Proc. Natl. Acad. Sci. USA Vol. 82, pp. 973-977, February 1985 Biochemistry Coding nucleotide sequence of rat NADPH-cytochrome P-450 oxidoreductase cDNA and identification of flavin-binding domains (amino acid sequence/flavoprotein/flavodoxin) TODD D. PORTER AND CHARLES B. KASPER McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI 53706 Communicated by James A. Miller, October 3, 1984 ABSTRACT The coding nucleotide sequence of the mRNA flavin binding. Segments homologous with flavin-binding for NADPH-cytochrome P-450 oxidoreductase (NADPH:fer- segments from Desulfovibrio vulgaris flavodoxin and Esch- ricytochrome oxidoreductase, EC 1.6.2.4) from rat liver was erichia coli fumarate reductase are identified. determined from two overlapping cDNA clones, pOR-7 and pOR-8, which together contain 2401 nucleotides complemen- MATERIALS AND METHODS tary to rat liver oxidoreductase mRNA. The single open cDNA Cloning. cDNA (pOR-8) was synthesized (21, 22) reading frame of 2034 nucleotides spanning these cDNAs codes from 5.5 pug of oxidoreductase-immunoenriched RNA; we for a-678 amino acid polypeptide with a molecular weight of replaced the oligo(dT) primer with 0.25 uxg of a denatured 76,962. The deduced amino acid composition is in excellent 42-base-pair Hinfl/Pvu II DNA fragment prepared from agreement with that determined by direct amino acid analysis pOR-7. Blunt-ended, double-stranded cDNA was ligated to of purified rat liver P-450 oxidoreductase, and the amino- BamHI linkers (New England Biolabs) and inserted into terminal region (residues 1-80) largely coincides with the pBR322. E. coli HB101 transformants were screened (23) amiflo-terminal sequence of the oxidoreductase isolated from with a nick-translated 5' Pst I fragment of pOR-7. rabbit liver. Comparison of the amino acid sequence to those DNA Sequencing. DNA was sequenced by the method of of other flavoproteins revealed two separate domains that are Maxam and Gilbert (24) with the adenosine plus guanosine likely to be involved in flavin binding: a long segment (residues modification of Krayev et al. (25). Radionucleotides were 77-228) homologous with Desulfovibrio vulgaris flavodoxin, an purchased from Amersham; polynucleotide kinase, Klenow FMN-containing protein, and a shorter segment (residues fragment of DNA polymerase, and terminal deoxynucleo- 452-477) homologous with the FAD-binding segment of tidyltransferase were from P-L Biochemicals; restriction fumarate reductase from Escherwchia coli. enzymes were from New England Biolabs and Promega Biotec (Madison, WI). NADPH-cytochrome P-450 oxidoreductase (NADPH:ferri- Computer Analysis. The sequence comparison programs cytochrome oxidoreductase, EC 1.6.2.4) is a membrane- of the University of Wisconsin Genetics Computer Group bound flavoprotein associated with the endoplasmic were used, including Bestfit (26) and Gap (27). Regions of reticulum (1, 2) and nuclear envelope (3) of a variety of cell similarity between proteins were identified by using a com- types and is the protein responsible for electron transfer parison program with a window of 30 residues and a match from NADPH to the cytochromes P-450 (4). This enzyme stringency of 8. The similar sequences were aligned using system, although present in a variety of tissues, is found in Bestfit, with gap weight set at 5.0 and gap-length weight set greatest abundance in the liver, where it is involved in the at 0.30. oxidative metabolism of numerous endogenous and foreign compounds and in the activation of chemical carcinogens RESULTS AND DISCUSSION to electrophilic derivatives (5, 6). In addition, the oxidore- Sequence Analysis. The sequencing strategy depicted in ductase is involved in electron transfer to heme oxygenase Fig. 1 was used to determine the cDNA nucleotide sequence (7) and cytochrome b5 (8) as well as other enzyme systems shown in Fig. 2. Clone pOR-8 is 706 base pairs long and has (9-11). The 77-kDa protein is unusual in that it con. a 108-base-pair overlap with the 5' end of pOR-7. Clone tains 1 mol each of FAD and FMN (12-15). It is anchored pOR-7 previously was shown to contain "1800 nucleotides to the microsomal membrane by its hydrophobic amino- terminal region (16, 17), the sequence of which has been 5' 3' determined for the rabbit protein (18). This hydrophobic which is from the by various > if .*I "tail", readily cleaved protein I t * I + | b I proteases including trypsin, is essential for the correct ,I .. .. interaction of oxidoreductase with the cytochromes P-450 Oil.TS H A + S N V TZ HS P (B) S DZZ tB) (16, 17); however, the protease-released flavoprotein retains '' the ability to reduce cytochrome c (14, 19). Electron transfer I proceeds from NADPH to FAD to FMN to cytochrome P-450 or other electron acceptor (20). e4 100 bp This report presents the cDNA sequence and derived amino acid sequence of the rat liver oxidoreductase. Com- FIG. 1. Sequencing strategy. pOR-7 (right) and pOR-8 (left) were parison of the amino acid sequence to other sequenced digested with the indicated restriction enzymes and end-labeled with reveals two separate domains within the [y-32P]ATP and polynucleotide kinase (open arrowheads) or [a- flavoproteins 32P]dNTP and the Klenow fragment of DNA polymerase (closed oxidoreductase polypeptide that are likely to be involved in arrowheads). The 5' Pvu II site of pOR-7 was 3' end-labeled with [a-32P]dATP and terminal deoxynucleotidyltransferase. More than 97% of the coding sequence was determined from both DNA The publication costs of this article were defrayed in part by page charge strands. Restriction sites: A, Aha II; B, BamHI; D, Dde I; H, Hinfl; payment. This article must therefore be hereby marked "advertisement" N, Not I; P, Pvu II; S, Sin 1; T, Taq I; V, Ava I; Z, BstNI. bp, Base in accordance with 18 U.S.C. §1734 solely to indicate this fact. pairs. 973 Downloaded by guest on September 26, 2021 974 Biochemistry: Porter and Kasper Proc. Natl. Acad. Sci. USA 82 (1985) C AAC ATG GGG GAC TCT CAC GAA GAC ACC AGT GCC ACC ATG CCT GAG GCC GTG GCT GAA GAA GTd TCT CTA TTC AGC ACG ACG GAC ATG 88 Met Gly Asp Ser His Glu Asp Thr Ser Ala Thr Met Pro Glu Ala Val Ala Glu Glu Val Ser Lou Phe Ser Thr Thr Asp Met 1 10 20 GTT CTG TTT TCT CTC ATC GTG GGG GTC CTG ACC TAC TGG TTC ATC TTT AGA AAG AAG AAA GAA GAG ATA CCG GAG TTC AGC AAG ATC CAA 178 Val Lou Phe Ser Lou Ile Val Gly Val Lou Thr Tyr Trp Phe Ile Phe Arg Lys Lys Lys Glu Glu Ile Pro Glu Phe Ser Lys Ile Gln 30 40 50 ACA ACGO CC CCA CCC GTC AAA GAG AGC AGC TTC GTG GAA AAG ATG AAG AAA ACG GGA AGG AAC ATT ATC GTA TTC TAT GGC TCC CAG ACG 268 Thr Thr Ala Pro Pro Val Lys Glu Ser Ser Phe Val Glu Lys Met Lys Lys Thr Gly Arg Asn Ile Ile Val Phe Tyr Gly Ser Gln Thr 60 70 80 GGA ACC GCT GAG GAG TTT GCC AAC CGG CTG TCC AAG GAT GCC CAC CGC TAC GGG ATG CGG GGC ATG TCC GCA GAC CCT GAA GAG TAT GAC 358 Gly Thr Ala Glu Glu Phe Ala Asn Arg Leu Ser Lys Asp Ala His Arg Tyr Gly Met Arg Gly Met Ser Ala Asp Pro Glu Glu Tyr Asp 90 100 110 TTG GCC GAC CTC AGC AGC CTG CCT GAG ATC GAC AAG TCC CTG GTA GTC TTC TGC ATG GCC ACA TAC GGA GAG GGC GAC CCC ACG GAC AAT 448 Lou Ala Asp Lou Ser Ser Lou Pro Glu Ile Asp Lys Ser Leu Val Val Phe Cys Met Ala Thr Tyr Gly Glu Gly Asp Pro Thr Asp Asn 120 130 140 GCG CAG GAC TTC TAT GAC TGG CTG CAG GAG ACT GAC GTG GAC CTC ACT GGG GTC AAG TTT GCT GTA TTT GOT CTT GGG AAC AAG ACC TAT 538 Ala Gln Asp Phe Tyr Asp Trp Leu Gln Glu Thr Asp Val Asp Lou Thr Gly Val Lys Phe Ala Val Phe Gly Lou Gly Asn Lys Thr Tyr 150 160 170 GAG CAC TTC AAT GCC ATG GGC AAG TAT CTG GAC CAG AGG CTG GAG CAG CTT GGC GCC CAG CGC ATC TTT GAG TTG GGC CTT GGT GAT GAT 628 Glu His Phe Asn Ala Met Gly Lys Tyr Val Asp Gln Arg Lou Glu Gln Lou Gly Ala Gln Arg Ile Phe Glu Lou Gly Lou Gly Asp Asp 180 190 200 GAC GGG AAC TTG GAA GAG GAT TTC ATC ACG TGG AGG GAG CAG TTC TGG CCA GCT GTG TGC GAG TTC TTT GGG GTA GAA GCC ACT GGG GAG 718 Asp Gly Asn Lou Glu Glu Asp Phe Ile Thr Trp Arg Glu Gin Phe Trp Pro Ala Val Cys Glu Phe Phe Gly Val Glu Ala Thr Gly Glu 210 220 230 GAG TCG AGC ATT CGC CAG TAT GAG CTC GTG GTC CAC GAA GAC ATG GAC GTA GCC AAG GTG TAC ACG GGT GAG ATG GGC CGT CTG AAG AGC 808 Glu Ser Ser Ile Arg Gln Tyr Glu Lou Val Val His Glu Asp Met Asp Val Ala Lys Val Tyr Thr Gly Glu Met Gly Arg Lou Lys Ser 240 250 260 TAC GAG AAC CAG AAA CCC CCC TTC GAT OCT AAG AAT CCA TTC CTG GCT GCT GTC ACC GCC AAC CGG AAG CTG AAC CAA GGC ACT GAG CGG 898 Tyr Glu Asn Gln Lys Pro Pro Phe Asp Ala Lys Asn Pro Phe Lou Ala Ala Val Thr Ala Asn Arg Lys Lou Asn Gln Gly Thr Glu Arg 270 280 290 CAT CTA ATG CAC CTG GAG TTG GAC ATC TCA GAC TCC AAG ATC AGG TAT GAA TCT GGA GAT CAC GTG GCT GTG TAC CCA GCC AAT GAC TCA 988 His Leu Met His Lou Glu Leu Asp Ile Ser Asp Ser Lys Ile Arg Tyr Glu Ser Gly Asp His Val Ala Val Tyr Pro Ala Asn Asp Ser 300 310 320 GCC CTG GTC AAC CAG ATT GGG GAG ATC CTG GGA GCT GAC CTG GAT GTC ATC ATG TCT CTA AAC AAT CTC GAT GAG GAG TCA AAC AAG AAG 1078 Ala Lou Val Asn Gln Ile Gly Glu Ile Leu Gly Ala Asp Leu Asp Val Ile Met Ser Lou Asn Asn Leu Asp Glu Glu Ser Asn Lys Lys 330 340 350 CAT CCG TTC CCC TGC CCC ACC ACC TAC CGC ACG GCC CTC ACC TAC TAC CTG GAC ATC ACT AAC CCG CCA CGC ACC AAT GTG CTC TAC GAA 1168 His Pro Phe Pro Cys Pro Thr Thr Tyr Arg Thr Ala Lou Thr Tyr Tyr Lou Asp Ile Thr Asn Pro Pro Arg Thr Asn Val Lou Tyr Glu 360 370 380 CTG GCA CAG TAC GCC TCA GAG CCC TCG GAG CAG GAG CAC CTG CAC AAG ATG GCG TCA TCC TCA GGC GAG GGC AAG GAG CTG TAC CTG AGC 1258 Lou Ala Gln Tyr Ala Ser Glu Pro Ser Glu Gln Glu His Lou His Lys Met Ala Ser Ser Ser Gly Glu Gly Lys Glu Lou Tyr Lou Ser 390 400 410 TGG GTG GTG GAA GCC CGG