacturer'sinstructions.PriortoRNA preparation, microsomes were prepared from a portion of the liver. to nitrocellulose in 25 mM Tris,pH 8.2, 192 mM glycine, 20% The androstenedione hydroxylase activity of the microsomes was methanol overnight a t 100 mA. After transfer, the membrane was blocked for 30 min in 3% non-fat dry milk in TTBS (20 mM Tris analyzed, to insure the presence of a WM-type IIB1. Purification of Enzymes-Liver microsomes from four PB-treated base, 0.5 M NaC1, 0.15% Tween 20). All washes were in TTBS. After WM ratswere pooled and used as starting material for the purification "Kedzie, K. M., Philpot, R. M., and Halpert, J . R., (1991) Arch. of P450IIBl-WM by the methodof Guengerich and Martin (20), with modifications described by Halpert et al. (21). P450IIB1 from outbred Biochem. Biophys. in press.

22517

A Functionally Unique P45OIIB1 Variant blocking, the membraneswere incubated with primary antibody (rabbit anti-rat IIBlIgG (10 pg/ml) in 3% non-fat dry milk in TTBS for 1 h. After washing three times for5 min, the second antibody in TTBS was added (goat anti-rabbit IgG conjugated to alkaline phosphatase) and the membrane incubated for 1 h. Proteins were visualized with nitro blue tetrazolium and5-bromo-4-chloro-3-indolylphosphate as substrates. The membrane was rinsed with water to stop the reaction and air-dried. Other Methods-Protein was determined by the method of Lowry (40). Cytochrome P450 was determined spectrally (41).

-

I**

strain

0 WM

m

WF LEW WKY

**

RESULTS 7a-OH 6P-OH 16P-OH 16a-OH Evaluation ofAndrostenedione Hydroxylase Activity in Liver $ ANDROSTENEDIONE Microsomes-Liver microsomes from PB-treated rats of four inbred strains, WM, LEW, WKY, and WF, were evaluated for their ability to hydroxylate androstenedione (Fig. U). The most striking straindifference was in the level of androstenedione 160-hydroxylase activity,a specific P450IIB1 marker (42), where a 10-fold range in activity was observed among the strains. In order to determine whether the 5 - to 10-fold lower 16P-hydroxylaseactivity in themicrosomes from the WM rats reflected a reduced level of P450IIB1 expression, immunoblots were performed using anti-IIB1 antibody. No major differences in immunoreactive IIBl protein among the microsomes from the four strains were observed (data not \\ shown), and the lack of expression of P450IIB2 in the WM 2' 0 2 4 6 8 10 strain was also confirmed (14). These datasuggested that the TIME (rnin) lower 16P-hydroxylase activity in microsomes from PBtreated WM rats is an intrinsicproperty of the P450IIB1. FIG. 1. A, androstenedione hydroxylase activities in liver microTo confirm this, theinactivation of the microsomal andro- somes from PB-treated ratsof four inbred strains. Liver microsomes stenedione 160-hydroxylase by certain mechanism-based in- (5 pg) from PB-induced rats were incubated for 3 min a t 37 "C with 25 PM androstenedione, and metaboliteswere identified and quantiactivators was monitored. The rationale was that the rate constant for inactivation is independent of the enzyme con- fied as described previously (22, 44). Values represent the mean 5 of duplicate analyses on multiple individual liver samples ( n = centration and would not be influenced by differences in 7S.D. for WM rats; n = 3 for all other strains). Results marked* and ** expression levels. As shown in Fig. 1B, preincubation of liver are significantly different from the others (*, p < 0.05; **, p < 0.01) with the selective as analyzed by the Newman-Keuls test. B , effect of preincubation microsomes from WKY, WF, and LEW rats on androP450IIB1 inactivator N-(2-p-nitrophenethyl)chlorofluoro- with 250 PM N-(2-p-nitrophenethyl)chlorofluoroacetamide acetamide (43) led to a rapid loss of 16P-hydroxylase activity stenedione 16~-hydroxylase activity in livermicrosomes from PBover time. In contrast,no time-dependent loss of 16P-hydrox- treated rats of four inbred strains. Samples were incubated with or inhibitor for 2 min at 37 "C. Reactions were started by the ylase activity was observed with the microsomes from the without addition of NADPH and were allowed to proceed for thetimes WM rats,although a time-independentloss of activity, indic- indicated, a t which point 80-pl aliquots were removed and added to ative of reversible inhibition, was observed (43, 44). In con- 20 pl of ["Clandrostenedione in buffer. The reactions were allowed trast to theresults with the chlorofluoroacetamide, chloram- to proceed for an additional 1.5 min and were quenched with 50 p1 of phenicol caused rapid inactivation of the androstenedione tetrahydrofuran. The concentrationof the various components of the 160-hydroxylase in microsomes from all four strains (data not incubation after the addition of androstenedione were: 250wg/ml 25 pM androstenedione, 1 mM NADPH, 50 mM HEPES, pH shown). Taken together, the results with the intact micro- protein, 7.6, 15 mM MgCI, and 0.1 mM EDTA. The inhibitor was added from somes suggested unique catalytic properties of the P450IIB1 a methanol stock solution. The controlis shown for the LEW microin the WM rats. somes only. In this typeof experiment, residual unmetabolized inacPurification of P4501IBl- WM and P45011Bl-SD-Two tivator is present during the assaysof substrate metabolism. Therepreparations of P4501IB1 were purified from liver microsomes fore, any time-dependent loss of activity (inactivation) is superimof PB-treated WM rats atospecific cytochrome P450 content posed upon a time-independent component (inhibition). The lines were drawn by linear regression analysis of the natural logaof 16 nmol/mg. One of these preparations was compared by shown rithm of the residual activity as a function of time. Rate constants SDS-polyacrylamide gel electrophoresis (Fig. 2 ) with a for inactivationare derived from the negativeslope of the lines, P450IIB1 isolated previously from outbred rats (22). This whereas the extent of inhibition is evident from the decrease in the outbred form will be referred to asP450IIB1-SD4 and appears extrapolated activity a t zero preincubation time ( y intercept) comto be identical to the form found in LEW rats based on pared with the controls.

The protein referred to as P450JIB1-SD has been purified from electrophoretic mobility on immunoblots (data notshown). A outbred Sprague-Dawley rats. It appears to he identicalto IIB1-LEW slight difference in mobility of the IIBl proteins was detected. based on one-dimensionalSDS-polyacrylamide gel electrophoresis By comparison to appropriate size standards, the M , was and immunoblotting. Thus, the electrophoreticmobility of the purified outbred P450IIB1-SD is identical to that of the P4501IB1 in determined to be 51,500 and 52,000 for IIB1-SD and IIB1WM, respectively. LEW microsomes and greater than the mobility of the IIBl in the WF, WKY, and WM strains. This is in agreement with the findings Catalytic Properties of P450IIBl Variants in Reconstituted of RampersaudandWalz (14, 15). The catalyticproperties of Systems-The susceptibility of P450IIB1-WM to inactivation P450IIBl-SD agree very well with those of other P450IIB1 preparaby chloramphenicol and two analogs was evaluated (Fig. 3, tions described in the literature including P-450b-LE (variant 6), especially with regard to the androstenedione 16D:16a hydroxylase Table I). As observed with WM microsomes, N-(2-p-nitroratio (45, 46). phenethy1)chlorofluoroacetamide did not reduce the 16P-hy-


22518

1

2

3

4

P45OIIB1 Variant TABLE I Inactivation constants ofpurified P4SOIIBI proteins Twoindependentpreparations each of IIB1-SD and IIB1-WM were used for these studies. Assays were performed as described in the legend to Fig. IR, except that microsomes were replaced by a or reconstitutedsystem containing 50 pmol/mlP450IIB1-WM P450IIB1-SD. The concentrations of the inactivators were 50 pM pN0,CIFA (N-(2-p-nitrophenethyl)chlorofluoroacetamide~,5 pM pN0,DCA (N-(2-p-nitrophenethyl)dichloroacetamide),and 50 p M chloramphenicol. The inhibitors were added from stock solutions in methanol. Methanol

SD 1 SD 2 WM 1 WM 2

DNO:XIFA

0.28 0.26 0.03 0.05

0.09 0.12 0.04 0.04

DNO,DCA

Chloramphenicol

k fmin”) 0.37 0.48 0.08 0.12

0.26 0.36 0.21 0.29

TABLE I1 Catalytic activities ofpurified P4SOIIBI proteins Two independent preparations each of IIB1-SD and IIB1-WM were used for these studies. Assays were performed as described under “Experimental Procedures.” The results represent the mean of duplicate analyses. Androstenedione hydroxylase

FIG. 2. SDS-polyacrylamide gel of purified P450IIB1 variants. Electrophoresis was performedaccording to the method of Laemmli (39) using a 10% gel.Size standards (Bio-Rad low molecular weight standards) were: rabbit muscle phosphorylaseB (97,400). bovine serum albumin (66,200), hen egg white ovalbumin (45,000), bovine carbonic anhydrase (31,000), soybean trypsin inhibitor (21,5001, and hen egg white lysozyme (14,400). The identity of the samples from left to right: lane 1, size standards; lane 2, 1 pg of P450IIBl-SD; lane 3, 1 pg of P45OIIB1-WM; lane 4, size standards. Proteins were stained with Coomassie Brilliant Blue.

Testosterone hydroxylase

7-Ethoxycoumarin

~

amine

~

nmol/min/nrnol

SD 1 SD 2 WM 1 WM 2

6.1 7.3 1.6 1.1

0.5 0.6 1.1

0.8

17.4 17.3 3.1 1.3

22.8 23.6 6.8 2.5

10.9 9.8 2.6 1.6

64 56 73 50

fifth as high for the SD enzyme. In contrast, the benzphetamine demethylase activitiesof the two variants are thesame, whereas the androstenedione16a-hydroxylase activity of the 00 * - . WM enzyme is higher than the SD variant. The ratios of A A androstenedione 16@:16a-hydroxylation are 12for theSD enzyme and 1.4 for the WM form,whereas theratios of testosterone 16@:16a-hydroxylationare 0.75 for the SD and 0.5 for the WM form of P450IIB1. Peptide Mapping-To confirm the high degree of structural lo A CONTROL similarity between the two variants of P450IIB1 and to search 0 pN02CIFA 5 A pN02DCA for any differences, tryptic peptide mapping was performed. CAP The proteins were subjected to proteolysis under identical 21 I conditions andanalyzed sequentiallyon HPLC. The two maps 0 2 4 6 a 10 were virtually indistinguishable (data not shown), and any TIME (min) structural uniqueness is not detectable by this method. It FIG. 3. Effects of preincubation with 50 p~ N-(a-p-nitro- should be noted that P450IIB1 and IIB2, which differ by phenethy1)chlorofluoroacetamide (pNOpCIFA),5 p~ N-(2-p- fourteen amino acid residues, are readily distinguishable by nitrophenethy1)dichloroacetamide (pNODCA), and 5 0 p~ chloramphenicol (CAP) on the androstenedione 160-hydrox- trypticpeptide mapping on HPLC (Ref. 47 and data not shown). ylase of purified P450IIBl-WM. Experiments were carried out Sequencing of ZZBl- W M cDNA-A cDNA encoding IIB1essentially as described in the legend to Fig. lR, except that the microsomes were replaced by areconstitutedsystem containing WM was generated by PCR (48) and sequenced by the dideP450IIR1-WM (50 pmol/ml). oxynucleotide chain-termination method (32). The deduced amino acid sequenceof IIB1-WM was compared withthe IIBl droxylase activity over time, although time-independent(re- sequence determined by Fujii-Kuriyama et al. (13). Three versible) inhibition is evident. Both chloramphenicol and N - codon differences were detected, one of which resulted in an (2-p-nitrophenethy1)dichloroacetamide inactivated IIB1- amino acid substitution (Table 111). This alteration, Gly-478 WM, although with very different rate constants. In contrast in IIBl versus Ala-478 in IIB1-WM,is presumably responsible to P450IIBl-WM, P450IIB1-SD was rapidly inactivated by for the altered functional properties of P450IIB1-WM. Heterologous Expression in Yeast-cDNAs encoding IIBl all three compounds (Table I). Theexperiments in reconstitutedsystems also revealed and IIB1-WM were subcloned intothe expression vector other catalytic differences between P450IIB1-WM and IIB1- YEp51 to form YEpIIBl and YEpIIB1-WM. The resulting SD. As noted in Table 11, the androstenedione 16P-hydrox- constructs were used to transform S. cerevisiae 334, creating cDNA encodingIIBl ylase, testosterone 16-hydroxylase, and 7-ethoxycoumarin de- strains 334:IIBl and 334:IIBl-WM. The ethylase activities of the WMenzyme are approximately one- was constructed by replacing a 374-base pair BglII-BglII frag’

~


P45OIIBl Variant

22519

TABLE 111 Co WM llBl Codin# nucleotide sequence differences between P45OIIBI and P450IIBI- WM A 1500-base pair cDNA encoding IIB1-WM was generated from WM rat liver mRNA by PCR (48). cDNA was generated from 1 pg of WM rat liver mRNA using oligo(dT),,o,, Moloney murine leukemia I1 L virus reverse transcriptase and deoxyribonucleotides. PCR was carried out using oligonucleotides which prime upstream of the start 16a' codon and downstream of the stop codon of the cDNA sequence of 16P P450IIB1. The primers contain convenient restriction sites for subcloning into the Bluescript vector. PCR was carried outfor 25 cycles. cDNAs generated by PCR were subcloned into BluescriptI1 KS- and I sequenced by double-stranded dideoxynucleotide chain-terminating was sequenced sequencing(32)using a Sequenase kit. One clone FIG. 4. Autoradiogram of androstenedione metabolites procompletely on both strands after nested deletions were (35) generated duced by transfected COS cells in situ. Cells were transfected using exonuclease 111. A second independent clone was sequenced with 20 pg of plasmid/lO-cmplate. After 72 h, the mediumwas 90% onthesecond completelyon onestrandandapproximately replaced with 2 ml of medium containing25 p~ ["Clandrostenedione. strand, after subcloning to obtain specific restriction fragments. In the regions where codon alterations from the published IIBl sequence After incubation for 1 h, the medium was removed and extracted were found, an additional independent clone was sequenced for veri- twice with 2.5 volumes of chloroform. The metabolites were dried, resuspended in methanol, and resolved on a TLC plate asdescribed fication. under "Experimental Procedures." The control (Co) represents samAmino acid ples from COS cells transfected with a pBC12BI plasmid with no IIBI-WM IIBl residue insert. 151 CAA CAG sequencing. Western blottingof microsomes from transfected 478 G~Y Ala COS cells confirmed the expression of both IIBl and IIB1GGA GCA WM, although no mobility differences were observed (Fig. T h r 483 Thr 5 A ) .The androstenedione hydroxylase activity of microsomes ACG ACA

m -dm7

Gln

ment from the IIB1-WM cDNA with the corresponding fragment from IIB1-3. This fragment, corresponding to codons 361-486, resulted in the alteration of amino acid 478 from Ala + Gly, yielding a construct identical in deduced amino acid sequence to the P450IIB1 of Fujii-Kuriyama et al. (13). Yeast microsomes from 334:IIBl and 334:IIBl-WM were evaluated by Western blotting (data not shown). Both IIBl and IIB1-WM were produced in yeast, although no mobility differences were detected on SDS-polyacrylamide gels. The androstenedione hydroxylase activity of 334:IIBl and 334:IIBl-WM microsomes was alsoevaluated (datanot shown). Use of this substrate obviated the need to establish precise initial rate conditions since the two phenotypes can be distinguished by their metabolic profiles. The activity of the 334:IIBl-WM microsomes was below the level of detection. However, the alterationof 1amino acid residue, Ala-478 -+ Gly by exchange of the BglII-23glII fragment, was sufficient to confer P450IIB1-like activity on the less active P450IIB1WM. Heterologous Expression in COS cells-Due to thedifficulty in monitoring androstenedione hydroxylase activity of IIB1WM in yeast microsomes, IIB1-WM and IIBlwere expressed in COS cells. In this case, the cDNA encoding IIBl was generated from the partialIIB1-3 cDNA by replacing a 1140base pair KpnI-KpnI fragment encompassing codons 3-378 with the fragment from the IIB1-WM cDNA encompassing codons 1-378. The generated cDNA is altered from IIB1-3 a t codon 282 (Val-282 in IIB1-3, Glu-282 in IIB1) and contains the complete IIBl coding sequence of Fujii-Kuriyama et al. (13). The cDNAs encoding IIBl andIIB1-WM were subcloned into pBC12BI and used to transfect COS cells. Androstenedione hydroxylase activities of transfected COS cells were determined in situ (Fig. 4). Expressed IIBl exhibited an 8fold higher androstenedione 16~-hydroxylase:16a-hydroxylase ratio than expressed IIB1-WM. The effect of the amino acid residue a t position 478 was further evaluated by generation of a P450IIB1 cDNA by sitedirected mutagenesis of the IIB1-WM cDNA. Mutants were produced by the method of Kunkel(33) and screened by

prepared from transfected COS cells was also evaluated (Fig. 523). This confirmed that alterationof one specific amino acid changes the androstenedione 16P-OH:16a-OH ratio from 0.95 (IIB1-WM microsomes) to 7.5 (IIB1 microsomes). The 16p0H:lGa-OH ratioof 7.5 obtained here is identical to theratio obtained from expression of IIBl in vaccinia virus-infected Hep G-2 cells (3). The %fold difference in the 16&OH:16aOH ratiobetween the two phenotypes is the same as observed in the reconstituted system (Table 11). DISCUSSION

The results of this investigation describe for the first time a functionallydistinct allelic variant of cytochrome P450IIB1 and pinpoint the critical amino acid substitution. Although two variant proteins have been isolated and characterized from well defined strains of rats (16) and a number of other variants purified from outbred animals (49, 50), no marked catalytic differences have been described previously. In addition, although two variant P450IIB cDNA sequences have been reported (3, 18), only one of the cDNAs (IIB1-3) has been expressed, and its catalytic properties appear to be the same as thoseof the cDNA of Fujii-Kuriyama et al. (13). The identification by Rampersaud and Walz (14) of inbred strains of rats expressing distinct P450IIB1 variants and theuse of a specific marker substrate and mechanism-based inactivator enabled us to rapidly identify the P450IIB1 in microsomes from PB-treated ratsof the WM strain functionally as unique. These findings led to the isolation of the protein and its cDNA and the identification of Ala at position 478 in the amino acid sequence as responsible for the unusual stereoselectivity of androstenedione 16-hydroxylation catalyzed by P450IIBl-WM. Although replacement of Gly-478 in P450IIB1 by Ala appearsto be sufficient to cause amarked decrease in the stereoselectivity of androstenedione 16-hydroxylation,inspection of other cytochrome P450IIB sequences reveals that this residue alone cannot be responsible for dictating attackat the /3 as opposed to thea face of the molecule. Thus, ratP450IIB1, rabbit P450IIB4, rabbit P450IIB5, and dog P450IIB11 all have a Gly a t residue 478 (13, 51, 52), yet the 16@-OH:16aOH ratios for androstenedione hydroxylation range from 0 in

A Functionally Unique P45OIIB1 Variant

22520

A 1

B

2

3

4

WM llBl i

region from a testosterone 16a-hydroxylase, a substitution of 17 amino acid residues, the laurate (0-1)hydroxylase acquired 16a-hydroxylase activity. Although the changes in substrate specificity observed with P450IIB1 and IIB1-WM do not include the acquisition of a new activity, the molecular basis for the functional change is better defined and identifies a portion of the molecule that bears further investigation. Perhaps the most novel finding of this investigation is the ability of the mechanism-based inactivator N-(2-p-nitrophenethy1)chlorofluoroacetamide to distinguish among allelic variants of P450IIB1. To our knowledge, this is the most striking example of the selectivity of a P450 inhibitor/inactivator reported to date. The lack of inactivation of P450IIB1WM by the compound appears to result from an inability of the enzyme to catalyze the oxidative dehalogenation reaction (43) required for reactive metabolite formation.s Another unusual feature of P450IIB1-WM is its slow inactivation by N-(2-p-nitrophenethyl)dichloroacetamidedespite rapid inactivation by chloramphenicol. These two compounds differ by the presence of a propanediol side chain in chloramphenicol in place of the ethyl side chain in the analog. As with the difference in androstenedione hydroxylation, the results suggest that alterations in secondary or tertiary structurechange the orientation of the chloramphenicol analogs in the active site of P450IIBl-WM, severely impairing the ability of the enzyme to activate the compounds. Regardless of the precise structural basis for the differences in inactivator selectivity, the results suggest that mechanism-based inactivators may be evenmore specific that previously realized. Therefore, such compounds may prove to be extremely valuable for distinguishing cytochromes P450 of different function but almost identical primary structure.

FIG. 5. A, immunological detection of heterologously produced P450IIB1 and P45OIIB1-WM in COS cell microsomes. COScells were transfected with IIB1-WM and IIBl (site-directed mutant of IIB1-WM) cDNAs. COS cell microsomal proteins were separated on an SDS-polyacrylamide gel, followed by electrotransfer toa nitrocellulose membrane. Proteins were detected with an anti-rat P450IIB1 IgG and an enzymatic color reaction catalyzed by alkaline phosphatase using nitro bluetetrazolium and 5-bromo-4-chloro-3-indolyl Acknowledgments-We thank Dr. Scott K. Boyer and Dr. Brian A. phosphate as substrates. The identity of the samples, from left to Larkins, Department of Plant Sciences, University of Arizona, Tucright: lane I, 0.3 pmol of P450IIB1-SD; lune 2, 50 pg of C0S:IIBl son, AZ, for the use of the PCR reagents and thermal cycler (Perkinmicrosomes; lane 3, 25 pg of C0S:IIBl-WM microsomes; lane 4, 0.3 Elmer Cetus Instruments) and for the use of the Microgenie DNA pmol of P450IIBl-WM. B, autoradiogram of androstenedione metab- Sequence Analysis Program (Beckman Instruments, Inc.). olites produced by microsomes from transfected COS cells. The samples were incubated with 25 p~ [“C]androstenedione for 30 min Note Added in Proof.-The cDNA sequence of Fujii-Kuriyama et a t 37 “C in 100 pl of 50 mM HEPES, pH 7.6, containing 1.5 mM al. (13) lacks the first five codons. In this region, the nucleotide MgCl?, 0.1 mM EDTA, 1 mM NADPH, and 20 pmol of rat.NADPH- sequence of the P450IIB1-WM cDNA is identical to thesequence of cytochrome P450 reductase. Samples were quenched by the addition the P450IIB1 genomic clone of Suwa et al. (Suwa, Y., Mizukami, Y., of 50 pl of tetrahydrofuran, and 5O-pl aliquots were analyzed for Sogawa, K., and Fujii-Kuriyama, Y. (1985) J. Biol. Chem. 260,7980metabolites by TLC. The amountsof protein incubated were 180 pg 7984) (exon 1 accession number M11251 in GenBank). for P450IIBl-WM and 225 pg for P450IIBl. REFERENCES the case of IIB5 to 15 in the case of IIB4 (45,46).’ In addition, 1. Gonzalez, F. G. (1989) Pharmacol. Rev. 40, 243-288 like IIB1-WM, rat IIB2 has an Ala residue at position 478 2. Kronbach, T., Larabee, T. M., and Johnson, E. F. (1989) Proc. (53) but exhibits the same stereoselectivity of androstenediNatl. Acad. Sci. U.S.A. 86,8262-8265 3. Aoyama, T., Korzekwa, K., Nagata, K., Adesnik, M., Reiss, A., one 16-hydroxylation as the IIBlwith Gly-478 (45). Clearly, Lapenson, D. P., Gillette, J., Gelboin, H. V., Waxman, D. J., other residues can compensate for the alterations in structure and Gonzalez, F. J. (1989) J . Biol Chem. 264,21327-21333 induced by a Gly + Ala substitution at residue 478. Ala is 4. Matsunaga, E., Zeugin, T., Zanger, U. M., Aoyama, T., Meyer, U. known to stabilize and Gly to destabilize an a-helix (54, 55). A., and Gonzalez, F. J. (1990) J . Biol. Chem. 265,17197-17201 Thus, therole of residue 478 in controlling the stereoselectiv5. Negishi, M., Lindberg, R., Burkhart, B., Ichikawa, T., Honkakoity of androstenedione hydroxylation by P450IIB1 may be ski, P., and Lang, M. (1989) Biochemistry 28,4169-4172 6. Kronbach, T., and Johnson, E. F. (1991) J . Biol. Chern. 266, mediated by alterations in secondary or tertiary structure, 6215-6220 leading to changes in the orientation of the substrate in the Hanoika, N., Korzekwa, K., and Gonzalez, F. J. (1990) Protein 7. active site. Eng. 3,571-575 Residue478 is located near the carboxyl terminus of 8. Uno, T., Yokota, H., and Imai, Y. (1990) Biochem. Biophys. Res. P450IIB1, whereas most studies have identified the amino Commun. 167,498-503 terminal third or half of cytochromes P450 as the region 9. Zvelebil, M. J. J. M., Wolf, C. R., and Sternberg, M. J. E. (1991) Protein Eng. 4, 271-282 responsible for defining the substrate binding site. In particular, residues 90-125 and 210-262 (56), residues 113-118 (2, 10. Laughton, C. A., Neidle, S., Zvelebil, M. J. J. M., and Sternberg,

6), residues 58 and 114 (3), and residue 209 (57) have been implicated. Incontrast, one study by Uno et al. (8) has identified the carboxyl terminus asan important determinant of substrate specificity. Byreplacing the carboxyl-terminal 28 residues of a laurate(0-1)hydroxylase with the corresponding

’Incubation of P450IIBl-SD with 250 p~ N-(2-p-nitrophenethy1)chlorofluoroacetamide underconditions which inactivate 8090% of the enzyme releases 1.11 0.16 ( n = 3) nmol F-/nmol P450. Under the same conditions P450IIBl-WM releases 0.14 0.04 nmol F-/nmol P450.

*


11. 12.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

27. 28. 29. 30.

P45OIIBI Variant

M. J. E. (1990) Biochem. Biophys. Res. Commun. 1 7 1 , 116031. 1167 Matsunaga,E., Zanger, U. M., Hardwick, J . P., Gelboin, H. V., Meyer, U. A,, and Gonzalez, F. J. (1989) Biochemistry 28, 32. 7349-7355 Nebert, D. W., Nelson, D. R., Coon, M. J., Estabrook, R. W., 33. Feyereisen, R., Fujii-Kuriyama, Y., Gonzalez, F. J., Guengerich, 34. F.P.,Gunsalus, I. C., Johnson, E. F., Loper, J . C., Sato,R., Waterman, M. R., and Waxman, D. J. (1991) DNA Cell Biol. 35. 10, 1-14 36. Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., and 37. Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79,279338. 2797 Rampersaud, A., and Walz, F. G. (1987) Biochem. Genet. 2 5 , 39. 527-534 Rampersaud, A., and Walz, F. G. (1983) Proc. Natl. Acad. Sci. 40. U. S. A. 80, 6542-6546 Ryan, D. E., Wood, A. W., Thomas, P. E., Walz, F. G., Yuan, P.- 41. M., Shively, J. E., and Levin, W.(1982) Biochim. Biophys. Acta 42. 709,273-283 43. Reik, L. M., Levin, W., Ryan, D. E., Maines, S. L., and Thomas, P. E. (1985) Arch. Biochem. Biophys. 2 4 2 , 365-382 44. Traber, P. G., Wang, W., McDonnell, M., and Gumucio, J . J. 45. (1990) Mol. Pharmacol. 37, 810-819 Chomczynski, P., andSacchi, N. (1987) Anal. Biochem. 1 6 2 , 156-159 46. Guengerich, F. P., andMartin, M. V. (1980) Arch. Biochem. Biophys. 205, 365-374 47. Halpert, J. R., Miller, N. E., and Gorsky, L.D. (1985) J. Biol. Chem. 260,8397-8403 48. Graves, P. E., Kaminsky, L. S., and Halpert, J. (1987) Biochemistry 26,3887-3894 Duignan, D. B., Sipes, I. G., Leonard, T. B., and Halpert, J. R. 49. (1987) Arch. Biochem. Biophys. 2 5 5 , 290-303 Hovland, P., Flick, J., Johnston, M., and Sclafani, R. A. (1989) 50. Gene (Amst.) 83,57-64 Rose, M. D., Winston, F., andHieter, P. (1990) Methodsin Yeast Genetics: ALaboratory Course Manual, pp. 177-187, Cold 51. Spring Harbor Laboratory Press, Cold Spring Harbor, NY Carter, B.L.A., Irani, M., MacKay, V. L., Seale, R. L., Slid- 52. A. (1987) in DNA Cloning: A ziewski, A.V., andSmith,R. Practical Approach (Glover, D.M., ed) Vol. 111, pp. 141-161, 53. IRL Press, Oxford Hanahan, D. (1983) J . Mol. Biol. 166, 557-580 BethesdaResearchLaboratories (1986) BRL Focus 8, 9 54. Sambrook, J., Fritsch, E. F., andManiatis, T. (1989) MolecularScience Cloning: A Laboratory Manual, 2nd Ed., pp. 1.74-1.84, Cold 55. Spring Harbor Laboratory Press,Cold Spring Harbor, NY 56. Cullen, B. R. (1987) Methods Enzymol. 152, 684-704 57.

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Broach, J. R., Li, Y.-Y., Wu, L.-C. C., and Jayaram, M. (1983) in Manipulation Experimental of Gene (Inouye, Expression M., ed) pp. 83-117, Academic Press, New York Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 Kunkel, T . A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82 ,488-492 Halpert, J. R., Naslund, B., and Betner, I. (1983) Mol. Pharmacol. 23,445-452 Henikoff, S . (1984) Gene (Amst.) 28, 351-359 Ciaccio, P.Halpert, J., and J. R. (1989) Arch. Biochem. Biophys. 2 7 1 , 284-299 Nash, T. (1953) Biochem. J . 5 5 , 416-421 Miller, N.E., and Halpert, J. (1986) Mol. Pharmacol. 2 9 , 391398 Laemmli, U. K. (1970) Nature 227,680-685 Lowry, 0.H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 Omura, T.,and Sato, R. (1964) J. Biol. Chem. 239,2379-2387 Waxman, D. J. (1988) Biochem. Pharmacol. 37, 71-84 Halpert, J., Jaw, J.-Y., Balfour,Kaminsky, C., and L. S. (1990) Drug Metab. Dispos. 18, 168-174 Stevens, J . C., and Halpert, J. (1988) Mol. Pharmacol. 33, 103110 Wood, A. W., Ryan, D. E., Thomas, P. E., and Levin, W. (1983) J. Biol. Chem. 258,8839-8847 Waxman, D. J., KO,A,, and Walsh, C. (1983) J. Biol. Chem. 2 5 8 , 11937-11947 Yuan, P.-M., Ryan, D. E., Levin, W., and Shively, J. E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 1169-1173 Innis, M. A,, Gelfand, H. D., Sninsky, J. J., and White, T. J. (1990) PCR Protocols: A Guide to Methods and Applications, pp. 21-27, Academic Press, San Diego Bansal, S . K., Love, J. H., andGurtoo, H. L. (1985) Eur. J. Biochem. 1 4 6 , 23-33 Oesch, F., Waxman, D. J., Morrissey, J. J., Honscha, W., Kissel, W., and Friedberg, T. (1989) Arch. Biochem. Biophys. 270,2332 Gasser, R.,Negishi,M., andPhilpot, R. M. (1988) Mol. Pharmacol. 3 2 , 22-30 Graves, P. E., Elhag, G. A., Ciaccio, P. J.,Bourque, D. P., and Halpert, J. R. (1990) Arch. Biochem. Biophys. 2 8 1 , 106-115 Mizukami, Y., Sogawa, K., Suwa, Y., Muramatsu, M., and FujiiKuriyama, Y. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 39583962 Lyu, P. C., Liff, M. I., Marky, L. A., andKallenbach,N. R. (1990) 250,669-673 O’Neil, K. T., and DeGrado, W. F. (1990) Science 2 5 0 , 646-651 Uno, T., and Imai, Y. (1989) J. Biochem. (Tokyo) 106, 569-574 Lindberg, R. L. P., and Negishi, M. (1989) Nature 339,632-634