APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2005, p. 621–628 0099-2240/05/$08.00⫹0 doi:10.1128/AEM.71.2.621–628.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 71, No. 2
Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis Zhi-Wei Chen,1† Cheng-Ying Jiang,1† Qunxin She,2 Shuang-Jiang Liu,1* and Pei-Jin Zhou1 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People’s Republic of China,1 and Institute of Molecular Biology, University of Copenhagen, Copenhagen, Denmark2 Received 11 July 2004/Accepted 15 September 2004
Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C31 and C101-X-X-C104; numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn2ⴙ strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have similar structures and that none of them form any disulfide bond. Thus, it is proposed that three cysteine residues, C31 and C101-X-X-C104, in the conserved domains constitute the putative binding and catalytic sites of SOR. Furthermore, enzymatic activity assays of the subcellular fractions and immune electron microscopy indicated that SOR is not only present in the cytoplasm but also associated with the cytoplasmic membrane of A. tengchongensis. The membrane-associated SOR activity was colocalized with the activities of sulfite:acceptor oxidoreductase and thiosulfate:acceptor oxidoreductase. We tentatively propose that these enzymes are located in close proximity on the membrane to catalyze sulfur oxidation in A. tengchongensis. the SORs are homoenzymes consisting of identical subunits of ⬃35-kDa peptides (14, 16, 26). (ii) Sulfide, sulfite, and thiosulfate are concomitantly produced during the oxidation of elemental sulfur catalyzed by SORs (5[S] ⫹ O2 ⫹ 4OH⫺ 3 HSO3⫺ ⫹ S2O32⫺ ⫹ 2HS⫺ ⫹ H⫹); however, thiosulfate is produced only from a spontaneous reaction of sulfite and elemental sulfur. (iii) Cofactors or external electron donors and acceptors for sulfur reduction were not required, and the two enzymatic activities (oxygenase and reductase) could not be separated. As SOR was the only elemental sulfur-oxidizing enzyme found in A. ambivalens, it was assumed that the enzyme catalyzes the initial step of the sulfur-oxidizing pathway in this organism. The structure and function of this type of enzyme, as well as its relationships, are largely unknown. We have developed a system to actively express the sor gene of A. tengchongensis in Escherichia coli cells and have established a simple procedure to efficiently purify the SOR protein from E. coli cells (10, 26). These developments have allowed us to circumvent the difficulties in the genetic manipulation of, and SOR purification from, an acidothermophilic archaeon like Acidianus. In this study, we conducted site-directed mutagenesis of all cysteine residues of the A. tengchongensis SOR protein and established that all of the cysteine residues are very important to SOR activities. Furthermore, the cooccurrence of SOR activities and the other sulfur-metabolizing enzymes on the cytoplasmic membrane suggests that these enzymatic reactions may be coupled on the membrane.
Biological oxidation and reduction of elemental sulfur are important reactions involved in the biogeochemical cycles of the element sulfur on the earth. Acidothermophilic archaea, such as species of the genus Acidianus, oxidize elemental sulfur through a pathway(s) that is different from that of sulfuroxidizing bacteria, such as Paracoccus pantotrophus. In the former, oxidation of elemental sulfur is initiated by sulfur oxygenase-reductase (SOR), and the reaction requires dioxygen molecules (O2) (7, 10, 14, 16), whereas in the latter, elemental sulfur or reduced sulfur compounds are oxidized through the catalysis of the SOX enzyme complex and dioxygen is not directly involved in the oxidation process (8, 22). Although there are a number of organisms that can metabolize elemental sulfur and reduced sulfurous compounds, the SOR pathway is limited to a few archaea so far. The sor genes have been identified and characterized only for Acidianus ambivalens and Acidianus tengchongensis (formerly Acidianus sp. strain S5 [11]) (10, 15). Studies of the native and recombinant SORs of Acidianus spp. revealed that they are distinctive in several respects. (i) Unlike the SOX enzyme complex with heterologous subunits,
* Corresponding author. Mailing address: Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People’s Republic of China. Phone: 86-10-62527118. Fax: 86-10-62652317. E-mail:
[email protected]. † Zhi-Wei Chen and Cheng-Ying Jiang contributed equally to this work. 621
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APPL. ENVIRON. MICROBIOL. TABLE 1. Bacterial strains, plasmids, and primers used in this study Characteristic(s) or sequencea
Strain, plasmid, or primer
Source or reference
Bacterial strains A. tengchongensis strain S5 E. coli strain HB101 E. coli strain XL1-Blue
From laboratory culture collection Expressing host for wild-type and mutated SOR Cloning host for SOR gene and for site-directed mutagenesis
11
Plasmids pBV220SOR pBV220SOR/C31A pBV220SOR/C31S pBV220SOR/C101S pBV220SOR/C104S
Contains entire wild-type SOR gene Harbors mutated SOR gene (C31A) Harbors mutated SOR gene (C31S) Harbors mutated SOR gene (C101S) Harbors mutated SOR gene (C104S)
10 This This This This
Primers C1F C1R C2F C2R C3F C3R
5⬘-GCTGTAGGACCAAAAGTGAGCATGGTAACAGCAAGAC-3⬘ 5⬘-GTCTTGCTGTTACCATGCTCACTTTTGGTCCTACAGC-3⬘ 5⬘-GAGTTACCTATTTAGACTTAGCTACTCATGCGCCTCAC-3⬘ 5⬘-GTGAGGCGCATGAGTAGCTAAGTCTAAATAGGTAACTC-3⬘ 5⬘-CTATTTAGACTTTGCTACTCAAGCGCCTCACAAATGGTTTGG-3⬘ 5⬘-CCAAACCATTTGTGAGGCGCTTGAGTAGCAAAGTCTAAATAG-3⬘
a
study study study study
All primers were designed in this study. Mutations are underlined.
MATERIALS AND METHODS Materials. Pyrobest DNA polymerase and deoxynucleoside triphosphates were purchased from TaKaRa Biotech. DpnI was obtained from New England Biolabs. Low-melting-point argarose, Tris base, and ammonium persulfate were products of Promega. A protein molecular mass marker (14.4 to 97.4 kDa) kit and a bicinchoninic acid protein assay kit were bought from Sigma. The Superdex ¨ kta 200, XL10/70 column, and fast protein liquid chromatography system (A FPLC) were from Amersham Biosciences. Alkaline phosphatase-conjugated antibodies were from Jackson Immunology Research Laboratory. All other reagents were of analytical grade. Cultivation, growth media, and determination of cell growth. All E. coli strains (Table 1) were cultivated in Luria-Bertani (LB) broth or on LB agar at 37°C unless otherwise indicated, and ampicillin was added to 100 g ml⫺1 when applicable. A. tengchongensis was grown at 70°C in Allen’s medium (1). The growth of cells was estimated either by monitoring the increase in optical density at 600-nm wavelength or by determining the increase in the protein contents of cultures. Site-directed mutagenesis and selection of mutated SOR genes. The primers used for generating mutated sor genes (C31S, C101S, and C104S) and the plasmids containing the generated mutated genes are listed in Table 1. Sitedirected mutagenesis was conducted on the SOR gene carried by the plasmid pBV220/sor according to the method of Sambrook and Russell (23). The mutations of the SOR gene were subsequently confirmed by DNA sequencing. Overexpression and purification of SOR and mutant proteins in Escherichia coli. E. coli strain HB101 was used as the host for overexpressing the A. tengchongensis sor gene and mutated sor genes. The E. coli strain containing a plasmid carrying the wild-type or a mutated sor gene was cultivated in 500-ml flasks filled with 200 ml of LB medium and supplemented with ampicillin to 100 g ml⫺1. Cultivation was carried out with shaking at 250 rpm at 30°C. When they had grown to an optical density at 600 nm of 0.6, the cultures were shifted from 30 to 42°C to induce protein synthesis. The cells were harvested by centrifugation after 8 h of induction, and the cell pellets obtained were stored at ⫺70°C until they were used. At the time of SOR protein purification, a 140-ml Superdex 200 gel filtration column was packed in our laboratory and preequilibrated with buffer B (10 mM KH2PO4–K2HPO4 buffer, pH 7.4). Crude cellular extract was prepared from the stored E. coli cells as described previously (10), and 1.4 ml of the crude cellular extract was incubated at 70°C for 10 min to denature E. coli heat-labile proteins, which were subsequently removed by centrifugation. The resulting supernatant was applied onto the Superdex 200 gel filtration column and fractionated using buffer B at a flow rate of 0.6 ml min⫺1. Fractions were collected at 0.6 ml per tube and assayed for SOR activity. Those that possessed SOR activity were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis for homogeneity. During the purification of the SOR mutant proteins (no SOR activity), the collected fractions were first analyzed by SDS-PAGE to check
for the presence of SOR mutant proteins. The mutant SORs were subsequently confirmed by immunoblot analysis with SOR-specific antibody. The purified proteins were concentrated by ultrafiltration with an Amicon Ultra-15 (100 kDa) and stored at ⫺20°C. SDS-PAGE and Western blotting. Proteins in fractions from chromatography were separated through SDS-PAGE (15% polyacrylamide) according to the method of Laemmli (18) and were visualized by staining them with Coomassie brilliant blue R-250. Western blotting was performed according to the method of Liu et al. (19). SOR, SOR mutant enzymes, and bovine serum albumin (negative control) were transferred from a gel onto a nitrocellulose membrane. The SOR proteins were then detected by a method described previously, using the antiserum raised against SOR in rabbits (26). Enzymatic activity assays. The oxygenase activity of SOR was determined as described by Kletzin (16), except that the assays were conducted at 70°C (26). One unit is defined as the amount of enzyme required for the formation of 1 mol of sulfite plus thiosulfate per min. Concentrations of SO32⫺ and S2O32⫺ were determined by basic fuchsin and methylene blue through colorimetry, respectively (16). The activities of sulfite:acceptor oxidoreductase (SAOR) and thiosulfate:acceptor oxidoreductase (TAOR) were determined as described previously (13, 28). Effects of chemicals on SOR activity. N-Ethylmaleimide (NEM), glutathione (GSH), dithiothreitol (DTT), EDTA, MgCl2, ZnCl2, CuCl2, CoCl2, NiCl2, and MnCl2 were added to the enzyme-buffer assay mixture and incubated for 15 min at room temperature. The enzymatic reaction was then started by raising the temperature to 70°C. The reactions without any chemical added and the reactions without the SOR enzyme added were run in parallel as double controls. Protein concentration assay. The protein concentration was determined using a bicinchoninic acid protein assay kit following the instructions of the manufacturer. Preparation of cellular fractions by centrifugation. Cells of A. tengchongensis were harvested by centrifugation at 5,000 ⫻ g for 15 min at room temperature. The cell pellets were suspended in distilled water with the pH adjusted to 7.5 with 1 M potassium phosphate buffer. Crude cell lysates were generated by sonication, and cell debris was removed by centrifugation at 12,000 ⫻ g for 20 min at 4°C. The resulting supernatant was then centrifuged at 120,000 ⫻ g for 5 h at 4°C, yielding the cytoplasm fraction (clear supernatant) and the pellet containing the membrane fraction. To avoid any possible contamination by the cytoplasm fraction, the pellet was washed twice with distilled water and finally suspended in 0.1 M potassium phosphate buffer (pH 7.5) to yield the membrane fraction. Localization of SOR at subcellular level by immunogold electron microscopy. SOR-specific antibody was raised in rabbits as described previously (26). Colloidal-gold (particle diameter, 10 nm)-cross-linked secondary antibody (goat antirabbit serum) was purchased from Sigma. A transmission electron microscope (TEM) and a scanning electron microscope (SEM) were used. The preparation of cells for immunogold TEM and SEM observation was conducted according to
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FIG. 1. Multiple alignment of SOR sequences showing the conserved cysteine residues (underlined in Aae) and other conserved regions. Ate, A. tengchongensis; Abr, A. brierleyi; Aam, A. ambivalens; Sto, S. tokodaii; Aae, A. aerolicus; Con, consensus.
a method described previously (6) and the operating instructions for the Critical Point Dryer CPD030 (BAL-TEC Inc., Balzers, Liechtenstein), respectively. CD measurements and secondary-structure analysis. The circular-dichroism (CD) spectra of wild and mutant SORs between 190 and 260 nm were acquired at 70°C in a 1-mm-path-length quartz cell with a JASCO model J-715 spectropolarimeter under the following conditions: response time, 2 s; scan speed, 20 nm min⫺1; 0.1-nm data acquisition interval; four accumulations; 2-nm bandwidth; and 100 g of SOR proteins ml⫺1 in buffer B (see above). The CD spectra were analyzed with the K2D and CDPro software packages, including SELCON3, CONTINLL, CDSSTR, and CLUSTER for determining the secondary-structure and tertiary-structure classes (2, 24, 25). Determination of fluorescence spectroscopy. The intrinsic fluorescence spectra of wild and mutated SORs were detected with a HITACHI Model F-2500 fluorescence spectrophotometer. Three hundred micrograms of the proteins/ml dissolved in buffer B was examined at 70°C with the following parameters: exciting wavelength, 295 nm; scanning range, 305 to 400 nm; scanning speed, 60 nm min⫺1; response time, 0.08 s. Sequence alignment and secondary-structure and hydrophobicity predictions. The amino acid sequences of SORs from A. tengchongensis, A. ambivalens, Sulfolobus tokodaii, and Aquifex aeolicus were retrieved from public databases. The amino acid sequence of Acidianus brierleyi was obtained directly from the
microbial genome project under way at the University of Copenhagen, Copenhagen, Denmark. Sequence alignments were performed with Clustal X software. Secondary-structure prediction based on the amino acid sequence was carried out with a software package including Antheprot version 5.2 and the Predict-
TABLE 2. Effects of various chemicals on SOR activity Chemical
Concn (mM)
Remaining sp act (%)
Mg2⫹ Zn2⫹ Co2⫹ Cu2⫹ Ni2⫹ Mn2⫹ GSH DTT EDTA NEM
1 1 1 1 1 1 0.05 0.05 10 0.1 1
77.2 27.0 10.8 0.7 12.5 34.5 110.8 109.3 108.8 0.02 0.03
FIG. 2. Purified wild and mutant SORs stained with Coomassie blue (A and C) and Western blotted (B). Lanes: M, protein molecular markers; 1 and 5, wild-type SOR; 2 and 6, mutant SOR (C31S); 3 and 7, mutant SOR (C101S); 4 and 8, mutant SOR (C104S). Lanes 5 to 8 were without -mercaptoethanol. Each lane was loaded with 2 g of protein.
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TABLE 3. Specific activities of SOR and its mutants Protein
Sp act (U/mg)
Activity lost (%)
SOR C31A C31S C101S C104S
4.85 0.00 0.00 0.08 0.04
Control 100 100 98.4 99.2
Protein server (http://cubic.bioc.columbia.edu/pp/). Hydrophobicity prediction was performed by the method of J. Kyte and R. F. Doolittle provided by the BioEdit software.
RESULTS Sequence analysis of SOR and SOR-like proteins and predictions of secondary structure and hydrophobicity-hydrophi-
licity of the SOR molecules. So far, only two SOR genes have been identified and characterized (10, 15). Another SOR gene has recently been identified from the ongoing A. brierleyi microbial genome project. Furthermore, two sor-like sequences were identified by BLAST searches of the public sequence databases, using the sor gene sequence of A. tengchongensis. These are NC_003106 from S. tokodaii and NC_000918 from A. aeolicus. A comparison of the amino acid sequences of the proteins revealed that the A. tengchongensis SOR shows 100, 88, 66, and 34% sequence identity with the SORs of A. brierleyi and A. ambivalens and the SOR-like proteins of S. tokodaii and A. aeolicus, respectively. Interestingly, all of these proteins carry three cysteine residues. Multiple alignments of their sequences revealed that the cysteine residues are located within two conserved motifs: C31 within V-G-P-K-V-C31 and C101 and C104 within C101-X-X-C104 (Fig. 1). A third conserved motif, D-H-E-E-M-H, was also observed.
FIG. 3. Circular dichroism spectra of wild-type and mutated SOR molecules. (A) BME did not exert a noticeable influence (2 mM BME was added to wild-type SOR, and the mixture was incubated at 4°C for 8 h). (B) Wild-type and mutant SOR molecules have identical CD spectra. Protein concentrations were adjusted to 100 g/ml, and the scanning wavelength range was 260 to 190 nm. mdeg, millidegree.
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FIG. 4. Fluorescence spectroscopy of wild-type and mutant SORs. Intrinsic tryptophan fluorescence emission spectrum analysis of wild-type SOR and its mutants was carried out as described in Materials and Methods. The excitation wavelength was set to 295 nm, and the range of emission wavelengths was set to 305 to 400 nm. Intensity is given in arbitrary units.
The secondary structure of the A. tengchongensis SOR was predicted by using the Antheprot version 5.2 software package and the PredictProtein server. It contained 26 to 29% ␣-helix and 21 to 23% -sheet. A putative extensive ␣-helix (positions 99 to 129), including a D-H-E-E-M-H box and a C101-X-XC104 motif, was identified. Hydrophobicity-hydrophilicity analysis revealed that the SOR was generally a hydrophilic molecule with one strongly hydrophilic segment and three moderately hydrophobic segments. The hydrophilic segment overlaps with the putative extensive ␣-helix, where the two conservative cysteine residues, C101 and C104, are located (positions 99 to 129). The C31 cysteine residue is located in a hydrophobic segment, and this region is largely -sheet or coil. Effects of various reagents on the catalytic properties of SOR. Different types of chemicals were tested for their effects on SOR activities (Table 2). All tested divalent metal ions strongly inhibited SOR activity, except magnesium, which showed moderate inhibition with a residual activity of 77.2%. The metal ions Cu2⫹, Co2⫹, Ni2⫹, and Mn2⫹ were strong inhibitors; the treated reaction mixture retained only 0.7 to 34.5% activity. Each of the reducing agents, GSH, DTT, and the nonspecific metal chelator EDTA, on the other hand, increased SOR activity slightly. Strikingly, the thio-binding reagent, NEM, showed very strong inhibition of SOR activity, and almost complete inhibition occurred at a concentration of 0.1 mM. This suggested that an SH group(s) of the enzyme
plays an important role in SOR catalysis. The SOR inhibitors NEM and Zn2⫹ must have interacted with the cysteine residues (C31, C101, and C104) to confer the strong inhibition of SOR activity. Site-directed mutagenesis of cysteines of SOR. There are three cysteine codons in the A. tengchongensis sor gene, and all of them were mutated as described in Materials and Methods, including individual replacement with a serine codon (C31S, C101S, and C104S), and once, a C31 codon replaced by an alanine codon (C31A). The mutations were then confirmed by DNA sequencing. After expression in E. coli, the SOR mutant proteins were purified and analyzed by SDS-PAGE and verified by Western blot analysis (Fig. 2B). Only a single band was observed in the wild-type and mutant SOR preparations, indicating the homogeneity of the recombinant proteins, and no degradation products of SOR were observed (Fig. 2A and C). Then, the mutant proteins were assayed for SOR oxygenase activity using the wild-type SOR as a control. Replacement of C101 or C104 by a serine resulted in 98.4 and 99.2% loss of activity, respectively, whereas replacement of C31 with either a serine or an alanine abolished SOR activity (Table 3). These results indicate that all the cysteine residues of SOR play a very important role in its activity. Structural analyses of SOR enzyme. To investigate whether cysteine replacements resulted in any structural changes, circular-dichroism spectra and SDS-PAGE were used to detect
TABLE 4. Distribution of SOR, SAOR, and TAOR activities in cytoplasm and membrane fractions of A. tengchongensis cells Activitya SOR
Site
Cellular lysates Supernatant (cytoplasmic fraction) Pellet (membrane fraction) a
SAOR
Total (mU)
Specific (mU/mg)
%
Total (U)
Specific (mU/mg)
12.7 9.94 4.5
70.4 107.8 476.0
100 78.3 35.2
ND 0.0 2.6
ND 0.0 482.8
TAOR %
0.0 100
Total (U)
Specific (mU/mg)
ND 0.7 12.7
ND 0.0 486.6
Percentage was calculated as follows: percentage ⫽ total activity of supernatant or pellet/total activity of cellular lysate ⫻ 100%. ND, not determined.
%
0.0 100
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FIG. 5. Cell morphology (A) and locations of SOR at subcellular level (B, C, and D). (A) SEM of A. tengchongensis. (B and C) TEM of immunogold-labeled A. tengchongensis. (D) TEM of recombinant E. coli that synthesized SOR. The thick and thin arrows indicate the cytoplasmic SOR and the cytoplasmic-membrane SOR, respectively. Preparation of SEM and TEM samples is described in Materials and Methods and reference 6.
the formation of any disulfide bond between SOR subunits. The distances of migrations of wild-type and mutant SORs during SDS-PAGE, in either the presence or the absence of -mercaptoethanol (BME) (Fig. 2A and C), were the same, indicating that no disulfide bonds were formed in SOR molecules. Furthermore, circular-dichroism (Fig. 3) and fluores-
cence (Fig. 4) spectra of the wild-type and mutant SOR molecules were very similar, and analyses of these spectra by using K2D and the CDPro software package did not reveal structural changes caused by site mutations of C31S, C101S, or C104S. Localization of SOR activity within A. tengchongensis cells. To investigate the cellular distribution of SOR activity in A.
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tengchongensis, crude cellular lysates of A. tengchongensis were fractionated (see Materials and Methods) to yield the cytoplasm and membrane fractions. Enzyme assays of the two fractions revealed that they both exhibited SOR activity. Whereas the cytoplasm fraction possessed 78.3% of the total activity, the cytoplasmic-membrane fraction exhibited 35.2% (Table 4). However, the specific activity in the membrane fraction was four times higher than that of the cytoplasm fraction (Table 4). This result could be interpreted in two ways: (i) the ratio of SOR to the total proteins in the membrane fraction was higher than that of the cytoplasm fraction, or (ii) the SORs associated with the membrane had higher specific activities. Besides SOR activity, the thiosulfate:acceptor oxidoreductase and sulfite:acceptor oxidoreductase activities were also found to be associated with the membrane in A. tengchongensis in our experiments (Table 4). Immune electron microscopy observations confirmed the subcellular distribution of SOR. Cells of A. tengchongensis appeared as irregular cocci or short rods under an electron microscope (Fig. 5A). To investigate the subcellular location of SOR, cells of A. tengchongensis buried in gels were sliced and treated with SOR-specific antibody and then labeled with colloidal-gold particles (6). The SOR locations were observed by transmission electron microscopy. The results clearly showed that the SORs were present in the cytoplasm and associated with the membrane (Fig. 5B). Closer examination revealed that SOR molecules were clustered on the membrane (Fig. 5C). Furthermore, the recombinant SOR molecules were similarly distributed in E. coli cells in which functional SORs were synthesized (Fig. 5D). DISCUSSION We have demonstrated, using site-directed mutagenesis, that all of the cysteine residues in the A. tengchongensis SOR, namely, C31, C101, and C104, are essential to its activity. These cysteine residues are located in two separately conserved domains, C31 at V-G-P-K-V-C31 and C101 and C104 at C101-X-XC104, both of which are conserved among the SORs identified in thermophilic archaea and bacteria, including A. ambivalens, A. brierleyi, A. tengchongensis, S. tokodaii, and A. aeolicus (Fig. 1). By investigating the catalytic properties of the A. tengchongensis wild-type and mutant SORs, we have shown that the C31 residue is more important than C101 or C104, since replacement of C31 by a serine or an alanine in the SOR resulted in complete loss of activity, whereas mutation of C101 or C104 retained 1.6 or 0.8% of the activity (Table 3). Bacterial sulfide:quinone oxidoreductase is another enzyme involved in sulfur metabolism (8, 22). It is a membrane-bound flavoprotein possessing three cysteines. Using site-directed mutagenesis, the enzyme activity has been shown to be completely or nearly completely abolished after mutation of each of the cysteines to serine (9, 21). However, since the sulfide:quinone oxidoreductase cysteines form disulfide bonds whereas the SOR cysteines do not, they must possess different catalytic mechanisms. There are several bacterial redox enzymes containing the C-X-X-C motif, and it has been demonstrated that the motif contributes to the redox activities of these enzymes (3, 5, 12, 22). For example, a single substitution of a cysteine residue in the C-X-X-C motif of protein disulfide isomerases resulted in a major loss of activity (4). Although protein disulfide isomer-
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ases and SOR are different types of enzymes, our results with SOR are in agreement with those obtained with bacterial redox enzymes. The possible functions of the SOR cysteines have been studied by structural analysis of the A. tengchongensis SOR. Predictions from secondary structure and hydrophobicity-hydrophilicity analyses indicated that C101 and C104 are located in a hydrophilic region with ␣-helix structure and that C31 is located in a hydrophobic region with -sheet structure. Taking the hydrophobic nature of the substrate (elemental sulfur [S8]) into account, we tentatively proposed that C31 possibly constitutes the substrate binding site and that C101 and C104, together with the previously identified ferric binding motif D-H-E-EM-H in the same ␣-helix segment (27), presumably constitute the catalytic site in SOR. Very recently, Urich et al. found that the A. ambivalens SOR resembles bacterial ferritins and proposed that the enzyme represents a new type of nonheme iron enzyme containing a mononuclear iron center coordinated by carboxylate and/or histidine ligands (27). Thus, these putative domains in SORs identified in this and other works (17, 27) provide a basis for continuing investigation of this largely unknown enzyme. The oxidation of elemental sulfur in A. tengchongensis proceeds in two steps. First, S0 is oxidized to sulfite and thiosulfate by SOR. Second, sulfite and thiosulfate are oxidized to sulfate by SAOR or TAOR. SOR had been purified from the cytoplasm fraction of A. ambivalens, and SAOR and TAOR are membrane-associated enzymes (reference 20 and this study). How the insoluble elemental sulfur crosses the barriers of the cell wall and cytoplasmic membrane is unknown. In this paper, we investigated the subcellular locations of three sulfur-metabolizing enzymes, SOR, TAOR, and SAOR, in the A. tengchongensis cell. Our finding that the activities of TAOR and SAOR and the partial activity of SOR were located together on the cytoplasmic membrane suggested that the functional coupling of the three activities possibly happens on the membrane. Thus, it will be intriguing to investigate whether SOR, TAOR, and SAOR interact physically with each other and whether the membrane-associated and cytoplasmic SOR enzyme has different physiological functions. ACKNOWLEDGMENTS This work was supported by grants from the National Nature Science Foundation of China and the Chinese Academy of Sciences. REFERENCES 1. Allen, M. B. 1959. Studies with Cyanidium caldarium, an anomalously pigmented chlorophyte. Arch. Mikrobiol. 32:270–277. 2. Andrade, M. A., P. Chaco ´n, J. J. Merelo, and F. Mora ´n. 1993. Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng. 6:383–390. 3. Bartlett, G. J., C. T. Porter, N. Borkakoti, and J. M. Thornton. 2002. Analysis of catalytic residues in enzyme active sites. J. Mol. Biol. 324:105– 121. 4. Chivers, P. T., M. C. A. Laboissoere, and R. T. Raines. 1996. The CXXC motif: imperatives for the formation of native disulfide bonds in the cell. EMBO J. 15:2659–2667. 5. Conway, M. E., N. Yennawar, R. Wallin, L. B. Poole, and S. M. Hutson. 2003. Human mitochondrial branched chain aminotransferase: structural basis for substrate specificity and role of redox active cysteines. Biochem. Biophys. Acta 1647:61–65. 6. Dorward, D. W., T. G. Schwan, and C. F. Garon. 1991. Immune capture and detection of Borrelia burgdorferi antigens in urine, blood, or tissues from infected ticks, mice, dogs, and humans. J. Clin. Microbiol. 29:1162–1170. 7. Emmel, T., W. Sand, W. A. Koenig, and E. Bock. 1986. Evidence for the
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8.
9.
10.
11. 12.
13. 14.
15. 16.
17.
CHEN ET AL. existence of a sulfur oxygenase in Sulfolobus brierleyi. J. Gen. Microbiol. 132:3415–3420. Friedriech, C. G., D. Rother, F. Bardischewsky, A. Quentmeier, and J. Fischer. 2001. Oxidation of reduced inorganic sulfur compounds by bacteria: emergence of a common mechanism? Appl. Environ. Microbiol. 67:2873– 2882. Griesbeck, C., M. Schutz, T. Schodl, S. Bathe, L. Nausch, N. Mederer, M. Vielreicher, and G. Hauska. 2002. Mechanism of sulfide-quinone reductase investigated using site-directed mutagenesis and sulfur analysis. Biochemistry 41:11552–11565. He, Z., Y. Li, P. Zhou, and S.-J. Liu. 2000. Cloning and heterologous expression of a sulfur oxygenase/reductase gene from the thermoacidophilic archaeon Acidianus sp. S5 in Escherichia coli. FEMS Microbiol. Lett. 193: 217–221. He, Z.-G., H. Zhong, and Y. Li. 2004. Acidianus tengchongensis sp. nov., a new species of acidothermophilic archaeon isolated from an acidothermal spring. Curr. Microbiol. 48:159–163. Heinzinger, N. K., S. Y. Fujimoto, M. A. Clark, M. S. Moreno, and E. L. Barrett. 1995. Sequence analysis of the phs operon in Salmonella typhimurium and the contribution of thiosulfate reduction to anaerobic energy metabolism. J. Bacteriol. 177:2813–2820. Kelly, D. P., and A. P. Wood. 1996. Enzymes involved in microbiological oxidation of thiosulfate and polythionates. Methods Enzymol. 243:501–510. Kletzin, A. 1989. Coupled enzymatic production of sulfite, thiosulfate, and hydrogen sulfide from sulfur: purification and properties of a sulfur oxygenase reductase from the facultatively anaerobic archaebacterium Desulfurolobus ambivalens. J. Bacteriol. 171:1638–1643. Kletzin, A. 1992. Molecular characterization of the sor gene, which encodes the sulfur oxygenase/reductase of the thermoacidophilic Archaeum Desulfurolobus ambivalens. J. Bacteriol. 174:5854–5859. Kletzin, A. 1994. Sulfur oxidation and reduction in Archaea: sulfur oxygenase/reductase and hydrogenases from the extremely thermophilic and facultatively anaerobic archaeon Desulfurolobus ambivalens. Syst. Appl. Microbiol. 16:534–543. Kletzin, A., T. Urich., F. Muller, T. M. Bandeiras, and C. M. Gomes. 2004. Dissimilatory oxidation and reduction of sulfur in thermophilic archaea. J. Bioenerg. Biomembr. 36:77–91.
APPL. ENVIRON. MICROBIOL. 18. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. 19. Liu, S.-J, H. Iwahashi, and Y. Komatsu. 1998. Novel post-translational modification system of NifF protein exists in Klebsiella pneumoniae which is different from that in Escherichia coli. Biotechnol. Lett. 20:963–967. 20. Mu ¨ller, F. H., T. M. Bandeiras, T. Urich, M. Teixeira, C. M. Gomes, and A. Kletzin. 2004. Coupling of the pathway of sulphur oxidation dioxygen reduction: characterization of novel membrane-bound thiosulphate:quinone oxidoreductase. Mol. Microbiol. 53:1147–1160 21. Rosato, V., N. Pucello, and G. Giuliano. 2002. Evidence for cysteine clustering in thermophilic proteomes. Trends Genet. 18:278–281. 22. Rother, D., H.-J. Henrich, A. Quentmeier, F. Bardischewsky, and C. G. Friedrich. 2001. Novel genes of the sox gene cluster, mutagenesis of the flavoprotein SoxF, and evidence for a general sulfur-oxidizing system in Paracoccus pantotrophus GB17. J. Bacteriol. 183:4499–4508. 23. Sambrook, J., and D. W. Russell. 2001. Molecular cloning, 3rd ed., vol 2, p. 13.19–13.25. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 24. Sreerama, N., S. Y. Venyaminov, and R. W. Woody. 2000. Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal. Biochem. 282:252–260. 25. Sreerama, N., S. Y. Venyaminov, and R. W. Woody. 2001. Analysis of protein circular dichroism spectra based on the tertiary structure classification. Anal. Biochem. 299:271–274. 26. Sun, C.-W., Z. Chen, Z.-G. He, P.-J. Zhou, and S.-J. Liu. 2003. Purification and properties of the sulfur oxygenase/reductase from the acidothermophilic archaeon, Acidianus strain S5. Extremophiles 7:131–134. 27. Urich, T., T. M. Banerras, S. S. Leal, M. Teixeira, C. M. Gomes, and A. Kletzin. 2004. The sulphur oxygenase reductase from Acidianus ambivalens is a multimeric protein containing a low-potential mononuclear non-haem iron center. Biochem J. 381:137–146. 28. Zimmermann, P., S. Laska, and A. Kletzin. 1999. Two modes of sulfite oxidation in the extremely thermophilic and acidophilic archaeon Acidianus ambivalens. Arch. Microbiol. 172:76–82.
