Vol. 265, No. 11. Issue of April 15, pp. 6066-6073, 1990 Printed in U.S.A. B 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Putidaredoxin Reductase and Putidaredoxin CLONING, SEQUENCE DETERMINATION, AND HETEROLOGOUS EXPRESSION OF THE PROTEINS*

(Received for publication, September 26, 1989)

Julian A. Petersoni, Matthew C. LorenceS, and Bilal Amarneh From the Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235.9038

The oxidation of camphor by cytochrome P-450,,, requires the participation of a flavoprotein, putidare- doxin reductase, and an iron-sulfur protein, putidare- doxin, to mediate the transfer of electrons from NADH to P-450 for oxygen activation. A 2.2-kilobase pair BarnHI-StuI fragment from whole cell DNA of cam- phor-grown Pseudomonas putida has been cloned and sequenced. Translation of the sequence revealed two open reading frames that could code for putidaredoxin reductase and putidaredoxin. In the case of putidare- doxin, the translated sequence matched the published sequence (Tanaka, M., Haniu, M., Yasunobu, K. T., DUS, K., and Gunsalus, I. C. (1974) J. Biol. Chem. 249, 3689-3701) with the exception of one amino acid. Codon usage in these proteins, like the proteins of other Pseudomonads, is strongly biased to G + C in the third nucleotide. A potential transcription termination site was found 3’ to the putidaredoxin coding region. The “FAD-binding” amino acid consensus sequence, present in other flavoproteins, was found in putidaredoxin reductase beginning at residue 11 and a second occur- rence of this sequence was found beginning with amino acid 156. The second sequence could represent the NAD-binding site. The regions encoding putidaredoxin reductase and putidaredoxin were subcloned and in- dependently expressed in Escherichia coli at the level of 0.4 and 4.8 mg of enzymatically active protein/g wet weight of cells, respectively. Site-directed muta- genesis was used to change the rare start codon, GTG, of putidaredoxin reductase to ATG which resulted in an 18-fold increase in the level of expression of this protein to 7.4 mg/g wet weight of cells. The construc- tion of these two clones, which express these important proteins, will facilitate studies of their interaction with each other and with P-450,,,.

The activation of molecular oxygen for incorporation into drugs, steroids, and carcinogens has been the focus of intense interest since the discovery of the role of cytochrome P-450 in the diverse set of reactions (1). The application of molecular

* This research was supported in part by Grant GM19036-20 from the National Institutes of Health and by Grant I-0405 from the Robert A. Welch Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “uduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 505406.

3 To whom correspondence should be addressed: Dept. of Biochem- istry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9038.

$ Supported by Postdoctoral Fellowship Training Grant 5- T32HD07190 from the United States Public Health Service.

cloning techniques to the study of these enzymes has resulted in the sequence determination of more than 100 to date (2, 3). The study of the three-dimensional structure of cyto- chromes P-450 received considerable impetus with the deter- mination of the structure of P-450,,, (4).

The study of the electron transfer reactions from reduced pyridine nucleotides to P-450 has also been rewarding. The oxidation of camphor by extracts from Pseudomonas putida requires the participation of three protein components. These have been identified as a FAD-containing flavoprotein, puti- daredoxin reductase, and an iron-sulfur protein, putidare- doxin as well as the P-450 (5). This electron transfer sequence is very similar to the one functional in adrenal cortex mito- chondrial steroid metabolism. Although these electron trans- fer components are formally similar, they will not substitute for one another in their respective assay systems (6, 7) indi- cating that the three-dimensional geometry of the groups that comprise the binding site for protein-protein interaction is different.

Our long-standing research into the mechanism of electron transfer from NADH to P-450,,, has been hindered by the limited availability of putidaredoxin reductase and the inher- ent difficulty associated with the chemical modification of these proteins to identify residues functional in these inter- actions (7). The ratio of putidaredoxin reductase to P-450,,, in P. putida is approximately 1% and these proteins are difficult to separate during their purification (8, 9). With the determination of the nucleotide sequence of the N-terminal 153 nucleotides of putidaredoxin reductase, it was found that the start codon for this protein was GTG rather than ATG (10). This low level of expression of putidaredoxin reductase was presumed to be due to the presence of this unusual start codon in the gene coding for this protein. To test this hypoth- esis and to obtain large amounts of the recombinant proteins, we have cloned, sequenced, and independently expressed both putidaredoxin reductase and putidaredoxin in Escherichia coli.

EXPERIMENTAL PROCEDURES

Materials-The E. coli strains DH5a (F-, endAl, hsdRl7 W, mk+), supE44, thi-1, X-, recA1, gyrA96, relA1, A(argF-laczya)U169, 480dlacZAM15) and DH~LuF’(F’, endAl, hsd R17 (rk-, mk’), supE44, thi-1, X-, recA1, gyrA96, relA1, $80dlacZAM15, A(lacZYAargF)U169) were obtained from Bethesda Research Laboratories, Life Technolo- gies, Inc. (BRL) as competent cells. The plasmids pIBI24 and pIBI25, which are derived from pEMBL plasmids, were obtained from Inter- national Biotechnologies, Inc. The restriction enzymes and the bac- teriophage, M13mp18 and M13mp19, were obtained from BRL. P. putida (ATCC17453) was obtained from the American Type Culture Collection. The cell line was stored in media containing 7.5% glycerol at -80 “C. The nucleotide sequencing kit Sequenase Ver. 1.0 was obtained from United States Biocheiicals, In;. The [““S]dATP-otS (500 Ci/mmol) was obtained from Du Pant-New England Nuclear. Ampicillin was obtained from Sigma. All other reagents and chemicals were of the highest purity available. Mutagenic oligonucleotides and DNA sequence primers were synthesized on an Applied Biosystems

6066

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380A oligonucleotide synthesizer and purified by Sep-Pak C,R (Waters Associates) column chromatography.

Bacterial Growth-Stock cultures of E. coli strain DH5o(, which harbored the anticipated plasmids, were grown in 2 X YT media containing 50 pg/ml ampicillin. For expression of either putidare- doxin reductase or putidaredoxin, the desired cell line was grown for 16 to 24 h in TB media (11) containing 50 rg/ml ampicillin. The cells were harvested by centrifugation at 8000 rpm in a Beckman 521 centrifuge. The cells were broken by gentle sonication in a Branson Sonifier for 60 s, and the cell debris and unbroken cells were removed by centrifugation. The amount of enzyme present in the extract was determined as described below.

Preparation of Whole Cell DNA from P. putida-The plasmids encoding catabolic enzymes of Pseudomonads are typically rather large and difficult to prepare (12). For the present studies, whole cell DNA was prepared by a variation of a published procedure (13). Cells of P. putida (1 liter), which had been grown on d-camphor to station- ary phase, were harvested by centrifugation. The cells were washed with 275 ml of 10 mM sodium phosphate buffer, pH 7.0, to remove media and excess camphor and its metabolites. The cells were resus- pended in 100 ml of 10 mM Tris buffer, pH 8.0, containing 1 mM EDTA (TE buffer) and recentrifuged. The cells were finally resus- pended in 100 ml of lysis buffer containing 50 mM Tris chloride, pH 8.0, 50 mM EDTA, and 20% sucrose and stored on ice. The cell suspension was made 2 mg/ml in lysozyme and the incubation on ice continued for 30 min. The cells were lysed by the addition of sodium dodecyl sulfate to a final concentration of 4.%, and the mixture was heated to 70 “C and mixed gentlv for 30 min. Proteinase K was added to a final concentration ofO.1 mg/mi and the heating continued for an additional 60 min. Potassium acetate was added to a final concen- tration of 0.5 M and the incubation at 70 “C continued for 15 min. After cooling to room temperature, the cell debris was removed by centrifugation at 17,000 rpm for 20 min in a JA20 rotor in a Beckman 521 centrifuge. The DNA was precipitated from the supernatant solution by the addition of PEG8000 to a final concentration of 10%. The solutions were mixed by gentle inversion and stored at 4 “C overnight. The precipitate was collected by centrifugation at 12,000 rpm in the JA20 rotor for 15 min at 4 ‘C, rinsed-with cold 95% ethanol. and resuspended in 8 ml of TE buffer. The suspension was made 0.1 mg/ml in RNase A and heated at 60 “C for 30 min. The sample was cooled and extracted with: I) I volume of phenol; 2) I volume of phenol, 0.5 volume of chloroform:isoamyl alcohol (24:l); and 3) 1 volume of chloroform:isoamyl alcohol. The DNA was precip- itated with 0.5 volume of 7.5 M ammonium acetate and 2 volumes of ethanol. The precipitate was collected by centrifugation and dissolved in 1 volume of TE buffer and the ethanol ammonium acetate precip- itation repeated. The precipitate was washed with a small volume of 70% ethanol and dried in a vacuum desiccator. The DNA was resus- pended in 2 ml of TE buffer.

Library Construction and Screening-Whole cell DNA was cleaved with BamHI and StuI, ligated into similarly cleaved pIBI25, and transformed into DH5n competent cells. Approximately 20,000 re- combinant cells were plated onto 2 x YT plates containing ampicillin (50 fig/ml), transferred to 85-mm nitrocellulose filter discs (Schleicher & Schuell, BA85), and screened by hybridization using a polynucle- otide kinase-labeled oligonucleotide as probe. The 21-mer that was used to identify the desired clones had the nucleotide sequence ATGGGGAATTACCGTCGCATC. Filter discs were washed usine empirically determined conditions to remove nonspecifically bound probe. Colonies that had hybridized to the probe were visualized by overnight exposure of x-ray film at -80 “C in the presence of a Du Pont Chronex Lightning Plus intensifying screen. Selected colonies were removed from plates with sterile toothpicks and grown overnight in 2 x YT media containing ampicillin (50 pg/ml). The identity of the clone was confirmed by restriction analysis or by Southern hybridization of the isolated plasmid DNA with the probe.

P&mid Constructions-Plasmid DNA was isolated for restriction enzyme analysis by the alkaline-lysis method of Birnboim and Doly (14). Selected restriction fragments were fractionated on a 1.5% low melting point agarose (FMC Marine Colloids) gel, the desired frag- ment was excised, melted at 65 “C in a final volume of 0.4 ml of TE (15) containing 100 mM NaCl, and extracted two times with TE- saturated phenol. The extracted fragment was precipitated twice with ethanol and analyzed by agarose gel electrophoresis. Purified frag- ments were ligated into similarly cleaved pIBI24 or pIBJ25 and transformed into DH5a competent cells according to the procedures recommended by the manufacturer. Recombinant clones were iden- tified as white colonies in the presence of the chromogenic substrate

X-gall and screened by restriction enzyme analysis. In most cases, the orientation of the insert in the ligation reaction was controlled by the nonidentical cohesive ends of the insert which matched the vector. The procedure used for the ligation of target DNA into either the plasmid vector or into the replicative form of the M13mp18 or M13mp19 bacteriophage was identical and has been described previ- ously (15).

The Southern hybridization procedure used to identify DNA frag- ments containing desired sequences was performed essentially as described (15). The washing conditions to remove excess radiolabeled oligonucleotide probe was determined empirically. The preparation of “‘P-labeled oligonucleotide probes has been described (15).

Nucleotide Sequence Determination-Selected restriction frag- ments were purified from low melting point agarose, ligated into similarly cleaved M13mp18 and M13mp19, and transformed into DH5aF’ competent cells. Recombinant clones were identified as clear plaques in the presence of X-gal and screened by restriction enzyme analysis of replicative form DNA isolated by the alkaline-lysis pro- cedure. Single-strand phage DNA was isolated from infected cell cultures by polyethylene glycol precipitation, followed by sodium dodecyl sulfate-proteinase K digestion, phenol extraction, and ethanol precipitation. The nucleotide sequence of the purified single- stranded DNA was determined by the dideoxynucleotide chain ter- mination method (16) using a modified T7 DNA polymerase (17). In those inst.ances where the sequences determined for both the coding and noncoding strand were not in agreement, the determinations were repeated with deaza-dGTP to resolve band compression artifacts (18-20). In each instance, the discrepancies were resolved by this procedure.

Oligonucleotide-directed Mutagenesis-Oligonucleotide-directed mutagenesis was performed using the two-primer method of Zoller and Smith (21). In each case, the single base change was inserted in the middle of a 21-mer which would hybridize with the single-stranded DNA at the desired location. The newly synthesized double-stranded DNA was used to transform DH5aF’ competent cells. Recombinant plaques were transferred and fixed to the filters as described (15), and mutants were identified by hybridization to the polynucleotide kinase-labeled mutagenic oligonucleotide. Rather than using the standard wash procedure, tetramethylammonium chloride was used to accentuate the difference in melting temperature between the probe and the wild-type and changed sequences (22). In each case the bacteriophage was purified until all of the plaques on a given plate would hybridize with the probe. To ascertain that only the desired base change occurred, each mutant was completely sequenced fol- lowing plaque purification.

P-450,,,, Putidaredoxin Reductase, and Putidaredoxin Determina- tions-The standard assay for the amount of putidaredoxin reductase and putidaredoxin takes advantage of their ability to catalyze the reduction of the cytochrome c. This reaction is dependent on the presence of the reducing agent, NADH, and both enzyme components (8). In the typical assay either putidaredoxin reductase or putidare- doxin was in excess while the other component was limiting. Under these conditions, the rate of reduction of cytochrome c was linearly dependent on the concentration of the limiting component. Although there is a slight background of NADH-dependent reduction of cyto- chrome c in whole cell extracts from E. coli, the increase in rate upon the addition of the limiting component was at least 4-fold greater than the background. The standard assay for putidaredoxin reductase contained the following components in 20 mM MOPS buffer, pH 7.4, 0.1 mM NADH, 10 KM cytochrome c, and 5.5 pM putidaredoxin. An appropriate dilution of the cell-free extract was added to the reaction mixture prior to the addition of the putidaredoxin. The rate of reduction of cytochrome c was compared with a standard curve to determine the amount of putidaredoxin reductase present in the cell extract. The standard assay for putidaredoxin contained the following components in 20 mM MOPS buffer, pH 7.4, 0.1 mM NADH, 10 PM cytochrome C, and 1 nM putidaredoxin reductase. In this instance, the cell extract was added prior to the addition of putidaredoxin reductase. The concentration of putidaredoxin in the whole cells was also determined by EPR spectroscopy and the signal obtained com- pared with a known standard. The concentration of cytochrome P- 450,,, in cell-free extracts was determined by standard procedures (23).

’ The abbreviations used are: X-gal, 5-bromo-4-chloro-3-indoyl p- D-galactoside; MOPS, 4-morpholinepropanesulfonic acid; Kbp, kilo- base pair(s); bp, base pair(s).

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6068 Putidaredoxin Reductase and Putidaredoxin

RESULTS

Putidaredoxin Reductase and Putidaredoxin Cloning and Sequence Determination-The cloning of putidaredoxin re- ductase and putidaredoxin was aided by the publication in 1986 (10) of the nucleotide sequence of P-450,,, which in- cluded the N-terminal 153 nucleotides of putidaredoxin re- ductase. Publication of the preliminary restriction map for the 4.4- and 2.6-kbp Hind111 fragments which contained the regulatory region, an alcohol dehydrogenase, P-450,., and both of these proteins (24) indicated their general position within these fragments. To assist in clone selection, an oli- gonucleotide was synthesized that was complementary to a portion of the N-terminal sequence of putidaredoxin reduc- tase between the BamHI and Hind111 sites. The whole cell DNA from P. putida was cleaved sequentially with BamHI and StuI, and the digested DNA was ligated into BamHI- SmaI-cleaved pIBI25. The resulting reaction mixture was used to transform E. coli strain DH5a. The oligonucleotide probe was used to select clones that contained the 2.2-kbp BamHI-StuI fragment. The preliminary restriction map pub- lished by Unger et al. (24) indicated that the coding sequence for putidaredoxin should span the StuI restriction site.

The 2.2-kbp BamHI-StuI fragment was subcloned into both M13mp18 and M13mp19 for sequence determination. The BamHI-SalI, SalI-NruI, and NruI-StuI (the EcoRI site from the polylinker region of the plasmid vector was actually used to clone the 3’ end of this piece of DNA) fragments shown in Fig. 1 were cloned into the appropriate bacteriophage. Either the universal primer or synthetic oligonucleotides were used to prime the synthesis of the complementary strand of DNA for the sequence determination. As indicated in Fig. 1, essen- tially all of the DNA was sequenced on both strands. The restriction map shown in this figure was deduced from the determined nucleotide sequence.

The nucleotide sequence for the BamHI-StuI fragment is shown in Fig. 2. The deduced amino acid sequences for putidaredoxin reductase and putidaredoxin are also shown in this figure. There are several points to be made about these sequences. 1) The amino acid sequence of the N terminus of putidaredoxin reductase is in perfect agreement with that reported (10, 24, 25). 2) The deduced amino acid sequence of putidaredoxin exactly matches the published sequence (26, 27) except for one amino acid. The reported sequence had a glutamine residue at amino acid position 15 while the nucleo-

b 4 m 4 *

b G- 4 I b

FIG. 1. Restriction map of the BamHI to StuI fragment containing the genes encoding putidaredoxin reductase and putidaredoxin. The region encoding putidaredoxin reductase is indicated by the longer of the two heavy bars, and the short heavy bar represents the region coding for putidaredoxin. The fragments that were subcloned for sequencing are represented by the light bars from BamHI to SalI, Sal1 to NruI, and NruI to Std. The actual segments that were sequenced and the direction of sequencing are represented by the light bars with arrows.

FIG. 2. Nucleotide and deduced amino acid sequence of pu- tidaredoxin reductase and putidaredoxin. As indicated in Fig.

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TABLE I

Deduced amino acid composition of putidaredorin reductase and putidaredoxin

Putidaredoxin Putidaredoxin Amino acid reductase

Deduced Reported Deduced Reportedb

Leucine :A ir: : 6 5

40 38 14 14 Alanine Isoleucine Aspartic acid Glutamic acid Glutamine Glycine Threonine Proline Asparagine s;;n;ne

Lysine Phenylalanine Histidine Methionine Cysteine TrvDtoDhan

a Ref. 8. b Refs. 9 and 10.

50 28 26 23

it 20 20 17 11 20 11

8 7 s 6 2

E4 45 45

37 23 20

2: 15 11

7

:: 3

4 6 1

tide sequence indicates that this amino acid is really a glu- tamic acid residue. 3) There are no open reading frames in the region 3’ to putidaredoxin which might code for a protein of greater than 60 amino acids.

The deduced amino acid compositions of putidaredoxin reductase and putidaredoxin are shown in Table I. As would be expected for putidaredoxin, the differences between the deduced and reported sequences are minor and explainable on the basis of the removal of the initiating methionine residue in post-translational processing of the protein. There is one additional glutamic acid residue and one fewer gluta- mine residue. The calculated molecular mass of this protein including the two iron and two acid labile sulfur atoms of the active site is 11,726 daltons which is in excellent agreement with the reported value (26, 27). The deduced and reported composition of putidaredoxin reductase are similar enough to lead one to the conclusion that this is probably the same protein. The calculated molecular mass of 46,215 agrees well with the reported value of 48,500 (8).

Expression of Putidaredoxin Reductase-To ascertain the validity of the identification of the coding region as putida- redoxin reductase, this fragment was subcloned into pIBI24 in the correct orientation for transcription directed by the la& promoter. To obtain a clone of E. coli which would express only putidaredoxin reductase, the BamHI-StuI/EcoRI frag- ment was digested with both BamHI and MluI and the re- sulting 1.5-kbp fragment of DNA was purified by electropho- resis in low melting point agarose. The fragment was re- covered and ligated into the plasmid pIBI24 as shown in Fig. 3, which resulted in the insertion of this fragment in the appropriate orientation with respect to the la& promoter. This particular piece of DNA was chosen because it created a termination codon in the P-galactosidase reading frame which would prevent the formation of a fusion protein between the fi-galactosidase and putidaredoxin reductase. The putative Shine-Dalgarno site is located 3’ to this termination codon (see Fig. 2). The MluI restriction site was chosen for the 3’ termination of this DNA fragment because it was within the

1, putidaredoxin reductase is the first protein sequence and putida- redoxin is the second. Other regions of the sequence are described in the text.

BS7IttHi

BELIllHI MIUI

XhOI

-

FIG. 3. Subcloning strategy for the expression of putidare- doxin reductase. The relative position of the 1acZ promoter is indicated by the heauy arrow on the circumference of the plasmid.

TABLE II

Expression of putidaredoxin reductase and putidaredoxin in E. coli The amount of each of these proteins is expressed as the number

of milligrams of protein/g wet weight of cells. ND, not determined.

Clone PdR Pd” P-450,.,

PdR (GTG) 0.4 0 0 PdR (ATG) 7.4 0 ND

Pd 0 4.8 0

’ Putidaredoxin reductase and putidaredoxin are designated by PdR and Pd, respectively.

coding region of putidaredoxin. The presence of both putida- redoxin reductase and putidaredoxin in the same E. coli cell line might result in cell death due to leakage of electrons from NADH through the reductase to putidaredoxin which has a greater sensitivity to oxidation by molecular oxygen than do the other components of this electron transfer system. In fact, when both putidaredoxin reductase and putidaredoxin were subcloned together in the correct orientation for transcription, no colonies were found (data not shown). As can be seen in Table II, the E. coli clone, which was isolated following transformation with the plasmid construct, would express active putidaredoxin reductase. This level of putidaredoxin reductase is similar to that observed in wild-type P. putida (8).

Effect of the Start Codon on the Level of Expression of Putidaredoxin Reductase-As has been noted previously, the start codon for putidaredoxin reductase is the rare initiation codon GTG (10, 24). This codon has been presumed to be important in the post-transcriptional regulation of protein adundance. To test this hypothesis, the G at position 1 was changed to an A by site-directed mutagenesis in the single- stranded becteriophage M13mp18 which contained the BarnHI-Sal1 fragment. This mutant clone was isolated and completely sequenced to ascertain whether there were any changes other than the desired one. The BamHI-XhoI frag- ment containing the desired nucleotide change was purified, from low melting point agarose, from the replicative form of the bacteriophage, and ligated into the plasmid described above for putidaredoxin reductase expression as shown in Fig. 3. Previously, the wild-type BamHI to XhoI fragment had

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6070 Putidaredoxin Reductase and Putidaredoxin

been removed from this plasmid by restriction digestion and low melting point agarose gel electrophoresis. The plasmid was transformed into E. coli DH5a, and clones that were able to grow in the ampicillin-containing media were selected. Samples of the cells were grown, and the sequence of the double-stranded DNA was determined with the expected change in nucleotide sequence present (data not shown). These cells were grown in TB media and their putidaredoxin reductase content was determined as shown in Table II. The average of three determinations gave a content of 7.4 mg of putidaredoxin reductase per g wet weight of cells, which is approximately an l&fold increase over the level in E. coli cells containing the wild-type gene.

Expression of Putidaredoxin-To obtain the expression of putidaredoxin independent of putidaredoxin reductase, the 856-bp NurI-SmaI fragment of the original BamHI-StuI clone was subcloned into the AccI and SmaI sites in the polylinker region of M13mp18. A single base change of a G to a T results in the formation of a Hind111 site at about 1160 bp in the putidaredoxin reductase coding sequence (see Fig. 2). The mutagenic oligonucleotide was used as a probe for the clones with the anticipated base change as described under “Exper- imental Procedures” (22). The 580-bp fragment of DNA from the new Hind111 site to the SmaI site was purified from the replicative form of M13mp18 by low melting point agarose gel electrophoresis and ligated into pIBI25 which had been cleaved with the same restriction enzymes. The level of expression of putidaredoxin in this cell line and control cells is shown in Table II.

DISCUSSION

The electron transfer systems associated with monooxygen- ation reactions catalyzed by P-450s fall into two general classes: 1) those requiring a flavoprotein dehydrogenase, an iron-sulfur protein, and the P-450; and 2) those requiring a FAD and FMN containing flavoprotein dehydrogenase and the P-450. The former class is usually associated with mito- chondrial steroidogenesis and P-450,., while the latter is associated with microsomal drug or steroid metabolism and P-450BM.3. P-450,,, has been cloned, sequenced, and expressed in an active form in E. coli (lo), thus providing a useful tool for studies of the electron transfer and monooxygenation reaction utilizing site-directed mutagenesis (28).

Studies of the interaction between components of the mi- tochondrial-like P-450 electron transfer systems have been informative in the definition of electrostatic effects on the reaction and the role of the iron-sulfur protein as a mobile electron transfer component (6-9, 29-34). Water-soluble car- bodiimides have proven useful in chemically modifying car- boxylate residues on the iron-sulfur proteins and altering or destroying their ability to transfer electrons (6, 7). Certain regions and residues on either the flavoprotein electron trans- fer donor (35) or P-450 acceptor (36) are attractive candidates for interacting with the iron-sulfur protein; however, to more accurately define the molecular structure and reaction mech- anism of these electron transfer events, an excellent source of these proteins will be required. One of the goals of this research was to obtain expression systems for the production of putidaredoxin reductase and putidaredoxin for use in the study of their interaction with each other and with P-450,,, during electron transfer.

Although putidaredoxin reductase can be purified from the wild-type strain of P. putida, there are several drawbacks to this procedure (9): 1) it is very difficult to separate putidare- doxin reductase and P-450,,, because of their similar size and ionic charge; and 2) the level of putidaredoxin reductase in

this cell line is significantly below that of P-450,,,. In this paper we report the expression of wild-type putida-

redoxin reductase in E. coli at a level which is comparable with that obtained in P. putida (8). Although this would probably be satisfactory for the production of this protein, we decided to test the hypothesis that the rare initiation codon GTG would influence the level of expression of putidaredoxin reductase (10, 24). Changing the ATG initiation codon of plasmid-encoded P-galactosidase to a GTG resulted in a 2-3- fold decrease in the level of expression of this enzyme in E. coli (37). In the present studies, changing the initiation codon from GTG to ATG, with no other changes in the nucleotide sequence or expression system, results in an 18-fold increase in the level of putidaredoxin reductase. We have estimated that under these conditions putidaredoxin reductase accounts for 7-10% of the total soluble protein of these cells. Although this recombinant protein has not been purified to homoge- neity, the protein present in the soluble cell extract behaves like the enzyme from P. putida when subjected to ammonium sulfate fractionation, DE52 ion exchange chromatography, and Sephadex gel exclusion chromatography. We believe that this recombinant form of the protein will prove to be an extremely useful tool for biophysical studies.

The iron-sulfur protein of the mitochondrial-like electron transfer system, adrenodoxin, has been cloned, sequenced (38), and expressed (39) in E. coli. This expression system results in the synthesis of a fusion protein between the amino- terminal fragment of the bacteriophage hcI1 protein and ad- renodoxin (39). Following purification of the fusion protein, the bacteriophage N-terminal portion was cleaved to yield the mature recombinant adrenodoxin which was fully active in the reconstitution of the appropriate P-450 hydroxylation systems. The level of expression of adrenodoxin in this clone was approximately 2-3% of the soluble protein.

The preparation of the clone that would express putidare- doxin independent of either putidaredoxin reductase or P- 450,,, required the insertion of a new Hind111 restriction site in the putidaredoxin reductase coding sequence (Fig. 2). This resulted in the creation of a termination codon in-frame with the @-galactosidase gene; thus, the putidaredoxin synthesized by this clone should have the same sequence as the native protein. Although we have not determined the amino acid sequence of this protein, the activity in the coupled assay is consistent with the synthesis of native protein. In this case, the level of synthesis of putidaredoxin is about 5% of the soluble protein.

A number of authors (40) have speculated that codon usage patterns in a given organism may reflect the level of expres- sion of various proteins. While the number of proteins of P. putzda (ATCC 17453) which have been sequenced to date is limited to only three (P-450,., (24) and the two proteins in the present report), their level of expression in viuo varies by a factor of 8-10. Thus, the potential for exploring whether codon usage is dramatically different in these cases exists. In addition to giving in Table III the absolute usage of each codon for the three proteins of the P-450,,, electron transfer system, we have also given the “Relative Synonymous Codon Usage” (RSCU) index for each of these codons (41). It can be seen from some of these entries, such as those for glycine, that there is little difference between the RSCU of low and highly expressed proteins. In other instances, such as leucine, there are dramatic differences between the RSCU index for these proteins. It remains to be determined whether the trend that has been observed for this very limited number of pro- teins is valid for other proteins. Clearly in the case of puti- daredoxin reductase, the use of the rare initiation codon GTG

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TABLE III Codon usage in P. putida

The values in parentheses are the Relative Synonymous Codon Usage (27) index which is the ratio of the actual usage of the codon to its expected usage if all codons for a given amino acid had an equal probabilitv of usage.

Amino Codon PdR” Pd” P-450 (Pd +

acid P-450\b Total

GlY Gly GlY GlY Glu Glu Asp Asp Val Val Val Val

Ala Ala Ala Ala

Arg Arg Ser Ser

LYS LYS Asn Asn

Met Ile Ile Ile

Thr Thr Thr Thr

Trp stop CYS CYS stop stop TY~ Tyr Leu Leu Phe Phe

Ser Ser Ser Ser

Arg Arg Arg Arg an Gln His His

Leu Leu Leu Leu

Pro Pro Pro Pro

GGG GGA GGT GGC

GAG GAA GAT GAC

GTG GTA GTT GTC

GCG GCA GCT GCC

AGG AGA AGT AGC

AAG AAA AAT AAC

ATG ATA ATT ATC

ACG ACA ACT ACC

TGG TGA TGT TGC

TAG TAA TAT TAC

TTG TTA TTT TTC

TCG TCA TCT TCC

CGG CGA CGT CGC

CAG CAA CAT CAC

CTG CTA CTT CTC

CCG CCA CCT ccc

5 (0.56) 7 (0.78) 4 (0.44)

20 (2.22)

12 (1.04) 11 (0.96) 13 (1.00) 13 (1.00)

15 (1.46) I (0.68)

10 (0.97) 9 (0.88)

12 (0.96) 6 (0.46) 6 (0.46)

26 (2.06)

1 (0.20) 2 (0.40) 5 (1.50) 4 (1.20)

1 (1.27) 4 (0.73) 2 (0.24)

15 (1.76)

5 (1.00) 3 (0.32) 7 (0.75)

18 (1.93)

5 (1.00) 5 (1.00) 1 (0.20) 9 (1.80)

2 (1.00) 1 2 (0.66) 4 (1.33)

0 0 3 (0.55) 8 (1.45)

10 (1.36) 1 (0.14) 3 (0.75) 5 (1.25)

4 (1.20) 1 (0.30) 3 (0.90) 3 (0.90)

9 (1.80) 5 (1.00) 5 (1.00) 8 (1.60)

13 (1.44) 5 (0.56) 1 (0.29) 6 (1.71)

18 (2.45) 7 (0.95) 2 (0.27) 6 (0.82)

6 (1.20) 8 (1.60) 1 (0.20) 5 (1.00)

0 1 (0.12) 1 3 (0.48) 2 3 (0.61) 5 18 (2.80)

3 20 (1.24) 4 13 (0.76) 8 12 (1.29) 1 10 (0.71)

6 9 (1.58) 1 3 (0.42) 1 5 (0.63) 6 1 (1.37)

2 1 1 5

2 0 0 4

2 1 1 3

4 0 1 5

4 0 0 1

1 0 2 4

0 1 2 1

0 0 0 1

0 1 1 1

1 0 1 1

2 1 2 0

5 0 0 1

1 0 0 3

4 (0.60) 4 (0.50) 2 (0.30)

21 (2.60)

3 0 2

11

(0.97) (0.00) (0.39) (2.90)

8 (1.33) 4 (0.67) 5 (0.70) 8 (1.29)

11 3 5

18

3 0 4

13

(1.00) (0.28) (0.56) (2.16)

(1.12) (0.00) (0.64) (2.24)

(1.00)

(0.43) (1.57)

(0.67) (1.33)

1 (0.15) 1 (0.15) 4 (0.42)

14 (1.58)

4 (0.77) 2 (0.58) 2 (0.58) 3 (0.77)

3 (0.77) 1 (0.19) 6 (1.35)

13 (2.71)

15 8 4 9

21 4 2 1

18 3 4 5

(1.31) (0.69) (0.80) (1.20)

(4.92) (0.62) (0.31) (0.15)

11 (0.81) 5 (0.37)

(0.94) 13 (0.96)

(2.24) (0.35) (0.47)

6 (0.35) 11 (0.64)

9 (0.52) 43 (2.49)

35 (1.11) 28 (0.89) 33 (1.16) 24 (0.84)

30 (1.50) 11 (0.55) 16 (0.80) 23 (1.15)

18 (0.80) 11 (0.49)

9 (0.40) 52 (2.31)

6 (0.59) 2 (0.20) 1 (0.82)

19 (2.24)

17 (1.31) 9 (0.69) 8 (0.47)

26 (1.53)

20 (1.00) 6 (0.30)

13 (0.65) 41 (2.05)

12 (1.07) 5 (0.44) 5 (0.44)

23 (2.04)

8 (1.00)

: (0.50) 15 (1.50)

0 2 7 (0.61)

16 (1.39)

11 (0.72) 2 (0.13) I (0.52)

20 (1.48)

8 (0.94) 4 (0.47) 6 (0.71) 7 (0.82)

13 (1.28) 6 (0.59)

12 (1.18) 22 (2.16)

30 (1.36) 14 (0.64)

7 (0.64) 15 (1.36)

50 (3.26) 11 (0.72)

4 (0.26) 14 (0.91)

25 (1.85)

a Putidaredoxin reductase and putidaredoxin are designated with the abbre- viations PdR and Pd, respectively.

bThis is the RSCU index for putidaredoxin plus P-450,.,. Both of these proteins are expressed at a high level in P. putida.

FIG. 4. Comparison of potential FAD and/or NAD-binding sequences of putidaredoxin reductase to the consensus se- quence for this region. The consensus for this sequence was taken from the paper of Hanukoglu and Gutfinger (54). This figure contains a listing of the amino acids that are present in putidaredoxin reduc- tase beginning with residues 6 and 151 on the first two lines. On the remaining lines of this figure are the amino acids that are present in this consensus sequence and the number at each of the positions. The consensus amino acids are indicated by the shaded bars. Those amino acids in putidaredoxin reductase that are functionally conserved are indicated by an asterisk. The proteins that were used to compile this consensus were adrenodoxin reductase (bovine (51, 54) and human (55)), D-amino acid oxidase (56), fumarate reductase (571, glutamate dehydrogenase (E. coli (58), N. crassa (59), and yeast (60, 61)), glutathione reductase (E. coli (62) and human (6311, lipoamide dehy- drogenase (E. coli (64), human (65), and porcine (65, 66)), mercuric reductase (67), NADH dehydrogenase (68), and p-hydroxybenzoate hydroxylase (69).

regulates the level of its expression in E. coli rather than being strictly dependent on the use of rare internal codons for modulating the levels of this protein. Codon usage in Pseudomonas aeruginosa has been studied in detail (42) and the authors were not able to correlate a particular codon usage pattern with predicted levels of mRNA expressivity. However, codon usage was extremely biased. Those synonymous codons with the strongest codon-anticodon interaction were prefer- entially used. This is in contrast to codon usage in E. coli or yeast (43-46). The results presented in Table III indicate that the strong codon bias observed with P. aeruginosa (42) is also present in all three proteins discussed in this report.

With the availability of the nucleotide sequence for more than 500 bases to the 3’ end beyond the gene encoding putidaredoxin, the sequence was examined for the presence of other open reading frames. There are no sequences that would code for a protein of greater than approximately 60 amino acids. This sequence of nucleotides was also examined for the presence of transcription terminators (47). As can be seen in Fig. 2, a variation of the E. coli transcription termi- nation consensus sequence CGGGC followed by an AT string and then a TCTG is present. In this case, the sequence is GCCCG followed by a short AT string ending in C, followed by TCTC. This type of sequence would form a stem-loop which is characteristic of procaryotic rho factor independent transcription termination (48). It remains to be determined whether this terminator sequence functions in P. putida.

Many different FAD-containing flavoproteins have been examined for the presence of a consensus sequence which could be used to characterize FAD-binding domains in other proteins (49-54). Although only a few flavoproteins have been described at the atomic level, the amino acid sequence GXGXXGXXXA or GXGXXGXXXG, which is present at the FAD-binding site, has been found to be present in all FAD-containing proteins (49-54). This consensus sequence is shown on the third line of Fig. 4. In addition, this sequence has been found to be located in a region of protein secondary structure characterized as TPPPPPPTaaaaaaaaaaaaaaTT- /?/3&3/3pT. The N-terminal sequence of putidaredoxin reduc- tase was found to have the consensus sequence GXGXX- GXXXA beginning with residue 11 (25) as shown in Fig. 4.

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6072 Putidaredoxin Reductase and Putidaredoxin

We have scanned the remainder of the sequence of putidare- doxin reductase and were somewhat surprised to find another exact match beginning with amino acid residue 156. It is interesting to note that the NAD-binding domain of lipoamide dehydrogenase has a similar sequence requirement (66). Whether either of these sequences truly represents the FAD- or NAD-binding sites or whether it is some altogether differ- ent sequence will only be known when the three-dimensional structure of this protein has been determined.

With the availability of the linear sequence of putidare- doxin reductase, we screened this protein for the presence of the NADP-binding sequence, GXGXXAXXXA (54,66). This was done as a test for the specificity of the NADP consensus sequence as well as the algorithm used for the search because putidaredoxin reductase is specific for NADH rather than NADPH as the electron donor. No matches for this sequence were found in putidaredoxin reductase.

Recently, the sequence of the peptide from adrenodoxin reductase which is cross-linked to adrenodoxin was deter- mined (35). Although there are several sequences within the overall putidaredoxin reductase sequence which will “align” with some of the amino acids within the published sequence, it remains to be determined whether a consensus sequence can be established for the regions that will define the electro- static interactions that control the binding between these electon transfer proteins.

In summary, putidaredoxin reductase and putidaredoxin in addition to P-450,,, are now available in large quantities as recombinant proteins. We believe that these proteins will be extremely useful for structure/function analyses of protein- protein interaction and electron transport.

Acknowledgments-J. A. P. would like to thank the faculty and students of the Department of Biochemistry for their patience and helpfulness while he mastered the molecular cloning techniques nec- essary to conduct the research described in this manuscript. J. A. P. would especially like to acknowledge the contributions of the members of the research groups of Drs. M. Waterman, M. J. Gething, and J. Sambrook.

Note Added in Proof-We became aware of the sequence of putida- redoxin reductase and putidaredoxin in Koga et al. (70) after we had submitted this paper for publication.

REFERENCES

1. Estabrook, R. W., Cooper, D. Y., and Rosenthal, 0. (1963) Biochem. J. 336, 741-755

2. Nebert, D. W., Adesnik, M., Coon M. J.. Estabrook, R. W.. Gonzalez, F. J., Guengerich, F. P., Gunsalus, I. C., Johnson, E. F., Kemper, B., Levin, W., Phillips, I. R., Sato, R., and Water- man, M. R. (1987) DNA (N. Y.) 6, l-11

3. Nebert, D. W., Nelson, D. R., Adesnik, M., Coon, M. J., Esta- brook. R. W.. Gonzalez. F. J.. Guenaerich. F. P.. Gunsalus. I. C., Johnson, E. F., Kemper, B.; Levin, W., Phillips, I. R., Saton, R., and Waterman, M. R. (1989) DNA (N. Y.) 8, l-13

4. Poulos, T. L., Finzel, B. C., Gunsalus, I. C., Wagner, G. C., and Kraut, J. (1985) J. Biol. Chem. 260, 16122-16130

5. Katagiri, M., Ganguli, B. N., and Gunsalus, I. C. (1968) J. Biol. Chem. 243,3543-3546

6. Lambeth, J. D., Geren, L. M., and Millett, F. (1984) J. Biol. Chem. 259.10025-10029

7. Geren, L., Tuls, J., O’Brien, P., Millett, F., and Peterson, J. A. (1986) J. Biol. Chem. 261. 15491-15495

8. Roome, P. W., Philley, J., and Peterson, J. A. (1983) J. Biol. Chem. 258,2593-2598

9. Roome, P. W., Jr., and Peterson, J. A. (1988) Arch. Biochem. Biophys. 266,32-40

10. Unger, B. P., Gunsalus, I. C., and Sligar, S. G. (1986) J. Biol. Chem. 261,1158-1163

11. Tartof, K. D., and Hobbs, C. A. (1987) Focus (Idaho) 9:2, 12 12. Hansen, J. B., and Olsen, R. H. (1978) J. Bacterial. 135, 227-

238

13.

14.

15.

16.

17.

18.

19. 20.

21. 22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45. 46.

47.

48. 49.

50.

51.

Lorence, M. C. (1984) Ph.D. dissertation, University of Texas at Dallas

Birnboim, H. C., and Doly, J. (1979) Nucleic Acids Res. 7, 1513- 1523

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., pp. 1.98-1.99, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Sanger, F., Miklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,5463-5467

Tabor, S., and Richardson, C. C. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,4767-4771

Barnes, W. M., Bevan, M., and Son, P. H. (1983) Methods Enzymol. 101,98-122

Gough, J. A., and Murray, N. E. (1983) J. Mol. Biol. 166, 1-19 Mizusawa, S., Nishimura. S., and Seela, F. (1986) Nucleic Acids

Res. 14; 1319-1324 Zoller, M. J., and Smith, M. (1984) DNA (N. Y.) 3,479-488 Wood, W. I., Gitschier, J., Lasky, L. A., and Lawn, R. M. (1985)

Proc. Natl. Acad. Sci. U. S. A. 62, 1585-1588 O’Keeffe, D. H., Ebel, R. E., and Peterson, J. A. (1978) Methods

Enzymol. 51,151-157 Unger, B. P., Sligar, S. G., and Gunsalus, I. C. (1986) in The

Bacteria: A Treatise on Structure and Function (Sokatch, J. R.. ed) Vol. X, pp. 557-589, Academic Press, Inc., San Diego

Romeo, C., Moriwaki, N., Yasunobu, K. T., Gunsalus, I. C., and Koga, H. (1987) J. Protein Chem. 6, 253-261

Tsai, R. L., Dus, K., and Gunsalus, I. C. (1971) Biochem. Biophys. Res. Commun. 45, 1300-1306

Tanaka, M., Haniu, M., Yasunobu, K. T., Dus, K.. and Gunsalus, I. C. (1974) J. Biol. km. 249, 3689-3701

Atkins. W. M.. and Sliear. S. G. (1988) J. Biol. Chem. 263. 18842-18849’

” I

Roome, P. W., Jr., and Peterson, J. A. (1988) Arch. Biochem. Biophys. 266, 41-50

Lambeth, J. D., Seybert, D. W., and Kamin, H. (1980) J. Biol. Chem. 255,4667-4672

Lambeth, J. D., and Kriengsiri, S. (1985) J. Biol. Chem. 260, 8810-8816

Hanukoglu, I., and Jefcoate, C. R. (1980) J. Biol. Chem. 255, 3057-3061

Hintz, M. J., and Peterson, J. A. (1981) J. Biol. Chem. 256, 6721-6728

Brewer, C. B., and Peterson, J. A. (1988) J. Biol. Chem. 263, 791-798

Hamamoto, I., Kurokohchi, K., Tanaka, S., and Ichikawa, Y. (1988) Biochim. Biophys. Acta 953,207-213

Stayton, P. S., Fisher, M. T., and Sligar, S. G. (1988) J. Biol. Chem. 263,13544-13548

Khudyakov, Yu.E., Neplyueva, V. S., Kalinina, T. I., and Smir- nov, V. D. (1988) FEBS Z&t. 232, 369-371

Okamura, T., John, M. E., Zuber, M. X., Simpson, E. R., and Waterman, M. R. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 570555709

Coghlan, V. M., and Vickery, L. E. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,835-839

Sharp, P. M., Cowe, E., Higgins, D. G., Shields, D. C., Wolfe, K. H., and Wright, F. (1988) Nucleic Acids Res. 16. 8207-8211

Sharp, P. M.,Tuohy, T. ‘M. F., and Mosurski,~ K. R. (1986) Nucleic Acids Res. 14, 5125-5143

West, S. E. H., and Iglewski, B. H. (1988) Nucleic Acids Res. 16, 9323-9335

Grantham, R., Gautier, C., Gouy, M., Jacobzone, M., and Mercier, R. (1981) Nucleic Acids Res. 9, 43-74

Bennetzen, J. L., and Hall, B. D. (1982) J. Biol. Chem. 257, 3026-3031

Grosjean, H., and Fiers, W. (1982) Gene (Am&.) 18, 199-209 Gouy, M., and Gautier, C. (1982) Nucleic Acids Res. 10, 7055-

7074 Brendel, V., and Trifonov, E. N. (1984) Nucleic Acids Res. 12,

4411-4427 Platt, T. (1986) Annu. Reu. Biochem. 55,339-372 Porter, T. D., and Kasper, C. B. (1986) Biochemistry 25, 1682-

1687 Powell, J. F., Hsu, Y.-P. P., Weyler, W., Chem, S., Salach, J.,

Andrikopoulos, K., Mallet, J., and Breakefield, X. 0. (1989) Biochem. J. 259,407-413

Sagara, Y., Takata, Y., Miyata, T., Hara, T., and Horiuchi, T. (1987) J. Biochem. (Tokyo) 102, 1333-1336

by guest on July 12, 2020http://w

ww

.jbc.org/D

ownloaded from

Putidaredoxin Reductase and Putidaredoxin 6073

52. Haniu, M., Iyanagi, T., Miller, P., Lee, T. D., and Shively, J. E. (1986) Biochemistry 25, 7906-7911

53. Yabusaki, Y., Murakami, H., and Ohkawa, H. (1988) J. Biochem. (Tokyo) 103, 1004-1010

54. Hanukoglu, I., and Gutfinger, T. (1989) EUF. J. Biochem. 180, 479-484

55. Solish, S. B., Picado-Leonard, J., Morel, Y., Kuhn, R. W., Mo- handas, T. K., Hanukoglu, I., and Miller, W. L. (1988) PFOC. Natl. Acad. Sci. U. S. A. 85, 7104-7108

56. Ronchi, S., Minchiotti, L., Curti, B., Zapponi, M. C., and Bridgen, J. (1976) Biochim. Biophys. Acta 427,634-643

57. Cole, S. T. (1982) EUF. J. Biochem. 122,479-484 58. McPherson, M. J., and Wootton, J. C. (1983) Nucleic Acids Rex

11,5257-5266 59. Kinnaird, J. H., and Fincham, J. R. S. (1983) Gene (Amst.) 26,

253-260 60. Moye, W. S., Amuro, N., Rao, J. K. M., and Zalkin, H. (1985) J.

Biol. Chem. 260.8502-8508

61. Naeasu, T., and Hall, B. D. (1985) Gene (Am&) 37, 247-253 62. Greer, S., and Perham, R. N. (1986) Biochemistry 25, 2736-2742 63. Krauth-Sieeel. R. L., Blatterspiel. R., Saleh. M.. Schiltz. E..

64.

65.

66.

67.

68.

69.

70.

SchirmeryR: H., and UntuchtlGrau, k. (1982) Eu;. J. Biochem: 121,259-267

Stephens, P. E., Lewis, H. M., Darlison, M. G., and Guest, J. R. (1983) EUF. J. Biochem. 121, 259-267

Otulakowski, G., and Robinson, B. H. (1987) J. Biol. Chem. 262, 17313-17318

Rice, D. W., Schultz, G. E., and Guest, J. R. (1984) J. Mol. Biol. 174,483-496

Brown, N. L., Ford, S. J., Pridmore, R. D., and Fritzinger, D. C. (1983) Biochemistry 22,4089-4095

Young, I. G.. Rogers, B. L.. Campbell, H. D., and Jaworowski, A., andShaw, D. c. (1981) EUF. j. Biochem. 116, 165-170

Weijer, W. J., Hofsteenge, J., Vereijken, J. M., Jekel, P. A., and Beintema, J. J. (1982) Biochim. Biophys. Acta 704, 385-388

Koga, H., Yamaguchi, E., Matsunaga, K., Aramaki, H., and Horiuchi, T. (1989) J. Biochem. (Tokyo) 106, 831-836

by guest on July 12, 2020http://w

ww

.jbc.org/D

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J A Peterson, M C Lorence and B Amarnehheterologous expression of the proteins.

Putidaredoxin reductase and putidaredoxin. Cloning, sequence determination, and

1990, 265:6066-6073.J. Biol. Chem. 

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