An Active Site Tyrosine Influences the Ability of the Dimethyl Sulfoxide Reductase Family of Molybdopterin Enzymes to Reduce S-Oxides
2001; Elsevier BV; Volume: 276; Issue: 16 Linguagem: Inglês
10.1074/jbc.m010965200
ISSN1083-351X
AutoresKimberly E. Johnson, K.V. Rajagopalan,
Tópico(s)Metal-Catalyzed Oxygenation Mechanisms
ResumoDimethyl sulfoxide reductase (DMSOR), trimethylamine-N-oxide reductase (TMAOR), and biotin sulfoxide reductase (BSOR) are members of a class of bacterial oxotransferases that contain the bis(molybdopterin guanine dinucleotide)molybdenum cofactor. The presence of a Tyr residue in the active site of DMSOR and BSOR that is missing in TMAOR has been implicated in the inability of TMAOR, unlike DMSOR and BSOR, to utilizeS-oxides. To test this hypothesis, Escherichia coli TMAOR was cloned and expressed at high levels, and site-directed mutagenesis was utilized to generate the Tyr-114 → Ala and Phe variants of Rhodobacter sphaeroides DMSOR and insert a Tyr residue into the equivalent position in TMAOR. Although all of the mutants turn over in a manner similar to their respective wild-type enzymes, mutation of Tyr-114 in DMSOR results in a decreased specificity for S-oxides and an increased specificity for trimethylamine-N-oxide (Me3NO), with a greater change observed for DMSOR-Y114A. Insertion of a Tyr into TMAOR results in a decreased preference for Me3NO relative to dimethyl sulfoxide. Kinetic analysis and UV-visible absorption spectra indicate that the ability of DMSOR to be reduced by dimethyl sulfide is lost upon mutation of Tyr-114 and that TMAOR does not exhibit this activity even in the Tyr insertion mutant. Dimethyl sulfoxide reductase (DMSOR), trimethylamine-N-oxide reductase (TMAOR), and biotin sulfoxide reductase (BSOR) are members of a class of bacterial oxotransferases that contain the bis(molybdopterin guanine dinucleotide)molybdenum cofactor. The presence of a Tyr residue in the active site of DMSOR and BSOR that is missing in TMAOR has been implicated in the inability of TMAOR, unlike DMSOR and BSOR, to utilizeS-oxides. To test this hypothesis, Escherichia coli TMAOR was cloned and expressed at high levels, and site-directed mutagenesis was utilized to generate the Tyr-114 → Ala and Phe variants of Rhodobacter sphaeroides DMSOR and insert a Tyr residue into the equivalent position in TMAOR. Although all of the mutants turn over in a manner similar to their respective wild-type enzymes, mutation of Tyr-114 in DMSOR results in a decreased specificity for S-oxides and an increased specificity for trimethylamine-N-oxide (Me3NO), with a greater change observed for DMSOR-Y114A. Insertion of a Tyr into TMAOR results in a decreased preference for Me3NO relative to dimethyl sulfoxide. Kinetic analysis and UV-visible absorption spectra indicate that the ability of DMSOR to be reduced by dimethyl sulfide is lost upon mutation of Tyr-114 and that TMAOR does not exhibit this activity even in the Tyr insertion mutant. dimethyl sulfoxide reductase trimethylamine-N-oxide reductase biotin sulfoxide reductase bis(molybdopterin guanine dinucleotide)molybdenum extended X-ray absorption fine structure spectroscopy trimethylamine-N-oxide dimethyl sulfoxide dimethyl sulfide biotin sulfoxide isopropyl-β-d-thiogalactopyranoside methionine sulfoxide adenosine-1N-oxide phenazine methosulfate 2,6-dichlorophenolindophenol. Unless otherwise noted, DMSOR refers to R. sphaeroides DMSOR and TMAOR refers to E. coli TMAOR nitrilotriacetic acid Rhodobacter sphaeroides and Rhodobacter capsulatus dimethyl sulfoxide reductase (DMSOR),1Escherichia coli and Shewanella massiliatrimethylamine-N-oxide reductase (TMAOR), and R. sphaeroides biotin sulfoxide reductase (BSOR) are members of a class of bacterial oxotransferases that all contain the bis(molybdopterin guanine dinucleotide) form of the molybdenum cofactor (bis(MGD)Mo) seen in Fig. 1 (1Hilton J.C. Rajagopalan K.V. Arch. Biochem. Biophys. 1996; 325: 139-143Crossref PubMed Scopus (46) Google Scholar, 2Schindelin H. Kisker C. Hilton J. Rajagopalan K.V. Rees D.C. Science. 1996; 272: 1615-1621Crossref PubMed Scopus (442) Google Scholar, 3Temple C.A. George G.N. Hilton J.C. George M.J. Prince R.C. Barber M.J. Rajagopalan K.V. Biochemistry. 2000; 39: 4046-4052Crossref PubMed Scopus (39) Google Scholar, 4Czjzek M. Dos Santos J.-P. Pommier J. Giordano G. Mejean V. Haser R. J. Mol. Biol. 1998; 284: 435-447Crossref PubMed Scopus (160) Google Scholar). These enzymes are ideal targets for spectroscopic and kinetic studies of the molybdenum center since they do not contain additional cofactors. In contrast, in all other molybdoproteins studied to date, the low energy absorption bands of the molybdenum atom are overshadowed by prosthetic groups such as hemes, iron-sulfur centers, and flavins. Mechanistic studies are aided by the extensive structural information reported for this family, including several x-ray crystal structures (2Schindelin H. Kisker C. Hilton J. Rajagopalan K.V. Rees D.C. Science. 1996; 272: 1615-1621Crossref PubMed Scopus (442) Google Scholar, 4Czjzek M. Dos Santos J.-P. Pommier J. Giordano G. Mejean V. Haser R. J. Mol. Biol. 1998; 284: 435-447Crossref PubMed Scopus (160) Google Scholar, 5Hung-Kei L. Temple C.A. Rajagopalan K.V. Schindelin H. J. Am. Chem. Soc. 2000; 122: 7673-7680Crossref Scopus (171) Google Scholar, 6McAlpine A.S. McEwan A.G. Shaw A.L. Bailey S. J. Biol. Inorg. Chem. 1997; 2: 690-701Crossref Scopus (153) Google Scholar, 7McAlpine A.S. McEwan A.G. Bailey S. J. Mol. Biol. 1998; 275: 613-623Crossref PubMed Scopus (171) Google Scholar, 8Schneider F. Löwe J. Huber R. Schindelin H. Kisker C. Knäblein J. J. Mol. Biol. 1996; 263: 53-69Crossref PubMed Scopus (256) Google Scholar), analysis by extended x-ray absorption fine structure spectroscopy (EXAFS) (9Baugh P.E. Garner C.D. Charnock J.M. Collison D. Davies E.S. McAlpine A.S. Bailey S. Lane I. Hanson G.R. McEwan A.G. J. Biol. Inorg. Chem. 1997; 2: 634-643Crossref Scopus (61) Google Scholar, 10George G.N. Hilton J. Rajagopalan K.V. J. Am. Chem. Soc. 1996; 118: 1113-1117Crossref Scopus (127) Google Scholar, 11George G.N. Hilton J. Temple C. Prince R.C. Rajagopalan K.V. J. Am. Chem. Soc. 1999; 121: 1256-1266Crossref Scopus (153) Google Scholar), EPR (10George G.N. Hilton J. Rajagopalan K.V. J. Am. Chem. Soc. 1996; 118: 1113-1117Crossref Scopus (127) Google Scholar, 11George G.N. Hilton J. Temple C. Prince R.C. Rajagopalan K.V. J. Am. Chem. Soc. 1999; 121: 1256-1266Crossref Scopus (153) Google Scholar, 12Bastian N.R. Kay C.J. Barber M.J. Rajagopalan K.V. J. Biol. Chem. 1991; 266: 45-51Abstract Full Text PDF PubMed Google Scholar, 13Bastian N.R. Foster M.J. Pope J.C. Biofactors. 1995; 5: 5-10PubMed Google Scholar, 14Bennett B. Benson N. McEwan A.G. Bray R.C. Eur. J. Biochem. 1994; 225: 321-331Crossref PubMed Scopus (98) Google Scholar), and resonance Raman spectroscopy (15Garton S.D. Hilton J.C. Oku H. Crouse B.R. Rajagopalan K.V. Johnson M.K. J. Am. Chem. Soc. 1997; 119: 12906-12916Crossref Scopus (114) Google Scholar,16Garton S.D. Temple C.A. Dhawan I.K. Barber M.J. Rajagopalan K.V. Johnson M.K. J. Biol. Chem. 2000; 275: 6798-6805Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Resonance Raman studies on BSOR and DMSOR indicate that both enzymes function by an oxo transfer mechanism whereby the oxo group from the substrate is directly transferred to the molybdenum atom (Fig.2) (15Garton S.D. Hilton J.C. Oku H. Crouse B.R. Rajagopalan K.V. Johnson M.K. J. Am. Chem. Soc. 1997; 119: 12906-12916Crossref Scopus (114) Google Scholar, 16Garton S.D. Temple C.A. Dhawan I.K. Barber M.J. Rajagopalan K.V. Johnson M.K. J. Biol. Chem. 2000; 275: 6798-6805Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). This process is reversible in DMSOR, and the ability of this enzyme to be reduced by dimethyl sulfide (Me2S) has been extensively studied (7McAlpine A.S. McEwan A.G. Bailey S. J. Mol. Biol. 1998; 275: 613-623Crossref PubMed Scopus (171) Google Scholar, 11George G.N. Hilton J. Temple C. Prince R.C. Rajagopalan K.V. J. Am. Chem. Soc. 1999; 121: 1256-1266Crossref Scopus (153) Google Scholar, 15Garton S.D. Hilton J.C. Oku H. Crouse B.R. Rajagopalan K.V. Johnson M.K. J. Am. Chem. Soc. 1997; 119: 12906-12916Crossref Scopus (114) Google Scholar,17Adams B. Smith A.T. Bailey S. McEwan A.G. Bray R.C. Biochemistry. 1999; 38: 8501-8511Crossref PubMed Scopus (51) Google Scholar). However, resonance Raman analysis has indicated that BSOR is unable to be reduced with either Me2S or by biotin (16Garton S.D. Temple C.A. Dhawan I.K. Barber M.J. Rajagopalan K.V. Johnson M.K. J. Biol. Chem. 2000; 275: 6798-6805Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Other than mutation of the protein ligands to the molybdenum in DMSOR and BSOR (18Hilton J.C. Temple C.A. Rajagopalan K.V. J. Biol. Chem. 1999; 274: 8428-8436Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 19Pollock V.V. Barber M.J. J. Biol. Chem. 2000; 275: 35086-35090Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), no mutagenic studies have been reported for TMAOR, BSOR, or Rhodobacter DMSOR, and there is little information about the roles of other amino acids in enzymatic activity. Alignment of R. sphaeroides and R. capsulatusDMSOR to E. coli and S. massilia TMAOR andR. sphaeroides BSOR indicates 22% sequence identity and 48% sequence similarity between all five enzymes. Despite this similarity, there are striking differences in their physiological roles, electron donors, and substrate specificity. TMAOR functions as the final enzyme in the anaerobic electron transport pathway that utilizes Me3NO as the terminal electron acceptor (20Silvestro A. Pommier J. Pascal M.C. Giordano G. Biochim. Biophys. Acta. 1989; 999: 208-216Crossref PubMed Scopus (54) Google Scholar). Although DMSOR also functions as a terminal enzyme during anaerobic respiration, it is able to utilize a greater variety of substrates including Me3NO and dimethyl sulfoxide (Me2SO) (21Satoh T. Kurihara F.N. J. Biochem. (Tokyo). 1987; 102: 191-197Crossref PubMed Scopus (116) Google Scholar). BSOR, a cytoplasmic protein whose possible roles include scavenging biotin sulfoxide (BSO) to generate biotin and protecting the cell from oxidative damage (22Pierson D.E. Campbell A. J. Bacteriol. 1990; 172: 2194-2198Crossref PubMed Scopus (68) Google Scholar), has also been shown to use a variety of S- and N-oxides (23Pollock V.V. Barber M.J. J. Biol. Chem. 1997; 272: 3355-3362Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The x-ray crystallographic structure of DMSOR has generated great interest in the role of the Tyr at position 114 during catalytic turnover of the enzyme. In the 1.3-Å crystal structure of R. sphaeroides DMSOR, the bis(MGD)Mo active site exhibits two different coordination geometries (5Hung-Kei L. Temple C.A. Rajagopalan K.V. Schindelin H. J. Am. Chem. Soc. 2000; 122: 7673-7680Crossref Scopus (171) Google Scholar). In the catalytically active, hexa-coordinated molybdenum site, the single oxo group is coordinated by Trp-116 (Fig. 3B), whereas in the inactive, penta-coordinated molybdenum environment, Tyr-114 is hydrogen-bonded to one of two oxo groups (Fig. 3C). Although sequence alignments have shown that BSOR contains a residue equivalent to Tyr-114, this residue is missing in E. coli and S. massilia TMAOR (Fig. 3, A and D). Since both BSOR and DMSOR are able to reduce a wide variety of S- andN-oxides whereas TMAOR shows a more limited specificity forN-oxides (24Iobbi-Nivol C. Pommier J. Simala-Grant J. Méjean V. Giordano G. Biochim. Biophys. Acta. 1996; 1294: 77-82Crossref PubMed Scopus (42) Google Scholar), Tyr-114 has been postulated to be responsible for this variance in substrate specificity (4Czjzek M. Dos Santos J.-P. Pommier J. Giordano G. Mejean V. Haser R. J. Mol. Biol. 1998; 284: 435-447Crossref PubMed Scopus (160) Google Scholar, 25Buc J. Santini C.L. Giordani R. Czjek M. Wu L.F. Giordano G. Mol. Microbiol. 1999; 32: 159-168Crossref PubMed Scopus (85) Google Scholar). Although R. sphaeroides DMSOR and BSOR were previously cloned and heterologously expressed in E. coli (3Temple C.A. George G.N. Hilton J.C. George M.J. Prince R.C. Barber M.J. Rajagopalan K.V. Biochemistry. 2000; 39: 4046-4052Crossref PubMed Scopus (39) Google Scholar, 23Pollock V.V. Barber M.J. J. Biol. Chem. 1997; 272: 3355-3362Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 26Hilton J.C. Rajagopalan K.V. Biochim. Biophys. Acta. 1996; 1294: 111-114Crossref PubMed Scopus (19) Google Scholar), TMAOR has not been cloned previously. In the studies reported here,E. coli TMAOR has been cloned and the recombinant protein purified, setting the stage for a comprehensive study of the role of Tyr-114 in this family of enzymes. This residue has been mutated to both Ala and Phe in DMSOR, and a Tyr has been inserted into TMAOR in an equivalent position. The molybdenum coordination environment of the wild-type and mutant proteins has been probed using UV-visible absorption spectroscopy. The activities of both wild-type and mutant proteins have been analyzed by steady-state kinetics with both S- and N-oxides in the forward direction, and the efficiency of Me2S reduction of these enzymes has been measured. The presence of these mutations does not appear to affect stability or cofactor incorporation. The mutation of Tyr-114 in DMSOR does increase the specificity for N-oxides while decreasing the specificity for S-oxides, and insertion of the Tyr residue in TMAOR increases the specificity forS-oxides with a concomitant decrease in specificity forN-oxides. Mutation of Tyr-114 in DMSOR also results in inefficient reduction of the enzyme by Me2S. The first 117 nucleotides of theE. coli torA sequence encode a 39-amino acid N-terminal signal sequence that targets TMAOR to the periplasm and is cleaved to form the mature enzyme (27Mejean V. Iobbi-Nivol C. Lepelletier M. Giordano G. Chippaux M. Pascal M.C. Mol. Microbiol. 1994; 11: 1169-1179Crossref PubMed Scopus (221) Google Scholar). The structural gene for TMAOR without the periplasmic signal sequence was isolated from DH5α genomic DNA using PCR primers created from the E. coli K12 torAgene sequence (GenBankTM accession number X73888). One primer inserted an NdeI site and a new start codon immediately 5′ to the codon for the first amino acid of the mature protein, and the second created a HindIII site 36 nucleotides downstream from the stop codon. The resulting DNA segment containing the torA sequence was ligated directly into the pCR2.1 cloning vector using the Topo TA cloning kit (Invitrogen). The consensus sequence of three independent clones confirmed the previously published K12 sequence (27Mejean V. Iobbi-Nivol C. Lepelletier M. Giordano G. Chippaux M. Pascal M.C. Mol. Microbiol. 1994; 11: 1169-1179Crossref PubMed Scopus (221) Google Scholar). Primers were obtained from Life Technologies, Inc., and automated sequencing was accomplished by the Duke University DNA Analysis Facility. One plasmid clone containing a single polymerase error that changed Trp-576 to Ala was selected for further manipulations. Due to difficulties in growing cells containing the pCR2.1 cloning vector during mutagenesis, the TMAOR coding sequence was released with HindIII and transferred into the pBluescript II Ks(+) cloning vector (Stratagene). The polymerase error was repaired by site-directed mutagenesis on double-stranded DNA using the CLONTECH Transformer Site-directed Mutagenesis Kit to obtain pKJ125. This plasmid was digested with NdeI and HindIII, and the coding sequence was ligated into the pET-29a(+) expression vector (Novagen) to form pKJ525 (Table I) which encodes for mature TMAOR. To aid in purification of the protein, the TMAOR coding region of pKJ125 was released with HindIII andNdeI and ligated into pET-28a(+) (Novagen) to create pKJ725, which encodes an N-terminal His6-tagged version of TMAOR. The coding sequence containing the structural gene, and the N-terminal His tag from pKJ725 was excised using HindIII andNcoI and ligated into the pTrc 99 A expression vector (Amersham Pharmacia Biotech) to form pKJ825.Table IE. coli expression plasmidsConstructVectorInsertKJ525pET-29a(+)TMAOR without periplasmic signal sequenceKJ725pET-28a(+)TMAOR with N-terminal His6 tagKJ825pTrc 99 ATMAOR with N-terminal His6 tagKJ830pTrc 99 ATMAOR+Y with N-terminal His6 tagJH720aHilton et al.(18).pET-28a(+)DMSOR with N-terminal His6 tagJH820bTemple and Rajagopalan (28).pTrc 99 ADMSOR with N-terminal His6 tagJN711pET-28a(+)DMSOR-Y114A with N-terminal His6 tagJN712pET-28a(+)DMSOR-Y114F with N-terminal His6taga Hilton et al.(18Hilton J.C. Temple C.A. Rajagopalan K.V. J. Biol. Chem. 1999; 274: 8428-8436Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar).b Temple and Rajagopalan (28Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Open table in a new tab Site-directed mutagenesis was used to insert a Tyr residue between amino acids 119 and 120 of the TMAOR coding sequence in pKJ725. The sequence for the structural gene and the N-terminal His tag was excised using HindIII and NcoI and ligated into pTrc 99 A to form pKJ830. This plasmid expresses the TMAOR+Y variant of TMAOR, which contains a Tyr residue in a location equivalent to Tyr-114 in DMSOR. Site-directed mutagenesis was performed on pJH118 (18Hilton J.C. Temple C.A. Rajagopalan K.V. J. Biol. Chem. 1999; 274: 8428-8436Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) to change the Tyr at position 144 to either Ala or Phe. The mutated coding sequences were subsequently liberated using HindIII and NdeI and ligated into pET-28a(+) in frame with the N-terminal His6 tag to produce pJN711 and pJN712, the expression vectors for DMSOR-Y114A and DMSOR-Y114F, respectively. All of the pTrc 99 A-based expression vectors (pJH820 (28Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), pKJ825, and pKJ830) were transformed into RK4353E. coli cells (29Stewart V. MacGregor C.H. J. Bacteriol. 1982; 151: 788-799Crossref PubMed Google Scholar). Growth and expression in these cells were done as described previously (3Temple C.A. George G.N. Hilton J.C. George M.J. Prince R.C. Barber M.J. Rajagopalan K.V. Biochemistry. 2000; 39: 4046-4052Crossref PubMed Scopus (39) Google Scholar) with the exception that isopropyl-β-d-thiogalactopyranoside (IPTG) was present at 10 μm. The RK4353 strain was DE3-lysogenized using the λ DE3 lysogenization kit from Novagen to create the RK4353(DE3) strain. All of the pET-based expression plasmids (pJH720, pKJ725, pKJ525, pJN711, and pJN712) were transformed into these cells. The resulting strains were subsequently transformed a second time with pLysS (Novagen). Conditions for growth and expression were as described previously (3Temple C.A. George G.N. Hilton J.C. George M.J. Prince R.C. Barber M.J. Rajagopalan K.V. Biochemistry. 2000; 39: 4046-4052Crossref PubMed Scopus (39) Google Scholar), with the exception that 30 μg/ml chloramphenicol and 34 μg/ml kanamycin were used as the sole antibiotics, and expression was induced with 40 μm IPTG. Cells expressing TMAOR were harvested by centrifugation at 5,000 × g and resuspended in 50 mm sodium phosphate buffer, pH 8.0, containing 300 mm NaCl (PN buffer). Cell lysis was achieved by three passages through the 112-μm interaction chamber of a Microfluidics M110L Microfluidizer Processor at 16,000–18,000 pounds/square inch. The resulting extract was stirred at room temperature for 20 min with ∼10 μg/ml DNase I and then centrifuged at 11,000 ×g for 25 min at 4 °C. The supernatant was adjusted to pH 6.5 with HCl, heated in a boiling water bath to 65 °C, maintained at 65 °C for 1 min, cooled rapidly in an ice bath, and then centrifuged at 11,000 × g for 25 min. Imidazole was added to a final concentration of 10 mm; the pH was adjusted to 7.5 with NaOH, and the solution was centrifuged again. The supernatant was then equilibrated with 40 ml of Ni2+-NTA affinity resin (Qiagen) by gentle stirring at 4 °C for 15 min. This slurry was loaded onto a gravity flow column and subsequently washed with 2 column volumes of PN buffer at pH 7.5 containing 10 mm imidazole, 8 column volumes of the same solution at pH 8.0, and 3 column volumes of PN buffer, pH 8, containing 20 mm imidazole. TMAOR was eluted at pH 8.0 with PN buffer containing 200 mmimidazole. The fractions containing TMAOR were combined, dialyzed against 50 mm Tris, pH 7.5, and purified using a Q-Sepharose fast protein liquid chromatography column with a 23-ml bed volume. The column was washed with 1 column volume of 50 mmTris, pH 7.5, followed by a 2-column volume 0–300 mmgradient of NaCl, and TMAOR was then eluted with a 2-column volume wash at 300 mm NaCl. The fractions containing TMAOR were combined and buffer exchanged into 50 mm Tris, pH 7.5, using either dialysis or a 100-ml Superose-12 fast protein liquid chromatography column. Q-Sepharose fast flow and Superose-12 resin were obtained from Amersham Pharmacia Biotech. A more stable form of TMAOR was obtained by cycling the protein during purification. The clarified lysate was placed in a Coy anaerobic chamber containing a mixture of carbon dioxide, hydrogen, and nitrogen and reduced with dithionite in the presence of methyl viologen until a dark blue color was present. A 100 mm solution of Me3NO was then added in a dropwise fashion until the solution lost all blue color, and the process was repeated. The resulting solution was immediately brought to pH 6.5 for the heat step and further purification as already described. TMAOR+Y was purified in the same manner as the wild-type enzyme including the cycling step. Native DMSOR was purified fromR. sphaeroides (1Hilton J.C. Rajagopalan K.V. Arch. Biochem. Biophys. 1996; 325: 139-143Crossref PubMed Scopus (46) Google Scholar), and recombinant DMSOR, DMSOR-Y114A, and DMSOR-Y114F were purified from E. coli as described previously (18Hilton J.C. Temple C.A. Rajagopalan K.V. J. Biol. Chem. 1999; 274: 8428-8436Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) with the exception that a Microfluidics M110L Microfluidizer Processor was used to break open the cells. The molybdenum content of the purified proteins was analyzed using a PerkinElmer Life Sciences Zeeman 3030 atomic absorption spectrometer as described previously (18Hilton J.C. Temple C.A. Rajagopalan K.V. J. Biol. Chem. 1999; 274: 8428-8436Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Pure samples of DMSOR were quantitated spectrophotometrically at 280 nm using an extinction coefficient of 200,000m−1 cm−1or 2.3 ml mg−1 cm−1(1Hilton J.C. Rajagopalan K.V. Arch. Biochem. Biophys. 1996; 325: 139-143Crossref PubMed Scopus (46) Google Scholar). For quantitation of TMAOR or total protein, the Pierce BCA assay was used as described in the manufacturer's protocol, with purifiedR. sphaeroides DMSOR as the standard. Guanine analysis was performed as described previously (18Hilton J.C. Temple C.A. Rajagopalan K.V. J. Biol. Chem. 1999; 274: 8428-8436Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Absorption spectroscopy was carried out using a Shimadzu UV-2101 PC spectrophotometer. All spectra were recorded in 50 mm Tris-HCl, pH 7.5, and normalized to 5 mg/ml unless otherwise indicated. Dithionite-reduced spectra were recorded under anaerobic conditions as described previously (3Temple C.A. George G.N. Hilton J.C. George M.J. Prince R.C. Barber M.J. Rajagopalan K.V. Biochemistry. 2000; 39: 4046-4052Crossref PubMed Scopus (39) Google Scholar), and re-oxidized spectra were obtained by the injection of anaerobic substrate directly into the cuvette containing the dithionite-reduced enzyme. To obtain Me2S-reduced spectra, purified recombinant DMSOR, DMSOR-Y114F, and DMSOR-Y114A were reduced under anaerobic conditions in the Coy chamber, re-oxidized with substrate, and dialyzed against 2× 1 liter of 50 mm Tris, pH 7.5. TMAOR and TMAOR+Y spectra were obtained using enzyme that had been cycled during purification. Enzyme was transferred in the Coy chamber to a cuvette and sealed before removal from the anaerobic environment. Anaerobic stock solutions of 2 m Me2S in ethanol and 2m Me2SO in water were added to the cuvette using a gas-tight syringe. Kinetic constants for DMSOR, DMSOR-Y114A, DMSOR-Y114F, TMAOR, and TMAOR+Y using Me2SO, Me3NO, methionine sulfoxide (MetSO), and adenosine-1N-oxide (ANO) as substrates were determined as described previously (18Hilton J.C. Temple C.A. Rajagopalan K.V. J. Biol. Chem. 1999; 274: 8428-8436Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Three to ten activity assays were performed for each of 5–10 different substrate concentrations.Km and kcat values were determined by direct fit to the Michaelis-Menten equation. Kinetic constants for reduction of recombinant DMSOR, TMAOR, DMSOR-Y114A, DMSOR-Y114F, and TMAOR+Y using Me2S were determined using the method of Adams et al. (17Adams B. Smith A.T. Bailey S. McEwan A.G. Bray R.C. Biochemistry. 1999; 38: 8501-8511Crossref PubMed Scopus (51) Google Scholar). Initial activity was measured aerobically after the addition of Me2S from a gas-tight syringe to a cuvette containing enzyme in the presence of 50 mm Tris, pH 8.0, 0.2 mm phenazine methosulfate (PMS), and 0.04 mm 2,6-dichlorophenolindophenol (DCPIP). Background activity, obtained by the addition of anaerobic Me2S to a cuvette containing no enzyme, was subtracted to obtain all final activity numbers. All substrates were obtained from Sigma. Whereas E. coliTMAOR has previously been purified from source (30Silvestro A. Pommier J. Giordano G. Biochim. Biophys. Acta. 1988; 954: 1-13Crossref PubMed Scopus (49) Google Scholar, 31Yamamoto I. Okubo N. Ishimoto M. J. Biochem. (Tokyo). 1986; 99: 1773-1779Crossref PubMed Scopus (48) Google Scholar) and a similar enzyme has been purified from S. massilia (32Dos Santos J.P. Iobbi-Nivol C. Couillault C. Giordano G. Mejean V. J. Mol. Biol. 1998; 284: 421-433Crossref PubMed Scopus (93) Google Scholar), the yield in both cases was substantially less than that obtained by the heterologous expression of R. sphaeroides DMSOR and BSOR inE. coli (3Temple C.A. George G.N. Hilton J.C. George M.J. Prince R.C. Barber M.J. Rajagopalan K.V. Biochemistry. 2000; 39: 4046-4052Crossref PubMed Scopus (39) Google Scholar, 28Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). To facilitate purification of the large quantities of enzyme required for comprehensive studies on the role of the Tyr residue in DMSOR and TMAOR, E. coli TMAOR was cloned, homogeneously expressed, and purified. For overexpression, the gene for mature E. coli TMAOR with an added N-terminal His6 tag was placed into the pTrc 99 A vector. Three mutants were created to elucidate the role of Tyr-114. In DMSOR, this residue was mutated to either Ala or Phe. The latter retains the large phenyl ring but cannot form a hydrogen bond to the molybdenum oxo ligand. The mutated sequences were transferred into a vector to generate His-tagged versions of DMSOR-Y114A and DMSOR-Y114F. In TMAOR, a Tyr codon was inserted into the His-tagged coding sequence between residues 119 and 120 in a position corresponding to Tyr-114 of DMSOR (Fig. 3A). BL21(DE3), the standard E. coli strain used for expression of pET plasmids, appears to have difficulty expressing enzymes containing the molybdenum cofactor (33Temple C.A. Graf T.N. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar). RK4353 is the base strain for an extensive series of cofactor biosynthesis mu insertion mutants (29Stewart V. MacGregor C.H. J. Bacteriol. 1982; 151: 788-799Crossref PubMed Google Scholar), and it was chosen as an alternative expression strain. Since expression from pET-based plasmids requires the presence of T7 polymerase, the RK4353 strain was DE3-lysogenized. The plasmids expressing TMAOR reductase with and without a His tag (pKJ525 and pKJ725), DMSOR (pJH720), DMSOR-Y114A (pJN711), and DMSOR-Y114F (pJN712) were transformed into RK4353(DE3) cells. The resulting strains were subsequently transformed with pLysS. Because of problems with cell stability as a result of DE3 lysogenization, an alternative expression system was created. Expression of R. sphaeroides DMSOR in the pTrc 99 A expression construct has been shown to produce a substantial improvement in yield and permits expression in an alternative cell strain without the presence of the T7 polymerase (28Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The plasmids expressing His-tagged TMAOR, TMAOR+Y, and DMSOR in pTrc 99 A (pKJ825, pKJ830, and pJH820, respectively) were transformed into RK4353E. coli cells. All strains were grown and induced for 24 h under anaerobic conditions as described previously (3Temple C.A. George G.N. Hilton J.C. George M.J. Prince R.C. Barber M.J. Rajagopalan K.V. Biochemistry. 2000; 39: 4046-4052Crossref PubMed Scopus (39) Google Scholar) with the exception that kanamycin and chloramphenicol were used as antibiotics for all RK4353(DE3) pLysS strains containing pET-based plasmids, and ampicillin was used for all expression from RK4353 cells containing pTrc 99 A-based plasmids. The IPTG concentration had to be lowered to 10 μm for expression of proteins from the pTrc 99 A vectors to obtain a level of active protein equivalent to that expressed from the pET vectors when induced with 40 μm IPTG. Analysis of DMSOR has shown that the presence of a N-terminal His6 tag does not alter the activity of the protein (18Hilton J.C. Temple C.A. Rajagopalan K.V. J. Biol. Chem. 1999; 274: 8428-8436Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). To ascertain whether the same was true for TMAOR, both the native and His-tagged versions of the prote
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