Re-design of Rhodobacter sphaeroides Dimethyl Sulfoxide Reductase
ENHANCEMENT OF ADENOSINE N1-OXIDE REDUCTASE ACTIVITY*

James C. Hilton, Carrie A. Temple, and K. V. RajagopalanDagger

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The periplasmic DMSO reductase from Rhodobacter sphaeroides f. sp. denitrificans has been expressed in Escherichia coli BL21(DE3) cells in its mature form and with the R. sphaeroides or E. coli N-terminal signal sequence. Whereas the R. sphaeroides signal sequence prevents formation of active enzyme, addition of a 6× His-tag at the N terminus of the mature peptide maximizes production of active enzyme and allows for affinity purification. The recombinant protein contains 1.7-1.9 guanines and greater than 0.7 molybdenum atoms per molecule and has a DMSO reductase activity of 3.4-3.7 units/nmol molybdenum, compared with 3.7 units/nmol molybdenum for enzyme purified from R. sphaeroides. The recombinant enzyme differs from the native enzyme in its color and spectrum but is indistinguishable from the native protein after redox cycling with reduced methyl viologen and Me2SO. Substitution of Cys for the molybdenum-ligating Ser-147 produced a protein with DMSO reductase activity of 1.4-1.5 units/nmol molybdenum. The mutant protein differs from wild type in its color and absorption spectrum in both the oxidized and reduced states. This substitution leads to losses of 61-99% of activity toward five substrates, but the adenosine N1-oxide reductase activity increases by over 400%.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The majority of molybdoenzymes contain one or more strongly chromophoric prosthetic groups in addition to molybdenum (1, 2). Although the properties of their molybdenum centers have been studied by EPR and EXAFS,1 other techniques such as MCD, resonance Raman spectroscopy (RR), and UV-visible absorption spectroscopy have not been applicable to these enzymes because the weakly absorbing molybdenum centers are masked by the other stronger chromophores. Periplasmic Rhodobacter DMSO reductase is one among the few proteins containing only the molybdenum cofactor as a prosthetic group (3). The active enzyme is also a monomer of 85 kDa in contrast to other well characterized molybdoenzymes that are larger oligomeric molecules, many containing a membrane-anchoring subunit complicating molecular cloning and purification (4). All of these factors combine to make Rhodobacter DMSO reductase an ideal molybdoenzyme for cloning and structural studies of the molybdenum active site.

The purified Rhodobacter sphaeroides enzyme has been studied by several spectroscopic techniques including absorption spectroscopy (3), EPR (3, 5), VTMCD (5), EXAFS (6), and RR (7). EXAFS studies of the oxidized R. sphaeroides protein showed the molybdenum ligand field to contain 4 Mo-S, 1 Mo=O, and 1 Mo-O bonds (6), and chemical analysis of the purified enzyme revealed the bis(MGD)molybdenum form of the cofactor (Fig. 1A) (8). The long wavelength absorption bands in the visible spectrum of DMSO reductase have proven to be extremely useful for RR, enabling detailed and nearly complete assignments for all of the observed RR resonance lines. In particular, redox cycling of the enzyme in the presence of 18O-labeled Me2SO caused a downshift of 43 cm-1 in the bond assigned to Mo=O, providing unequivocal evidence for the single oxo group. This finding also demonstrated the mono-oxo molybdenum center is fully capable of carrying out an oxo transfer reaction (7).


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Fig. 1.   Structure of the molybdenum center. A, bis(molybdopterin guanine dinucleotide)molybdenum cofactor. B, bis(molybdopterin)molybdenum center with Ser, Cys, and selenocysteine serving as protein ligands to the molybdenum.

As mentioned above, EXAFS studies on R. sphaeroides DMSO reductase indicated that the ligand field of molybdenum includes an Mo-O bond presumably from a protein side chain (6). Subsequent x-ray crystallography of the enzyme identified this protein ligand as Ser-147 (9). Amino acid sequence alignment of the DMSO reductase family of proteins suggests that the protein ligand to molybdenum in diverse members of the group may be either Ser, Cys, or selenocysteine (Fig. 1B) (9) with the latter residue identified as the molybdenum ligand in Escherichia coli formate dehydrogenase H (10, 11). The reason for this diversity in protein molybdenum ligation is not known.

An exciting recent development in the molybdoenzyme field is the elucidation of the x-ray crystallographic structures of a number of proteins (9, 11-15), including at least one from each of the three major families, setting the stage for detailed structure-function studies relating to substrate specificities, identification of active site residues, and delineation of internal electron transfer pathways in proteins containing multiple prosthetic groups. In particular, the effect of site-directed mutagenesis on the electronic and chemical properties of the molybdenum centers can be examined using kinetic techniques, analysis of substrate specificities, and a variety of spectroscopic techniques including EXAFS, RR, EPR, and x-ray crystallography.

In order to carry out detailed structural and mechanistic studies on R. sphaeroides DMSO reductase, we have cloned the R. sphaeroides DMSO reductase gene (16) with the intent of expressing it heterologously in E. coli and generating site-directed mutants. Although other laboratories have also reported the cloning of the R. sphaeroides (17) and Rhodobacter capsulatus (18, 19) DMSO reductase structural genes, no successful heterologous expression and purification of the active enzyme has been reported. Knäblein et al. (20) have homologously expressed the cloned DMSO reductase structural gene in R. capsulatus in an active form, although the enzyme has not been purified or characterized. Since plasmids cannot be introduced into Rhodobacter by transformation, homologous expression requires transformation of a donor E. coli strain with a suicide plasmid followed by conjugation and subsequent crossover to integrate a mutated gene into the R. capsulatus cellular chromosome. These complications are eliminated by heterologous expression in E. coli, with the additional advantage of a multitude of molecular biology techniques tailored to this system. Furthermore, because of the availability of E. coli mutants defective in cofactor biosynthesis (21), heterologous expression provides the ability to examine the mechanism of incorporation of the complex cofactor into the apoprotein.

Initial attempts at heterologous expression of the cloned R. sphaeroides DMSO reductase gene in E. coli produced only inactive protein (16). Since other molybdoenzymes including the human and rat sulfite oxidases (22, 23) and E. coli DMSO reductase (24) have been successfully cloned and expressed in E. coli, the apparent lack of expression of active R. sphaeroides DMSO reductase was unexpected. The cloned R. sphaeroides DMSO reductase gene codes for a precursor protein that includes an N-terminal 42-residue signal sequence serving to target the mature protein to the periplasmic space. R. sphaeroides DMSO reductase shares approximately 50% amino acid sequence identity with mature E. coli TMAO reductase, another periplasmic molybdoenzyme (25). Although the mature enzymes share significant homology, the periplasmic signal sequences differ substantially (26). Since differences in the signal sequence requirements of the two organisms could result in improper processing, we have examined the effects of deletion of the N-terminal signal sequence as well as the effects of substitution of the R. sphaeroides DMSO reductase signal sequence with that of E. coli TMAO reductase on the expression of active DMSO reductase. These studies show that maximum expression of active enzyme is achieved without any signal sequence. Paradoxically, the inclusion of an N-terminal His-tag results in an even higher production of active enzyme and facilitates rapid and high yield purification of the recombinant protein.

To explore the effect of ligand substitution on catalytic activity and electrochemical properties such as oxidation-reduction potentials, we have created the S147C mutant of DMSO reductase and examined its properties. As described below, the mutant enzyme displays attenuated activities with several substrates but markedly elevated activity with adenosine N-oxide. These data as well as the results of spectroscopic analysis of the recombinant wild type and the S147C forms of DMSO reductase are presented in this paper.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- T4 polynucleotide kinase, T4 DNA ligase, calf intestinal alkaline phosphatase, 1-kilobase pair DNA ladder standards, Miller's LB broth, and competent DH5alpha and DM1 E. coli cells were from Life Technologies, Inc. XL1-Blue E. coli cells and pBluescript II KS(+) were from Stratagene. Restriction enzymes were from Stratagene and Life Technologies, Inc. AmpliTaq DNA polymerase was from Perkin-Elmer. Glycerol-tolerant DNA sequencing gel mix was from Amersham Pharmacia Biotech. The Transformer Site-directed Mutagenesis Kit and BMH 71-18 mutS cells were from CLONTECH. The pET-28 and pET-29 expression systems, BL21(DE3) E. coli cells, induction control plasmid F, pLysE, and Perfect Protein Standards were from Novagen. The TA cloning kit, including the pCRII vector and INValpha F' E. coli cells, was from Invitrogen. BCA protein assay reagents and 1-step chloronaphthol were from Pierce. Ultrafiltration devices and membranes were from Millipore. Q-Sepharose Fast Flow, Sephadex G-25, and Superose 12 resins were from Amersham Pharmacia Biotech. Ni-NTA-agarose resin was from Qiagen. Electrophoresis reagents, protein mini-gels, prestained low range SDS-polyacryamide gel electrophoresis standards, and gelatin were from Bio-Rad. Goat anti-rabbit horseradish peroxidase-conjugated IgG was from Boehringer Mannheim. The atomic absorption molybdenum standard was from J. T. Baker Inc. All other reagents were obtained from Sigma with the exceptions of IPTG, obtained from Research Products International, Me2SO, obtained from Mallinckrodt, and D-biotin D-sulfoxide, prepared as described by Pollock and Barber (27).

Recombinant DMSO Reductase Expression Constructs-- Oligonucleotides were synthesized at the Duke University DNA Core Facility on an Applied Biosystems DNA synthesizer model 394. Site-directed mutagenesis of R. sphaeroides DMSO reductase with the CLONTECH Transformer Site-directed Mutagenesis Kit was carried out on double-stranded DNA according to the manufacturer's protocol. The pJH115 construct (Table I), containing the DMSO reductase coding sequence, free of NcoI and NdeI restriction sites, in pBluescript II KS(+), was subjected to mutagenesis with selection primer BSXbaD677 (Table II) and mutagenic primers NdeA122 or NdeA-2 to create pJH118 and pJH119, respectively. The NdeI restriction site created at position 122 in pJH118 added a Met codon to the first position of the mature enzyme coding sequence, whereas the coding sequence of the precursor enzyme in pJH119 remained unaltered. The inserts were released with NdeI and HindIII and ligated into the pET-29 expression vector with the initiation codons for the mature or precursor peptides positioned at the translation start site of the vector, creating pJH520 and pJH521, respectively. Similar ligation of the insert from pJH118 into the pET-28 expression vector positioned the mature coding sequence in frame with the 6× His-tag coding sequence present in the vector, creating pJH720.

                              
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Table I
Cloning and expression constructs

                              
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Table II
Primers used in mutagenesis of R. sphaeroides DMSO reductase

Creation of the DMSO Reductase/TMAO Reductase Fusion Construct-- PCR primers TRNdeA-2 and TRBclA132 were designed to amplify the first 150 nucleotides of the torA coding sequence from E. coli DH5alpha genomic DNA, incorporating an NdeI site at position -2 and a BclI site at position 132, respectively. The 100-µl PCR reaction contained 0.8 µg of DNA, 4 mM MgCl2, 1× Taq PCR buffer II, 20 nmol of dNTP, 50 pmol of each primer, and 2.5 units of AmpliTaq DNA polymerase. The PCR parameters included an initial 2-min 94 °C denaturation, 15 amplification cycles (30 s denaturation at 97 °C, 30 s annealing at 45 °C, and 1-min extension at 72 °C), and a final 10-min 72 °C extension. The PCR product was ligated directly into the pCRII vector using the TA cloning kit, and the construct, pTT601, containing the insert, was isolated from E. coli INValpha F' cells that had been transformed with the ligated material. Mutagenesis with selection primer BSXbaD677 and mutagenic primer BclA123 on pJH115 incorporated a BclI site at position 123 of the gene creating pJH125. Unmethylated pJH125 and pTT601 constructs were propagated in dam-/dcm- DM1 cells, and the torA insert was released from pTT601 with SpeI and BclI and ligated into pJH125 that had been digested with the same enzymes, creating the fusion construct pJH126. The coding sequence for the fusion protein was released from pJH126 with NdeI and HindIII and ligated into the pET-29 expression vector with the initiation codon of the insert positioned at the translation start site of the vector, creating pJH527.

Preparation of S147C Constructs-- Construct pJH118 was subjected to mutagenesis with selection primer BSSpeD683 and mutagenic primer S147C to alter the Ser codon at position 147 to a Cys codon creating pJH130. The mutant coding sequence for the mature peptide was released from the construct with NdeI and HindIII and ligated into pET-28 in frame with the 6× His-tag coding sequence creating pJH731.

DNA Sequencing-- Inserts containing the DMSO reductase coding sequence were sequenced using the chain termination method on double-stranded DNA with the Oncor Fidelity DNA Sequencing System according to the manufacturer's protocol. Sequencing gels containing 6% acrylamide were prepared from Glycerol-tolerant DNA Sequencing Gel Mix and included 20% formamide to resolve compressions. Sequence analysis was accomplished using the Wisconsin Sequence Analysis Package, Version 8 (Genetics Computer Group, Inc.). Alternatively, automated sequencing was performed at the Duke University DNA Analysis Facility using a Perkin-Elmer Dye Terminator Cycle Sequencing system with AmpliTaq DNA polymerase combined with an Applied Biosystems 377 PRISM DNA Sequencing instrument.

Expression Analysis of Recombinant and S147C DMSO Reductase-- Expression constructs pJH520, pJH521, pJH527, pJH720, and pJH731 were transformed into BL21(DE3) E. coli cells for expression. Aerobic growth and expression conditions were explored in 3-ml LB cultures supplemented with 30 µg/ml kanamycin and 1 mM NaMoO4 in 14-ml Falcon 2057 tubes. Anaerobic growth and expression analysis were carried out in sterile 5-ml, additive-free blood collection tubes containing 5 ml of M9ZB medium (28) consisting of 10 g/liter N-Z-amine, 5 g/liter NaCl, 1 g/liter NH4Cl, 1.5 g/liter KH2PO4, 15 g/liter Na2HPO4, 0.25 g/liter MgSO4·7H2O, 0.4% glucose, supplemented with 40 mM fumaric acid, 10-1000 µM NaMoO4, and 30 µg/ml kanamycin. BL21(DE3) E. coli cells containing expression constructs were transformed with pLysE using the calcium chloride method (29). When cells also contained pLysE, 34 µg/ml chloramphenicol was included in the medium. Expression under both aerobic and anaerobic conditions was induced with up to 1 mM IPTG.

Purification of DMSO Reductase-- Mature DMSO reductase was purified from R. sphaeroides as described previously (8). Recombinant enzyme was purified from BL21(DE3) cells containing pLysE and either pJH720 or pJH731. Cells were grown aerobically at 37 °C overnight in 80-ml cultures containing LB supplemented with kanamycin and chloramphenicol and used to inoculate 40-liter cultures containing M9ZB medium supplemented as described above and including NaMoO4 at 0.5 mM and IPTG at 80 µM. The cells were grown for 24 h at room temperature and harvested by centrifugation at 5,000 × g. The cell pellets were resuspended in 500 ml of 50 mM sodium phosphate, 300 mM NaCl, pH 8.0, and frozen to lyse the cells. The frozen cells were thawed in the presence of 5 mg of DNase I and stirred at room temperature until the lysate was smooth and homogeneous. After centrifugation at 11,000 × g for 30 min, the pH of the supernatant was adjusted to 6.5, and the extract was heated rapidly to 50 °C in a boiling water bath, maintained at 50 °C for 1 min, cooled rapidly in an ice bath, and centrifuged at 11,000 × g for 20 min. Imidazole was added to the supernatant to a concentration of 10 mM. The pH was adjusted to 7.5 with NaOH, and the solution was centrifuged again. The supernatant was combined with 15-25 ml of Ni-NTA resin, and the slurry was equilibrated with gentle stirring at 4 °C for 15 min. The slurry was poured into a 2.5 × 10-cm chromatography column, and the protein solution was allowed to flow through the resin. The column was then washed with 2 column volumes of 10 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, pH 7.5, 8 column volumes of the same solution at pH 8.0, and 3 column volumes of the pH 8 solution at a 20 mM imidazole concentration. The DMSO reductase was eluted with 100 mM imidazole in 50 mM sodium phosphate, 300 mM NaCl, pH 8.0. Fractions containing the DMSO reductase were combined and made 20% saturated with solid ammonium sulfate. The protein was loaded on a 23-ml phenyl-Sepharose HR16/10 FPLC column equilibrated in 114 g/liter ammonium sulfate in 50 mM Tris-HCl, pH 8.5. The column was then washed with 1 column volume of this solution followed by a 1-column volume gradient that decreased the ammonium sulfate concentration from 114 to 34 g/liter (NH4)2SO4, a 1-column volume wash at 34 g/liter, a 2-column volume gradient further dropping the ammonium sulfate concentration to 17 g/liter, and finally a 4.4-column volume gradient decreasing the ammonium sulfate concentration from 17 to 0 g/liter. Selected fractions were combined, and the ammonium sulfate was removed by ultrafiltration using Amicon PM30 membranes or Centricon 30 devices.

Polyacrylamide Gel Electrophoresis and Western Blot Analysis-- Samples were heated at 95 °C in sample buffer containing 2% SDS and 5% beta -mercaptoethanol. Electrophoresis was carried out on 4-20% gradient polyacrylamide Ready Gels using the Bio-Rad Mini-PROTEAN II Electrophoresis System at 200 V, and proteins on the gels were stained with Coomassie Blue or transferred to nitrocellulose membranes using the Bio-Rad Mini Trans-Blot electrophoretic transfer cell. Western blot analysis was performed as described previously (16).

Activity and Protein Assays-- Pure samples of DMSO reductase were quantitated spectrophotometrically at 280 nm using an extinction coefficient of 200,000 M-1 cm-1 or 2.3 (mg/ml)-1 cm-1 (8). Total protein was also assayed using the Pierce BCA assay on trichloroacetic acid-precipitated samples as described in the manufacturer's protocol, with known concentrations of DMSO reductase purified from R. sphaeroides as standards. DMSO reductase activity was assayed as described previously (3) in a 2-ml anaerobic assay mixture containing up to 3 µg of the enzyme, 0.1 mM sodium dithionite-reduced methyl viologen, 50 mM Tris-HCl, pH 7.5, and 10 mM Me2SO. The assay was initiated by injecting sodium dithionite into a sealed anaerobic mixture containing all other assay components. The change in absorbance at 600 nm was monitored for 1 min, and the activity in micromoles of Me2SO reduced per min was determined by the formula U = 1/2[Delta A600/min][1/epsilon 600][v] where epsilon 600, the extinction coefficient of reduced methyl viologen at 600 nm, is 1.3 × 10-2 µM-1 cm-1 and v = 2 × 10-3 liter. Alternative substrates were assayed using enzyme concentrations that varied relative to the activity toward that substrate. The substrates TMAO, DL-MetSO, potassium chlorate, fumaric acid, and potassium nitrate were assayed at 10 mM substrate concentrations, whereas BSO was assayed at 1.7 mM (30). ANO was dissolved directly in the assay buffer at 3.4 mM (31).

DMSO Reductase Activity Stains-- Solid ammonium sulfate was added to soluble cell extracts to 45% saturation at 4 °C, and the precipitated material was removed by centrifugation at 23,000 × g for 15 min. The supernatant was heated rapidly to 60 °C, and immediately chilled in an ice bath, and the precipitated material was again removed by centrifugation. The ammonium sulfate concentration of the supernatant was increased to 70% saturation, and after centrifugation the supernatant was discarded. The precipitated protein was dissolved in 50 mM Tris-HCl, pH 7.5, and SDS-free samples containing 25-30 µg of total protein were loaded onto 4-20% Ready Gels. After non-denaturing electrophoresis at 200 V for approximately 30 min, the gels were stained in a 13-ml anaerobic solution of 150 mM Me2SO, 4 mM reduced methyl viologen, and 77 mM Tris-HCl, pH 7.5 (32). After bands containing Me2SO reducing activity became clear, the stained background was fixed with 2.5% tetrazolium red (33).

Absorption Spectroscopy-- Absorption spectroscopy was carried out using a Shimadzu UV-2101 PC spectrophotometer and quartz cuvettes. In order to record the spectrum of purified recombinant DMSO reductase that had been subjected to one or more catalytic cycles, concentrated samples of the enzyme in 0.8 mM methyl viologen, Tris-HCl, pH 7.5, were reduced dropwise with 10 mg/ml sodium dithionite under anaerobic conditions until the methyl viologen became dark purple in color. The enzyme was reoxidized by dropwise addition of 1 mM Me2SO until the solution cleared. Finally, to ensure complete oxidation of any remaining reduced enzyme, a few drops of 2 M Me2SO were added. The reoxidized enzyme was subsequently removed from the anaerobic environment, concentrated, and passed through an HR16/50 Superose 12 FPLC column to remove the methyl viologen and Me2SO while ensuring homogeneous enzyme samples. Selected fractions containing the DMSO reductase were combined and concentrated to at least 5 mg/ml. To obtain a reduced spectrum of the S147C variant, the enzyme was reduced in excess methyl viologen in the manner described above but was then quickly separated from the reductant on a 7-ml Sephadex G-25 column equilibrated with 10 mM Tris-HCl, pH 7.5. The 5-10 mg/ml red enzyme was collected and sealed in glass cuvettes before removal from the anaerobic environment to record the spectra.

Molybdenum Analysis-- The molybdenum content of DMSO reductase samples was determined using a Perkin-Elmer Zeeman 3030 atomic absorption spectrometer using protocols described previously (8, 34). Protein samples were passed through an HR 16/50 Superose 12 FPLC column immediately before quantitative analysis to ensure a homogeneous, molybdate-free sample. Concentrated protein samples and molybdenum standards were wet-ashed in 35% HNO3 at 100 °C for 6 h and diluted 1:50 in H2O to molybdenum concentrations of 8-12 parts/billion, where, with a molecular weight of 95.94, 1 parts/billion (1 µg/liter) molybdenum = 1.042 × 10-8 M.

Guanine Analysis-- DMSO reductase in 10 mM Tris, 0.2 N HCl was incubated at 100-105 °C for 1 h to release the guanine component of MGD. After cooling, the pH was adjusted to 8.5 with NaOH, and 400-µl aliquots were chromatographed on an Alltech C-18 reverse phase high performance liquid chromatography column as described previously (8). The absorbance of the eluent was monitored at 280 nm with a Hewlett-Packard 1040A diode array detector, and the amount of guanine in the sample was calculated from the integrated area of the absorbance peak associated with the eluting compound. A standard curve was calculated using 7, 14, and 21 µM guanine standards.

Kinetic Analysis-- Three to ten activity assays were performed as described above for each of 6-10 different substrate concentrations to determine the Km of the wild type and S147C variant of DMSO reductase for Me2SO and ANO. DMSO reductase activity was determined using 1.8 µg of enzyme purified from R. sphaeroides cells or 5.5 µg of the purified S147C variant in each reaction. ANO reductase activity was determined using 0.90 µg of enzyme purified from R. sphaeroides cells or 0.22 µg of the purified S147C variant per reaction. Km values were determined by direct fit of the data to the Michaelis-Menten equation.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although initial attempts to express active recombinant DMSO reductase were not successful (16), further investigation revealed that active enzyme could be produced under appropriate conditions. The coding sequences for mature and precursor DMSO reductase were ligated into the pET-29 expression vector incorporating the initiation sites of the mature and precursor coding sequences directly into the initiation sites present in the vector such that the expressed products of these constructs would consist of either the mature DMSO reductase sequence with only a Met residue added to the N terminus or the unaltered precursor protein. A pET29 construct containing the coding sequence for a fusion protein replacing the R. sphaeroides DMSO reductase periplasmic signal sequence with that of E. coli TMAO reductase was prepared in a similar manner. Attempts to express recombinant R. sphaeroides DMSO reductase in BL21(DE3) E. coli cells containing these expression constructs, grown aerobically in LB medium in the presence of excess molybdate, revealed that although more DMSO reductase protein appears to be produced when the precursor encoded by pJH521 was expressed (Fig. 2A), expression of the recombinant, mature form of the enzyme from pJH520 produced more active enzyme as revealed by activity-stained native polyacrylamide gels (Fig. 2B). Expression of DMSO reductase with the TMAO reductase signal sequence resulted in the production of active enzyme when expressed at 26 °C (Fig. 2B); however, this fusion protein was not investigated further due to an active enzyme yield that was lower than that of the much simpler mature, recombinant form. The expression results suggest that the 42-residue periplasmic signal sequence found on the precursor enzyme (16, 35) may actually interfere with formation of the active R. sphaeroides enzyme in the cytoplasm of E. coli. Further optimization attempts revealed that the cultures required molybdate supplementation in the LB medium to produce active enzyme2 and that expression of the active enzyme was much more effective at 26  than at 37 °C (Fig. 2B).


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Fig. 2.   Expression analysis of aerobic cultures of BL21(DE3) E. coli cells containing pET-29 expression constructs. A, Western blot analysis of 5 µg of whole cell extracts. Lanes 1 and 2 contain mature and precursor DMSO reductase, respectively, expressed from pJH520 and pJH521 at 37 °C. Lanes 4 and 5 contain mature and precursor DMSO reductase, respectively, expressed from pJH520 and pJH521 at 26 °C. Lanes 3 and 6 contain the DMSO reductase/TMAO reductase fusion protein expressed from pJH527 at 37 and 26 °C, respectively. The extract in lane 7 is from cells expressing LacZ, and the extract in lane 8 is from cells containing pET-29 with no insert. Lane 9 contains 20 ng of mature DMSO reductase purified from R. sphaeroides cells. Lane 10 contains prestained low range molecular weight standards. B, DMSO reductase activity stain of ammonium sulfate and heat-treated soluble extracts containing 25-30 µg of total protein. The samples in lanes 1-8 originated from extracts identical to those described in A. Lanes 9 and 10 contain 50 and 10 ng, respectively, of purified mature DMSO reductase from R. sphaeroides cells.

Because expression of the mature enzyme demonstrated that the signal sequence was not necessary for the production of active R. sphaeroides DMSO reductase in E. coli cells, a 6× His-tag was attached to the N terminus of the mature enzyme to facilitate purification. The coding sequence of the mature peptide was ligated into the pET-28 expression vector in frame with the His-tag encoded by the vector. BL21(DE3) cells expressing recombinant DMSO reductase fused with the His-tag appeared to produce more active enzyme than cells expressing the mature enzyme alone.2 It has been demonstrated that the proteins involved in synthesis of the molybdenum cofactor are present at higher levels under anaerobic conditions, since many of the prokaryotic molybdenum-containing enzymes are anaerobic respiratory enzymes (36, 37). Expression of recombinant E. coli DMSO reductase was shown to be more effective under anaerobic conditions in a glycerol/fumarate medium (24). The production of active R. sphaeroides DMSO reductase was enhanced when cells were grown at room temperature in M9ZB medium supplemented with 40 mM fumaric acid and 0.4% glucose. In addition, the pLysE plasmid was added to the cells to facilitate further purification by allowing freeze-thaw lysis of the cells. Small scale induction experiments indicated that the most efficient production of the active enzyme occurred when induction was carried out with 80 µM IPTG in the presence of at least 0.5 mM Na2MoO4.

The recombinant DMSO reductase used in these studies was purified from E. coli BL21(DE3) cells containing the pJH720 construct and the pLysE plasmid grown anaerobically at room temperature for 24 h in 40 liters of M9ZB medium supplemented with fumaric acid, glucose, NaMoO4, IPTG, chloramphenicol, and kanamycin at the concentrations listed above. This combination will support anaerobic growth without requiring any of the molybdenum-containing E. coli terminal reductases, including nitrate reductase, TMAO reductase, and DMSO reductase (38). Typically, 40 liters of cells grown under these conditions released more than 20 mg of soluble DMSO reductase upon lysis. The cells were harvested by centrifugation, resuspended, and frozen. The cells were lysed by thawing, and the DMSO reductase was purified 130-fold in a three-step purification procedure that includes a 50 °C heat step, metal affinity chromatography using Ni-NTA resin, and hydrophobic interaction chromatography on a phenyl-Sepharose FPLC column (Table III). This procedure results in recovery of 31% of the DMSO reductase activity originally present in the extracts, and the purified enzyme appears as a single band on a Coomassie-stained SDS-polyacrylamide gel (Fig. 3).

                              
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Table III
Purification of recombinant R. sphaeroides DMSO reductase
Protein purified from a 40-liter anaerobic culture.


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Fig. 3.   SDS-polyacrylamide gel electrophoresis analysis of steps in the purification of recombinant DMSO reductase. Lane 1, soluble cell extract. Lane 2, supernatant from 50 °C heat step. Lane 3, combined fractions from Ni-NTA column. Lane 4, combined fractions from phenyl-Sepharose. Lane 5, mature DMSO reductase purified from R. sphaeroides cells.

Whereas DMSO reductase purified from R. sphaeroides cells grown anaerobically in medium containing Me2SO as the terminal electron acceptor is dark brown in color, the recombinant mature R. sphaeroides DMSO reductase purified from the BL21(DE3) cells is green-colored. As reported previously, the absorption spectrum of the brown DMSO reductase purified from R. sphaeroides cells displays maxima at 355, 470, 550, and 720 nm (Table IV) (3). The spectrum of the green recombinant DMSO reductase purified from the BL21(DE3) cells also contains long wavelength bands (Fig. 4), but the absorption peaks are at 380, 470, and 650 nm (Table IV). Because the recombinant protein was purified from cells that were not dependent upon the reduction of Me2SO for growth and an endogenous reductant of the enzyme may not be present, it appeared that the recombinant enzyme may not have been subjected to catalytic turnover after assembly. Accordingly, the effect of redox cycling on the recombinant enzyme was examined. Complete reduction of the green recombinant DMSO reductase with excess reduced methyl viologen followed by controlled reoxidation by dropwise addition of dilute Me2SO generated a product with an absorption spectrum almost indistinguishable from that of the R. sphaeroides DMSO reductase (Fig. 4).

                              
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Table IV
Absorption maxima of recombinant, wild-type, and S147C DMSO reductase


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Fig. 4.   Absorption spectra of recombinant DMSO reductase. Top, 5 mg/ml recombinant R. sphaeroides DMSO reductase from BL21(DE3) cells as purified (- - -) and after reduction with reduced methyl viologen and reoxidation with Me2SO (---). Bottom, 5 mg/ml methyl viologen-reduced, Me2SO-reoxidized recombinant R. sphaeroides DMSO reductase from BL21(DE3) cells (- - -) and 5 mg/ml DMSO reductase purified from R. sphaeroides cells (---).

The substitution of a Cys residue for the molybdenum-ligating Ser-147 was accomplished through site-directed mutagenesis of pJH118. The coding sequence for this variant was ligated into pET-28 to create pJH731, such that the expression product of this construct consisted of the mature peptide containing the substituted Cys at position 147 and a 6× His-tag at the N terminus. BL21(DE3) cells containing the construct were grown anaerobically at room temperature on M9ZB supplemented with fumarate and glucose. Expression was induced with IPTG in the presence of NaMoO4, and the protein was purified using the conditions and procedures described above with a final yield comparable to that of the recombinant, wild type enzyme.

The purified S147C variant is salmon colored and retains a DMSO reductase specific activity of up to 14 units/mg. The absorption spectrum of the variant displays maxima at 405, 515, and 695 nm (Fig. 5). Upon reduction, the spectrum differs markedly from the wild type Mo(IV) spectrum which is light green in color and has peaks at 374 and 640 nm with a shoulder at 430 nm. The reduced S147C variant is pink in color, and the spectrum is dominated by a strong peak at 520 nm with an additional peak at 415 nm and a shoulder at 345 nm. Reduction and reoxidation of the S147C variant results in only slight changes to the oxidized spectrum.3


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Fig. 5.   Absorption spectra of the S147C R. sphaeroides DMSO reductase variant. Top, 5 mg/ml oxidized (<OVL>  </OVL>) and methyl viologen-reduced (- - -) S147C R. sphaeroides DMSO reductase from BL21(DE3) cells. Middle, 5 mg/ml oxidized S147C DMSO reductase and as-purified recombinant DMSO reductase from BL21(DE3) cells. Bottom, 5 mg/ml oxidized S147C DMSO reductase and methyl viologen-reduced, Me2SO-oxidized recombinant DMSO reductase from BL21(DE3) cells.

Using procedures described previously, the molybdenum and guanine contents of the recombinant proteins were determined by quantitative chemical analysis in order to demonstrate the incorporation of the bis(MGD)molybdenum cofactor into the recombinant and S147C enzymes (Table V). Atomic absorption spectroscopy of wet-ashed DMSO reductase samples revealed 0.66 to 0.87 atoms of molybdenum per DMSO reductase molecule expressed in BL21(DE3) cells compared with a ratio of 0.9 for a sample purified from R. sphaeroides cells. The guanine released from these samples ranged from 1.7 to 1.9 molecules per molecule of recombinant and S147C DMSO reductase, and the ratio was 1.9 for the wild type enzyme as well. Activity assays on these same samples demonstrated that when DMSO reductase activity is measured relative to the molybdenum content of the sample, the specific activity of the wild type enzyme is 3.7 units/nmol molybdenum. The specific activity of the recombinant enzyme is 3.4-3.7 units/nmol molybdenum (93-100% of wild type) and that of the S147C variant is 1.4-1.5 units/nmol molybdenum (37-41% of wild type).

                              
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Table V
Stoichiometry of the molybdenum and guanine structural components of the molybdenum cofactor from recombinant (rDR), S147C (mDR), and wild type (wtDR) DMSO reductase

The specific activity of the S147C mutant was examined with TMAO, MetSO, chlorate, BSO, and ANO (Table VI). The S147C molybdenum ligand substitution results in nearly a total loss of the ability to reduce MetSO, chlorate, and BSO as well as a 61% decrease in the ability to reduce TMAO. However, the mutation increases the ANO reducing activity of the enzyme by greater than 400%, exceeding the specific activity of the wild type enzyme toward any substrate measured. Surprisingly, the S147C substitution increases the Km for Me2SO from the wild type value of 7 µM to 1 mM and for ANO from the wild type value of 0.02 to 0.8 mM, despite the opposing effects on Vmax.

                              
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Table VI
Effect of the S147C substitution on specific activity with various substrates


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Several spectroscopic studies on R. sphaeroides DMSO reductase have resulted in significant advances in the understanding of the structure and chemistry of molybdenum enzymes. The recent discovery of the bis(MGD) form of the molybdenum cofactor (8) and the elucidation of the x-ray crystal structure of DMSO reductase (9) along with the information derived from other spectroscopic studies including EXAFS (6), EPR (3, 5), RR (7), and MCD (5) have laid the foundation for further advances in the understanding of the structure and function of more complex molybdoenzymes.

The formation of mature DMSO reductase involves two major processes: attachment of the complex bis(MGD)molybdenum cofactor to the protein and transport of the protein to the periplasm with removal of the N-terminal signal peptide. Knäblein et al. (20) resorted to homologous expression of R. capsulatus DMSO reductase in Rhodobacter on the basis that E. coli is unable to supply the complex bis(MGD)molybdenum cofactor. The data presented here show that their conclusion is incorrect and that Rhodobacter DMSO reductase can in fact be expressed in E. coli in sufficient amounts for extensive structural and spectroscopic characterization. We also find that the coding sequence of the mature protein is sufficient to produce fully functional enzyme, demonstrating that attachment of the cofactor does not require translocation of the protein into the periplasm. In fact, these data suggest the holoprotein is formed prior to translocation into the periplasmic compartment, a result consistent with recent work by Santini et al. (39) demonstrating that acquisition of the molybdenum cofactor in the cytoplasm of E. coli is a prerequisite for translocation of TMAO reductase into the periplasm.

E. coli cells contain both MPT and MGD. Earlier studies from our laboratory showed rat liver sulfite oxidase heterologously expressed in E. coli contained only MPT, its normal pterin, and no MGD (23). In the present studies we find that heterologously expressed DMSO reductase contains only MGD and no MPT. In combination, these observations show that even in the case of exogenous molybdoenzymes there is rigorous specificity with respect to the form of pterin incorporated. The stoichiometry of molybdenum and MGD in the recombinant R. sphaeroides DMSO reductase also compares very favorably with that of enzyme purified from R. sphaeroides cells. In contrast, purified recombinant E. coli DMSO reductase homologously expressed in E. coli was found to contain only about 28% of the expected amount of molybdenum cofactor (40). The much higher cofactor occupancy in our preparations is a definite advantage, and indeed a necessity, for high quality crystallographic data.

One surprising result of heterologous expression of DMSO reductase in E. coli was the obvious difference in color between the recombinant enzyme as purified and protein isolated from R. sphaeroides. Since a single turnover converts the isolated, recombinant enzyme to the native state and form, the difference between the two forms is not distinguishable by steady state kinetic analysis. However, detailed EXAFS studies indicate that the isolated green recombinant enzyme initially contains a dioxo-molybdenum structure with no Mo-O bond, suggesting that its conversion to the brown protein with the native absorption spectrum involves loss of one of the oxo groups concomitant with the mooring of the molybdenum to Ser-147 ligand (41). This finding demonstrates a plasticity of the molybdenum-active site that may explain the structural variations previously observed. Other heterologously expressed molybdenum enzymes may also require a reduction-oxidation cycle to convert an anomalous molybdenum coordination field to the native form.

In addition to the ability to produce recombinant R. sphaeroides DMSO reductase in E. coli cells, the work presented here involving the S147C variant demonstrates the anticipated suitability of DMSO reductase for the creation of selected mutations in the coding sequence of the enzyme followed by efficient expression in E. coli cells and rapid, activity-independent purification to homogeneity. The residue known or expected to provide a ligand to the molybdenum has been subjected to site-directed mutagenesis in other enzymes, providing a variety of results. An analogous Ser to Cys variant was recently created in the catalytic subunit of the E. coli DMSO reductase (42), but the purified enzyme was essentially inactive. The effect of the mutation on the absorption spectrum of the molybdenum center could not be assessed, since the protein also contains Fe/S centers. In addition, EPR studies on the mutant demonstrated a heterogeneous ligand field. Molybdenum ligands have also been altered in enzymes that do not contain the bis(MGD) form of the molybdenum cofactor with resultant loss of activity. The Cys-207 molybdenum ligand of human sulfite oxidase has been replaced with Ser, and although the resulting variant contained a novel trioxo Mo(VI) center, it possessed no activity (43, 44). The replacement of Cys-150 of Aspergillus nidulans nitrate reductase, expected to be a ligand to the molybdenum, with an Ala residue, yielded an inactive protein (45). On the other hand, the replacement of the selenocysteine ligand of E. coli formate dehydrogenase H with a Cys produced a variant that retained formate dehydrogenase activity, although the kcat was significantly decreased (10, 46). The purified variant described in this work retains about 40% of the DMSO reductase activity of the wild type enzyme and has reproducible oxidized and reduced absorption spectra that differ from those of the native enzyme. Additionally, EXAFS (41) and x-ray crystallography4 have shown that the side chain sulfur of the Cys-147 variant does in fact become a molybdenum ligand.

An interesting result of the S147C substitution is the variable effect on the activity of the enzyme toward alternative substrates. The substitution leads to losses of 61 and 79% of DMSO and TMAO reductase activities, respectively, and a nearly complete loss of ability to reduce MetSO, chlorate, and BSO. However, it increases the ANO-reducing activity of the enzyme by greater than 400%. In fact, the ANO reductase activity of the S147C variant exceeds the activity of the wild type enzyme toward any other substrate, exceeding the TMAO reductase activity 2-fold and the DMSO reductase activity 10-fold. These results demonstrate that this substitution does not simply attenuate the catalytic competence of the enzyme but actually modifies the reactivity of the molybdenum center in such a manner that the variant displays both decreases and increases in specific activity for various substrates. The reason for the unexpected large increase in the Km for the S147C variant with both Me2SO and ANO will need to be further explored especially in light of the opposing effects on the Vmax with each substrate.

As previously stated, the biochemistry of the molybdenum enzymes has progressed to the point that the ability to use biochemical analysis, molecular genetics, x-ray crystallography, and spectroscopy in combination will provide the greatest opportunity for significant advancement in determining the structure-function relationships that differentiate these diverse enzymes that share so many common structural features. Considerable progress has already been made toward this objective. In addition to the Rhodobacter DMSO reductases (9, 15, 47, 48), the crystal structures of Desulfovibrio gigas aldehyde oxidoreductase (14, 49), E. coli formate dehydrogenase H (11), chicken liver sulfite oxidase (13), Shewanella massilia TMAO reductase (12), and the tungsten-containing Pyrococcus furiosus aldehyde oxidoreductase (50) have all been solved. However, of these, only R. sphaeroides DMSO reductase has been cloned, heterologously expressed in an active form, and purified. A few other molybdenum enzymes have been cloned and heterologously expressed, including rat liver (23) and human liver sulfite oxidase (22), and R. sphaeroides BSO reductase (27, 30), whereas E. coli DMSO reductase (24) has been cloned and homologously overexpressed. However, crystal structures have not been determined for these other cloned and expressed molybdoenzymes. In addition sulfite oxidase and E. coli DMSO reductase contain additional prosthetic groups that complicate spectroscopic studies, and E. coli DMSO reductase is a multisubunit, membrane-bound enzyme. R. capsulatus DMSO reductase has been cloned and homologously expressed in an active form, but the difficulties associated with expression in Rhodobacter in combination with the presence of a wide range of cofactor biosynthesis mutants in E. coli make heterologous expression more versatile. The successful heterologous expression and site-directed mutagenesis of R. sphaeroides DMSO reductase should enable detailed structure-function studies, making the enzyme a prototype for the entire family of MGD-containing enzymes.

    ACKNOWLEDGEMENTS

We thank Ralph D. Wiley of this laboratory for assistance in enzyme purification and Dr. Margot Wuebbens of this laboratory for critical review of the manuscript.

    FOOTNOTES

* This work was supported by Grant GM00091 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 919-681-8845; Fax: 919-684-8919; E-mail: raj{at}biochem.duke.edu.

2 J. C. Hilton and K. V. Rajagopalan, unpublished results.

3 J. C. Hilton, C. A. Temple, and K. V. Rajagopalan, unpublished results.

4 Hermann Schindelin, personal communication.

    ABBREVIATIONS

The abbreviations used are: EXAFS, extended x-ray absorption fine structure; MCD, magnetic circular dichroism; RR, resonance Raman; Me2SO, dimethyl sulfoxide; MGD, molybdopterin guanine dinucleotide; VTMCD, variable-temperature MCD; TMAO, trimethylamine N-oxide; IPTG, isopropyl-beta -D-thiogalactoside; FPLC, fast protein liquid chromatography; MetSO, methionine sulfoxide; DMSO reductase, dimethyl sulfoxide reductase; BSO, D-biotin D-sulfoxide; ANO, adenosine N1-oxide; MPT, molybdopterin; PCR, polymerase chain reaction; Ni-NTA, nickel-nitriloacetic acid.

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DISCUSSION
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