Coding Nucleotidesequence of Rat NADPH-Cytochrome P-450

Home , Flavin group

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 AGG CAC ATC CTA GCC ATC CTC CAA GAC TAC CCA TCA CTG CGG CCA CCC ATC GAC CAC CTG TGT GAG CTG CTG 1348 Trp Val Val Glu Ala Arg Arg His Ile Lou Ala Ile Lou Gln Asp Tyr Pro Ser Lou Arg Pro Pro Ile Asp His Lou Cym Glu Lou Lou 420 430 440 CCA CGC CTG CAG GCC CGA TAC TAC TCC ATT GCC TCA TCC TCC AAG GTC CAC CCC AAC TCC GTG CAC ATC TGT GCC GTG GCC GTG GAO TAC 1438 Pro Arg Lou Gln Ala Arg Tyr Tyr Set Ile Ala Ser Ser Ser Lys Val His Pro Asn Ser Val His Ile Cys Ala Val Ala Val Glu tyr 450 460 470 GAA GCG AAG TCT GGC CGA GTG AAC AAG GGG GTG GCC ACT AGC TGG CTT CGG GCC AAG GAA CCA GCA GGC GAG AAT GGC GGC CGC 0CC CTG 1528 Glu Ala Lys Ser Gly Arg Val Asn Lys Oly Vol Ala Thr Ser Trp Lou Arg Ala Lys Glu Pro Ala Gly Glu Asn Gly Gly Arg Ala Lou 480 490 500 GTA CCC ATG TTC GTG CGC AAA TCT CAG TTC CGC TTG CCT TTC AAG TCC ACC ACA CCT GTC ATC ATG GTG GGC CCC GGC ACT GGG ATT GCC 1618 Val Pro Met Phe Val Arg Lys Ser Gln Phe Arg Lou Pro Phe Lys Ser Thr Thr Pro Val Ile Met Val Gly Pro Gly Thr Gly Ile Ala 510 520 530 CCT TTC ATG GGC TTC ATC CAG GAA CGA GCT TGG CTT CGA GAG CAA GGC AAG GAG GTG GGA GAG ACG CTG CTA TAC TAT GGC TGC COG CGC 1708 Pro Phe Met Gly Phe Ile Gln Glu Arg Ala Trp Lou Arg Glu Gln Gly Lys Glu Val Gly Glu Thr Lou Lou Tyr Tyr Gly Cys ArB Ar8 540 550 560 TCG OAT GAG GAC TAT CTG TAC CGT GAA GAG CTA GCC CGC TTC CAC AAG GAC GGT GCC CTC ACG CAG CTT AAT GTG GCC TTT TCC CGG GAG 1798 Ser Asp Glu Asp Tyr Lou Tyr Arg Glu Glu Leu Ala ArB Phe His Lys Asp Gly Ala Lou Thr Gln Lou Asn Val Ala Phe Ser Arg Glu 570 580 590 CAG 0CC CAC AAG GTC TAT GTC CAG CAC CTT CTG AAG AGA GAC AGG GAA CAC CTG TGG AAG CTG ATC CAC GAG GGC GGT GCC CAC ATC TAT 1888 Gln Ala His Lys Val Tyr Val Gln His LeU Leu Lys ArB Asp Arg Glu His Lou Trp Lys Lou Ile His Glu Gly Gly Ala His ho0 Tyr 600 610 620 GTG TGC GG0 GAT GCT CGA AAT ATG GCC AAA GAT GTG CAA AAC ACA TTC TAT GAC ATT GTG OCT GAG TTC GGG CCC ATG GAG CAC ACC CAG 1978 Val Cys Gly Asp Ala Arg Asn Met Ala Lys Asp Val Gin Asn Thr Phe Tyr Asp Ile Val Ala Glu Phe Gly Pro Met Glu His Thr Gln 630 640 650 OCT GTG GAC TAT GTT AAG AAG CTG ATG ACC AAG GGC CGC TAC TCA CTA GAT GTG TGG AGC TAG GAGCTACCACCCTCCCACCCCTCGCTCCCTGTAAT 2076 Ala Val Asp Tyr Val Lys Lys Lou Het Thr Lys Gly Arg Tyr Ser Lou Asp Val Trp Ser End 660 670 678 CACCTAACTTCTGCCGACCTCCACCTCTGdTGGTTCCTGCCTGGCCTGGACACAGGGAGGCCCAGGGACTGACTCCTCCTGGCCTGAGTGGTGCCCTCCTGGGCCCCTAGCCAGAGCCC 2195 GGTCCATTGTATCAGGCAGCCCAGCCCCAGGGCACAtGGCAAGAGGGACTGGACCCACCTTTGGGTGATGGGTGCCTTAGGTCCTCTGCAGCTGTACAGAAGGGGCTCTTCTCTCCAkCA 23114 GAGCTGGGGTGCAGCCCCCACACGTGATTTTGAATGAGTGTAAATAATTTTAAATAACCTGGCCCTTGGAATAAAGTTGTTTTCAGT(AAA.... ) 2401 FiG. 2. Coding nucleotide sequence for NADPH-cytochrome P-450 oxidoreductase with derived amino acid sequence. Nucleotides are numbered to the right of each line; amino acids, below the corresponding residue. of sequence complementary to the 3' end of rat liver is 2401 nucleotides long, in addition to a 3' poly(A) stretch of oxidoreductase mRNA (28). The complete cDNA sequence 27 nucleotides. Primer extension as described by Lee and Downloaded by guest on September 26, 2021 Biochemistry: Porter and Kasper Proc. Nati. Acad. Sci. USA 82 (1985) 975 Roeder (29) of pOR-8 with oxidoreductase-immunoenriched (38). Regions of similarity were identified on a computer- RNA indicated that -68 nucleotides of the nontranslated generated dot matrix plot and the sequences involved were region at the 5' end of the oxidoreductase message had not identified using an alignment algorithm, as described in been cloned (unpublished data). Materials and Methods. Those matches to NADPH- The open reading frame initiating at the amino-terminal cytochrome P-450 oxidoreductase occurring within regions methionine codon (Fig. 2) codes for a polypeptide of 678 of the flavoproteins reported to be involved in flavin binding amino acids with a molecular weight of 76,962. The amino or stabilization or in NADPH binding are indicated by acid composition of the derived polypeptide-alanine, 51; overlining in Fig. 4. The matches are clustered in two arginine, 36; asparagine, 23; aspartic acid, 41; half-cystine, discrete regions of the oxidoreductase sequence (residues 7; glutamine, 24; glutamic acid, 60; glycine, 45; histidine, 21; 84-242 and 393-588), suggesting the presence of two flavin- isoleucine, 27; leucine, 59; lysine, 38; methionine, 19; binding domains. Because the FAD-binding domain and the phenylalanine, 30; proline, 30; serine, 43; threonine, 35; NADPH-binding domain are likely to be in close proximity tryptophan, 9; tyrosine, 32; and valine, 48-is in close and probably have elements in common, it was not possible agreement with the published compositions of the rat liver to resolve these two regions by using this comparison oxidoreductase (30, 31). The amino terminus of the rat technique. Attempts to identify and separate these two protein is blocked to direct sequence determination; how- domains with "FAD-specific" or "NADPH-specific" se- ever, compositional analysis of the trypsin-released amino- quences were unsuccessful. terminal peptide is consistent with initiation of translation as The two matches to D. vulgaris flavodoxin present in the shown in Fig. 2 (unpublished data). Furthermore, the cDNA- first domain of oxidoreductase (residues 84-242, Fig. 4) derived sequence from Ile-57 to Arg-78 is in perfect agree- prompted further comparison of these two proteins. D. ment with the sequence reported by Black and Coon (18) for vulgaris flavodoxin is an FMN-containing, 148 amino acid the amino terminus of the trypsin-solubilized rat protein. protein that functions as an electron carrier and can replace Although the published carboxyl-terminal sequence Trp- transport reactions. Its (Leu,Val)-Asp-Ser-COOH (18) differs from the cDNA- ferredoxin in a variety of electron derived sequence Leu-Asp-Val-Trp-Ser-COOH, composi- primary and three-dimensional structures have been deter- tionally the amino acids released by carboxypeptidase Y mined (38, 39), and alignment of the complete amino acid digestion are in agreement with the cDNA-derived se- sequence of this protein with residues 77-228 of quence. oxidoreductase reveals a surprising degree of similarity Comparison to the Rabbit Oxidoreductase. The rat and between the two proteins (Fig. 5). The two boxed regions rabbit oxidoreductase amino-terminal amino acid sequences within this comparison correspond to the D. vulgaris are compared in Fig. 3. Although 8 residues longer at the amino terminus, the rat sequence shows 68% identity with 1 MGDSHEDTSA TMPEAVAEEV SLFSTTDMVL FSLIVGVLTY WFIFRKKKEE 50 the rabbit sequence, with the remainder mostly conservative amino acid differences. In the rat, residues 1-56 comprise 51 IPEFSKIQTT APPVKESSFV EKMKKTGRNI IVFYGSQTGT AEEFANRLSK 100 the membrane-binding portion of oxidoreductase, with the SADPEEYDLA DLSSLPEIDK SLVVFCMATY 150 uniquely sensitive trypsin-cleavable Lys-56/Ile-57 bond de- 101 DAHRYGMRGM GEGDPTDNAQ marcating the membrane-binding segment from the catalytic 151 DFYDWLQETD VDLTGVKFAV FGLGNKOEtH FNAMGKYVDQ RLEQLGAQRI 200 portion of the enzyme. Two regions appear highly conserved between the two species: rat residues 45-50, which, due to 201 FELGLGDDDG NLEEDFITWR EQFWPAVCEF FGVEATGEES SIRQYELVVH 250 their charged nature, may be involved in interactions with the polar phospholipid head-groups (18), and residues 64-80, 251 EDMDVAKVYT GEMGRLKSYE NQKPPFDAKN PFLAAVTANR KLNQGTERHL 300 found in the amino-terminal region of the catalytic portion of 301 MHLELDISDS KIRYESGDHV AVYPANDSAL VNQIGEILGA DLDVIMSLNN 350 the protein. Identification of Flavin-Binding Domains. The deduced 351 LDEESNKKHP FPCPTTYRTA LTYYLDITNP PRTNVLYELA QYASEPSEQE 400 amino acid sequence for rat oxidoreductase was compared to amino acid sequences of six other flavoproteins: human 401 HLHKMASSSG EGKELYLSWV VEARRHILAI LQDYPSLRPP IDHLCELLPR 450 erythrocyte glutathione reductase (33), Pseudomonas fluorescens p-hydroxybenzoate hydroxylase (34), pig kidney 451 LQARYYSIAS SSKVHPNSIO ICAVAVEYEA KSGRVNKGVA TSWLRAKEPA 500 D-amino acid oxidase (35), E. coli fumarate reductase (36), 501 GENGGRALVP MFVRKSQFRL PFKSTTPVIM VGPGTGIAPF MGFIQERAWL 550 Clostridium MP flavodoxin (37), and D. vulgaris flavodoxin 551 REQGKEVGET LLYYGCRRSD EDYLYREELA RFHKDGALTQ LNVAFSREQA 600 I MGDSHEDTSATMPEAVAEEVSLFSTTDMVLFSLIVGVLTY 40 601 HKVYVQHLLK RDREHLWKLI HEGGAHIYVC GDARNMAKDV QNTFYDIVAE 650 1 (PTHD)6EAAAQEASVFSMTDVVLFSLIVGLITN 32 651 FGPMEHTQAV DYVKKLMTKG RYSLDVWS 678 41 NFIFRKKKEEIPEFSKIO-TTAPPVKESSFVEKMKKTGRNI 80 FIG. 4. Identification of flavin-binding domains in NADPH- 33 YFLFRKKKEEVPNFTKIOAPTSSSVKESSFVEKMKKTGRN172 cytochrome P-450 oxidoreductase. The oxidoreductase amino acid sequence (shown) was compared to other flavoprotein sequences as FIG. 3. Comparison of the rat and rabbit oxidoreductase amino- described in Materials and Methods, and regions of similarity terminal sequences. The cDNA-derived rat amino acid sequence is occurring within the flavin- or NADPH-binding domains of the aligned above the rabbit sequence (18); an asterisk indicates identi- proteins are indicated on the oxidoreductase sequence by overlin- cal residues, a dot indicates a conservative substitution (comparison ing. The amino acid coordinates of the matches shown, followed by value >10, from ref. 32). The dash indicates a gap inserted to the appropriate flavoprotein and its matched sequence, are as optimize the alignment. The order of the first 4 residues of the rabbit follows: 84-95, D. vulgaris flavodoxin (D-fdx) 8-19; 143-172, sequence was undetermined. The single-letter amino acid abbrevia- Clostridium MP flavodoxin (C-fdx) 57-86; 165-217, D-fdx 85-137; tions used are A, alanine; C, cysteine; D, aspartic acid; E, glutamic 202-242, p-hydroxybenzoate hydroxylase (p-hbh) 250-290; 393-415, acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, p-hbh 303-325; 395-413, C-fdx 62-80; 452-485, fumarate reductase lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, 26-56; 488-509, glutathione reductase (gr) 27-48; 524-560, gr glutamine; R, arginine; S. serine; T, threonine; V, valine; W, 259-295; 558-588 D-fdx 85-115. The circled tyrosyl and histidyl tryptophan; and Y, tyrosine. residues are implicated in flavin binding, as discussed in the text. Downloaded by guest on September 26, 2021 976 Biochemistry: Porter and Kasper Proc. Natl. Acad. Sci. USA 82 (1985) flavodoxin/rat oxidoreductase matches present in the first the random average 4.9, arguing strongly against a chance flavin-binding domain of Fig. 4. The first region involves similarity of this extent. It is therefore likely that Tyr-178 of residues 3-25 of flavodoxin and residues 79-101 of the oxidoreductase, like Tyr-98 of flavodoxin, interacts directly oxidoreductase. X-ray crystallographic studies have shown with the isoalloxazine ring of FMN. In accord with this, that this portion of the flavodoxin molecule is hydrogen- Nisimoto et al. (41) have presented evidence obtained from bonded to the phosphate group of FMN (39); the specific nuclear polarization studies that a tyrosyl residue is involved residues involved are individually boxed in Fig. 5. Two of in FMN binding in NADPH-cytochrome P-450 oxidore- the four bonding residues, Ser-10 and Thr-12, are conserved ductase. in the oxidoreductase sequence (Ser-86 and Thr-88), and a In addition to the residues just discussed, x-ray crystal- third, Asn-14, is replaced by threonine (Thr-90), a residue lographic studies have identified a Ser-Thr-Trp sequence capable of hydrogen bonding. The conservation of these (residues 58-60, overlined in Fig. 5) as also being involved in specific residues suggests that this region of oxidoreductase FMN binding in D. vulgaris flavodoxin (39). Ser-58 is interacts with the phosphate group of FMN. Further evi- hydrogen-bonded to the FMN phosphate group; however, dence in support of this conclusion was gained from a this residue is replaced by Ala-138 in the oxidoreductase. statistical analysis of the similarity between these flavodoxin The adjacent residue Thr-59 of flavodoxin is hydrogen- and oxidoreductase sequences. Within this stretch of 23 bonded to the ribityl moiety of FMN and is conserved in residues there are 7 matches (designated by asterisks in Fig. oxidoreductase. From a three-dimensional analysis of the 5), and at least 9 of the differences are considered conserva- flavodoxin molecule, it becomes clear that the flavin group is tive substitutions (designated by dots). To test the likelihood situated in a pocket between Tyr-98 and Trp-60; the latter of this degree of similarity arising by chance, the residue is replaced by Tyr-140 in the oxidoreductase and by oxidoreductase sequence (residues 79-101) was randomly methionine in several other bacterial flavodoxins (37). shuffled 10 times (40) and each shuffled sequence was Hence, by analogy with flavodoxin, the oxidoreductase compared to the flavodoxin sequence (residues 3-25) using flavin would be situated between tyrosyl residues 140 and the Bestfit program (26). For each comparison a "quality 178. It is interesting to note that the distance separating the value" was assigned based on the number of amino acid two aromatic residues interacting with the flavin is the matches between the two sequences. The quality value of same-38 amino acids-in both proteins. Finally, the glycine the original comparison, 6.7, is 4 standard deviations from immediately following Trp-60 in D. vulgaris flavodoxin, the random average 3.9, arguing against a chance similarity although not implicated in FMN binding, is conserved in the of this extent. It thus appears likely that these two segments oxidoreductase as well as in all sequenced flavodoxins (see have a common evolutionary origin; furthermore, this find- ref. 42). ing, coupled with the conservation of the phosphate-bonding By insertion of a four-residue gap between the two FMN- residues, argues that this region of oxidoreductase interacts binding regions (Fig. 5), the flavodoxin and oxidoreductase with the phosphate group of FMN. sequences are optimally aligned. These two regions are The second region of similarity shown in Fig. 5 involves separated by a stretch of 63 residues in oxidoreductase that residues 85-122 of flavodoxin and residues 165-202 of the shows a rather low level of similarity to the corresponding oxidoreductase. X-ray crystallographic studies have shown flavodoxin sequence, excluding the Thr-Tyr-Gly sequence that Tyr-98 of flavodoxin stabilizes the FMN prosthetic (residues 139-141) discussed above. Since the number of group through stacking interactions between its aromatic residues separating these two regions of the FMN-binding side chain and the flavin isoalloxazine ring (39); this tyrosine domain in oxidoreductase and flavodoxin is 63 and 59, matches Tyr-178 of the oxidoreductase (inner box in Fig. 5 respectively, the preservation of this spacing may be of functional significance. For example, this intervening region and circled in Fig. 4), suggesting that Tyr-178 is involved in may be involved in interactions with electron acceptor flavin binding in the oxidoreductase. This comparison of 38 proteins, such as the cytochromes P-450 in the case of residues contains 13 matches and 11 conservative substitu- oxidoreductase. The overall similarity between these two tions; the quality value of 12.0 is 8 standard deviations from flavoproteins suggests that residues 77-228 of oxidoreductase originated from the same ancestral gene that gave 1 HPKALIVYG6TfGNTEYTAETIARELANAGYEVDSRDAAS 40 rise to the bacterial flavodoxins. As stated above, because the FAD-binding domain and 77 GR NIIVFYGSjTG TAJEEFANRLSKD AHRYGMRGHSADPEE 116 the NADPH-binding domain of NADPH-cytochrome P-450 oxidoreductase are likely to be in close proximity and share 41 VEAGGLFEGFD----LVLLGCSTWGDDSIELODDFIPLFD 76 a number of characteristics, we were not able to individually *.. * * * .. . * * . . . 117 YDLADLSSLPEIDKSLVVFCMATYGEGDPTDNAODFYDWL 156 resolve and characterize these two domains through sequence comparisons. Nonetheless, the match to E. coli fumarate reductase identified in the FAD/NADPH-binding 77 SLEETGAOGRKVACFGCGDSSf_]EYFCGAVDAIEEKLKNLG 116 ~~~.*1* *** * * *X,,. .** region of oxidoreductase (residues 393-588, Fig. 4) is of 157 QETDVDLTGVKFAVFGLGNKT YEHFNAMGKYVDQRLEQLG 196 interest. E. coli fumarate reductase is a 66-kDa, membrane- bound flavoprotein associated with the electron transport proteins of the cell membrane. It catalyzes the reduction of 117 AEIVQDGLRIDGDPRAARDDIVGWAHDVRGAI 148 fumarate to succinate under anaerobic conditions, and its 197 AQRIFELGLGDDDGNLEEDFITWREOFWPAVC 228 composition is similar to that of several succinate dehydrogenases (36). The FAD prosthetic group of fumarate FIG. 5. Alignment of NADPH-cytochrome P-450 oxidoreduc- reductase is covalently attached to a histidyl residue (43), tase and D. vulgaris flavodoxin. The complete amino acid sequence thought to be His-45 by sequence comparison to the flavin- of D. vulgaris flavodoxin is aligned above oxidoreductase residues binding peptide of beef heart succinate dehydrogenase (36). 77-228. This alignment was obtained using the Gap program (gap When amino acids 26-50 of fumarate reductase are aligned weight set at 3.0, gap-length weight set at 0.3, with ends weighted). with residues 452-477 of oxidoreductase (Fig. 6), His-45 of An asterisk indicates identical residues, a dot indicates a conserva- fumarate reductase matches His-470 of oxidoreductase tive substitution (comparison value >10, from ref. 32). The dashes in 6 and circled in Fig. 4). In this stretch of 26 indicate a gap inserted to optimize the alignment. The boxed (boxed Fig. sequences and overlined residues identify regions directly involved residues, there are 12 matches and 1 conservative substitu- in FMN binding. tion. The quality value for this match of 8.5 is 7 standard Downloaded by guest on September 26, 2021 Biochemistry: Porter and Kasper Proc. Natl. Acad. Sci. USA 82 (1985) 977

26 OANPNAKIALISKVYPMRSH --TVAAE 50 8. Enoch, H. G. & Strittmatter, P. (1979) J. Biol. Chem. 254, 8976-8981. 452 QA-RYYS IASSSKVHPNSVH I CAVAVE 477 9. Ilan, Z., Ilan, R. & Cinti, D. L. (1981) J. Biol. Chem. 256, 10066-10072. FIG. 6. Comparison of NADPH-cytochrome P-450 oxidoreduc- 10. Ono, T., Ozasa, S., Hasegawa, F. & Imai, Y. (1977) Biochim. tase to the flavin-binding segment of E. coli fumarate reductase. Biophys. Acta 486, 401-407. Amino acid residues 26-50 of E. coli fumarate reductase are aligned 11. Masters, B. S. S., Baron, J., Taylor, W. E., Isaacson, E. L. & above oxidoreductase residues 452-477. This alignment was ob- LoSpalluto, J. (1971) J. Biol. Chem. 246, 4143-4150. tained using the Gap program, as described in the legend to Fig. 5. 12. Iyanagi, T. & Mason, H. S. (1973) Biochemistry 12, 2297-2308. The histidine discussed in the text with regard to flavin binding is 13. Vermilion, J. L. & Coon, M. J. (1974) Biochem. Biophys. Res. boxed. The length of the comparison shown here differs from the Commun. 60, 1315-1322. length of the corresponding match shown in Fig. 4 due to the use of 14. Masters, B. S. S., Prough, R. A. & Kamin, H. (1975) Bio- a different comparison program (Gap vs. Bestfit). chemistry 14, 607-613. 15. Dignam, J. D. & Strobel, H. W. (1975) Biochem. Biophys. Res. Commun. 63, 845-852. deviations from the random average 4.5, making it unlikely 16. Black, S. D., French, J. S., Williams, J. H., Jr., & Coon, that this degree of similarity arose by chance. It is interesting M. J. (1979) Biochem. Biophys. Res. Commun. 91, 1528-1535. that this sequence is conserved in the oxidoreductase, which 17. Gum, J. R. & Strobel, H. W. (1981) J. Biol. Chem. 256, has a noncovalently attached FAD group (1, 2). The func- 7478-7486. tional significance ofthe covalent or noncovalent attachment 18. Black, S. D. & Coon, M. J. (1982) J. Biol. Chem. 257, is unclear, as the flavin group is capable of functioning in 5929-5938. both states. One possible explanation is that the covalent 19. Lu, A. Y. H., Junk, K. W. & Coon, M. J. (1969) J. Biol. attachment in the oxidoreductase has been lost relatively Chem. 244, 3714-3721. recently, while being preserved in E. coli fumarate reductase 20. Vermilion, J. L., Ballou, D. P., Massey, V. & Coon, M. J. (1981) J. Biol. Chem. 256, 266-277. and beef heart succinate dehydrogenase. It is perhaps sig- 21. Hardwick, J. P., Gonzalez, F. J. & Kasper, C. B. (1983) J. nificant in this regard that succinate dehydrogenase is a Biol. Chem. 258, 10182-10186. mitochondrial enzyme, in light of the possible bacterial 22. Gonzalez, F. J. & Kasper, C. B. (1981) J. Biol. Chem. 256, origin of mitochondria. We note that a sequence similar to 4697-4700. the FAD-binding segment of fumarate reductase can also be 23. Thayer, R. E. (1979) Anal. Biochem. 98, 60-63. identified in D-amino acid oxidase at residues 21-45; how- 24. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, ever, the position corresponding to the proposed flavin- 499-560. binding histidine of fumarate reductase is occupied by 25. Krayev, A. S., Kramerov, D. A., Skryabin, K. G., Ryskov, threonine. Perhaps not surprisingly, the FAD group of this A. P., Bayev, A. A. & Georgiev, G. P. (1980) Nucleic Acids Res. 8, 1201-1215. cytosolic protein is not covalently attached. 26. Smith, T. F. & Waterman, M. S. (1981) Adv. Appl. Math. 2, Comparison of D. vulgaris flavodoxin as well as other 482-489. bacterial flavodoxins to fumarate reductase reveals little 27. Needleman, S. B. & Wunsch, C. D. (1970) J. Mol. Biol. 48, similarity between these two types of flavoproteins, indicat- 443-453. ing that they are not closely related. Comparison of the 28. Gonzalez, F. J. & Kasper, C. B. (1982) J. Biol. Chem. 257, oxidoreductase to itself reveals no broad internal similarities 5962-5968. suggestive of a gene duplication event responsible for the 29. Lee, D. C. & Roeder, R. G. (1981) Mol. Cell. Biol. 1, 635-651. generation of the two flavin-binding domains, although it is 30. Vermilion, J. L. & Coon, M. J. (1978) J. Biol. Chem. 253, that this event occurred very and is no 2694-2704. possible early longer 31. Gum, J. R. & Strobel, H. W. (1979) J. Biol. Chem. 254, recognizable. Clearly, the FMN-binding domain is homolo- 4177-4185. gous with the bacterial flavodoxins; the homology of the 32. Staden, R. (1982) Nucleic Acids Res. 10, 2951-2%1. FAD-binding domain with a second, apparently unrelated 33. Krauth-Siegel, R. L., Blatterspiel, R., Saleh, M., Schiltz, E., protein (fumarate reductase) presents the intriguing possibil- Schirmer, R. H. & Untucht-Grau, R. (1982) Eur. J. Biochem. ity that the oxidoreductase gene is the result of a fusion of 121, 259-267. two different genes coding for flavin-binding proteins. 34. Weijer, W. J., Hofsteenge, J., Vereijken, J. M., Jekel, P. A. & Beintema, J. J. (1982) Biochim. Biophys. Acta 704, 385-388. 35. Ronchi, S., Minchiott, L., Galliano, M., Curti, B., Swenson, We thank Dr. Harold Deutsch for the amino acid analyses and Dr. R. P., Williams, C. H., Jr., & Massey, V. (1982) in Flavins and Richard Burgess for use of his computer facilities. Helpful discus- Flavoproteins, eds. Massey, V. & Williams, C. H. (Elsevier, sions with Edwin Madison during the course of this work were much New York), pp. 66-72. appreciated. This research was supported by Grants CA09230 and 36. Cole, S. T. (1982) Eur. J. Biochem. 122, 479-484. CA22484 from the National Institutes of Health. 37. Tanaka, M., Haniu, M., Yasunobu, K. T. & Mayhew, S. G. (1974) J. Biol. Chem. 249, 4393-4396. 1. Williams, C. H., Jr., & Kamin, H. (1962) J. Biol. Chem. 237, 38. Dubourdieu, M. & Fox, J. L. (1977) J. Biol. Chem. 252, 587-595. 1453-1463. 2. Phillips, A. H. & Langdon, R. G. (1962) J. Biol. Chem. 237, 39. Watenpaugh, K. D., Sieker, L. C. & Jensen, L. H. (1973) 2652-2660. Proc. Natl. Acad. Sci. USA 70, 3857-3860. 3. Kasper, C. B. (1971) J. Biol. Chem. 246, 577-581. 40. Fitch, W. M. & Smith, T. F. (1983) Proc. Natl. Acad. Sci. 4. Lu, A. Y. H., Junk, K. W. & Coon, M. J. (1969) J. Biol. USA 80, 1382-1386. Chem. 244, 3714-3721. 41. Nisimoto, Y., Hayashi, F., Akutsu, H., Kyogoku, Y. & 5. Miller, E. C. & Miller, J. A. (1974) in The Molecular Biology Shibata, Y. (1984) J. Biol. Chem. 259, 2480-2483. ofCancer, ed. Busch, H. (Academic, New York), pp. 377-402. 42. Tanaka, M., Haniu, M., Yasunobu, K. T. & Yoch, D. C. 6. Gelboin, H. V. (1980) Physiol. Rev. 60, 1107-1166. (1977) Biochemistry 16, 3525-3537. 7. Schacter, B. A., Nelson, E. B., Marver, H. S. & Masters, 43. Weiner, J. H. & Dickie, P. (1979) J. Biol. Chem. 254, B. S. S. (1972) J. 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