Characterization of a Two-component Alkanesulfonate Monooxygenase from Escherichia coli*

The Escherichia coli ssuEADCB gene cluster is required for the utilization of alkanesulfonates as sulfur sources, and is expressed under conditions of sulfate or cysteine starvation. The SsuD and SsuE proteins were overexpressed and characterized. SsuE was purified to homogeneity as an N-terminal histidine-tagged fusion protein. Native SsuE was a homodimeric enzyme of M r 58,400, which catalyzed an NAD(P)H-dependent reduction of FMN, but it was also able to reduce FAD or riboflavin. The SsuD protein was purified to >98% purity using cation exchange, anion exchange, and hydrophobic interaction chromatography. The pure enzyme catalyzed the conversion of pentanesulfonic acid to sulfite and pentaldehyde and was able to desulfonate a wide range of sulfonated substrates including C-2 to C-10 unsubstituted linear alkanesulfonates, substituted ethanesulfonic acids and sulfonated buffers. SsuD catalysis was absolutely dependent on FMNH2 and oxygen, and was maximal for SsuE/SsuD molar ratios of 2.1 to 4.2 in 10 mm Tris-HCl, pH 9.1. Native SsuD was a homotetrameric enzyme of M r 181,000. These results demonstrate that SsuD is a broad range FMNH2-dependent monooxygenase catalyzing the oxygenolytic conversion of alkanesulfonates to sulfite and the corresponding aldehydes. SsuE is the FMN reducing enzyme providing SsuD with FMNH2.

In Escherichia coli, sulfate starvation causes increased synthesis of several proteins involved in scavenging sulfur from alternative sulfur sources (1). Among these proteins are the tauABCD-encoded proteins required for uptake and desulfonation of taurine (2-aminoethanesulfonic acid) (2,3) and the proteins SsuE and SsuD of the ssuEADCB gene cluster. We have shown that the ssuEADCB gene cluster, located at 21.4 min on the E. coli chromosome, is specifically involved in the utilization of alkanesulfonates as a source of sulfur for growth (4). Deletion of ssuEADCB resulted in the loss of the ability to utilize alkanesulfonates as a sulfur source but did not affect the utilization of taurine for this purpose. The amino acid sequences of SsuABC exhibit similarity to components of ABCtype transport systems (4,5). SsuA has a putative signal sequence, indicating that it functions as a periplasmic binding protein, and the sequences of SsuB and SsuC are significantly similar to those of ATP-binding proteins and membrane com-ponents, respectively, of members of the ABC transporter superfamily. It thus appears that the proteins encoded by ssuABC constitute an uptake system for alkanesulfonates.
The ssuD gene product shows 25% sequence identity to a characterized nitrilotriacetate two-component monooxygenase of Chelatobacter heintzii (6) and to the pristinamycin II A synthase subunit A of Streptomyces pristinaespiralis (7), suggesting that SsuD is involved in the oxygenolytic release of sulfite from alkanesulfonates. Here we report the purification of the SsuD and SsuE proteins, describe their biochemical properties, and demonstrate that SsuD is a monooxygenase that catalyzes the desulfonation of alkanesulfonates and requires reduced FMN, which is provided by the NAD(P)H:flavin oxidoreductase SsuE.
Construction of ssuE Expression Plasmids-For the production of wild type SsuE, the ssuE gene was placed under the control of the T7 RNA polymerase promoter of vector pET-24a(ϩ) (Novagen). The ssuE gene was amplified by PCR 1 from plasmid pME4180 (4) with the oligonucleotide primers EE7 (5Ј-AAGGAGAGCATATGCGTGTCAT-3Ј), and EE4 (5Ј-CTATACGTAAAGCTTCAGGCGAG-3Ј), with the changes to introduce NdeI and HindIII restriction sites, respectively, underlined. The 672-base pair PCR product was digested with NdeI and HindIII and the resulting 655-base pair fragment was ligated in pUC19 (10), generating plasmid pME4145. The NdeI-HindIII fragment from pME4145 was subsequently cloned in pET-24a(ϩ) resulting in plasmid pME4146.
For the production of SsuE as an N-terminal histidine-tagged fusion protein, the NdeI-HindIII SsuE-encoding fragment from plasmid pME4145 was ligated into NdeI-HindIII digested pET-28a(ϩ) (Novagen) leading to plasmid pME4287. The ssuE sequence of plasmid pME4287 was sequenced to confirm that no changes had been introduced during PCR amplification.
Construction of a ssuD Expression Plasmid-The complete ssuD gene was PCR-amplified with TaqPlus DNA polymerase from genomic DNA prepared from E. coli EC1250 (11) as described elsewhere (12). The oligonucleotide primers used were EE8 (5Ј-GGAAAACACATATGAGT-CTGA-3Ј) and EE10 (5Ј-ATGCTGCCAAGCTTCGCCGCTG-3Ј) with the changes to introduce, respectively, the NdeI and HindIII restriction * This work was supported by a grant of the Swiss Federal Institute of Technology, Zü rich, Switzerland. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the Swiss Protein Database under Swiss-Prot accession no. P80644.
Protein Production-For the production of SsuE and SsuD, E. coli BL21(DE3) containing the appropriate overexpression plasmid was grown at 30°C and 180 rpm in a 5-liter Erlenmeyer flask containing 1000 ml of growth medium. To minimize the formation of insoluble protein aggregates, which were observed when protein production was carried out at 30°C, cultures grown to an A 650 of 0.5 were cooled to 16°C, induced by the addition of isopropyl-␤-D-1-thiogalactopyranoside to a final concentration of 50 M and incubated for a further 5 h at 16°C with constant shaking (180 rpm). Cells were collected by centrifugation for 20 min at 5800 ϫ g and 4°C, washed in an excess of 20 mM Tris-HCl buffer, pH 8.0, and stored at Ϫ20°C as frozen pellets until further use. About 4.5 g fresh weight of cells were collected from a 1000-ml culture.
Purification of SsuE-For the production of crude extracts containing the histidine-tagged SsuE fusion protein, 1.8 g of induced E. coli BL21(DE3)(pME4287) cells were resuspended in 4 ml of binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) supplemented with 25 g/ml DNase I and disrupted by three passages through a French Pressure cell at 5.5 megapascals. After clarification by ultracentrifugation for 30 min at 110,000 ϫ g and 4°C, the volume of the crude extract was adjusted to 10 ml with binding buffer and supplemented with 10 l of Nonidet P-40 (Fluka). The crude extract was then loaded at a flow rate of 25 ml/h onto a 2.5-ml HisBind Resin column (Novagen), which had been activated with NiSO 4 and equilibrated with binding buffer as described by the manufacturer. The sample was washed with 10 column volumes of binding buffer, followed by washing with 6 column volumes of wash buffer (60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). An additional wash step with a buffer containing 100 mM imidazole was performed for 6 column volumes prior to SsuE elution with 200 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, 20% glycerol, pH 7.9. The protein solution was stored at Ϫ20°C until further use.
Purification of SsuD-For the production of crude extracts containing SsuD, 2.5 g of induced E. coli BL21(DE3)(pME4282) cells were resuspended in 4 ml of 20 mM sodium phosphate, pH 6.0, supplemented with 25 g/ml DNase I and disrupted by three passages through a French Pressure cell at 5.5 megapascals. After clarification by ultracentrifugation (40 min, 110,000 ϫ g, 4°C), the SsuD enzyme was purified on a BioCAD apparatus using 20 mM sodium phosphate, pH 6.0, and 20 mM sodium phosphate, pH 6.0, containing either NaCl or (NH 4 ) 2 SO 4 at the concentrations indicated below. The crude extract was first loaded at a flow rate of 5 ml/min on a 3.5-ml Fractogel EMD SO 3 Ϫ (S) cation exchange column (Merck) equilibrated with 20 mM sodium phosphate, pH 6.0. The sample was washed for 2 column volumes with 70 mM NaCl and SsuD was eluted with 160 mM NaCl. The SsuD fractions were pooled, diluted twice with 20 mM sodium phosphate, pH 6.0, and loaded at a flow rate of 4 ml/min on a 4.5-ml Fractogel EMD TMAE (S) anion exchange column (Merck) equilibrated with the same buffer. After a wash step with 20 mM sodium phosphate, pH 6.0, for 2 column volumes followed by washing with 90 mM NaCl (6 column volumes), SsuD was eluted with 150 mM NaCl. In a third step, the pooled SsuD fractions collected from the anion exchange column were diluted twice with 1 M (NH 4 ) 2 SO 4 and loaded onto a 7.5-ml phenyl-Sepharose Fast Flow 66 hydrophobic interaction column (Amersham Pharmacia Biotech) at a flow rate of 5 ml/min and washed with 2 column volumes of 500 mM (NH 4 ) 2 SO 4 . Proteins were eluted with a linear gradient from 500 to 0 mM (NH 4 ) 2 SO 4 in 5 column volumes, followed by washing with 20 mM sodium phosphate, pH 6.0. SsuD eluted between 100 and 0 mM (NH 4 ) 2 SO 4 . NAD(P)H:FMN Oxidoreductase Assays-Appropriate amounts of SsuE were incubated at 30°C in a reaction mixture (1 ml final volume) containing either 500 M NADPH and 3 M FMN, or 1 mM NADH and 0.5 M FMN in 20 mM imidazole, 50 mM NaCl, 20 mM Tris-HCl, pH 7.9. Reactions were started by addition of enzyme to the reaction mixture; activity was followed online with a Uvikon 900 spectrophotometer. An assay mixture without FMN was used as a blank. One unit was defined as the amount of enzyme catalyzing the oxidation of 1 mol of NAD(P)H/min at 30°C under aerobic conditions, calculated from the slope of the absorbance decrease at 340 nm using ⑀ 340 ϭ 6.2 mM Ϫ1 cm Ϫ1 for NAD(P)H.
When kinetic parameters for FMN and FAD were investigated, reductase activity was followed spectrophotometrically under anaerobic conditions in a reaction mixture containing 1 mM NADH, varying FMN or FAD concentrations and appropriate amounts of SsuE in 20 mM imidazole, 50 mM NaCl, 20 mM Tris-HCl, pH 7.9. All reagents and buffers were therefore made anaerobic by at least 10 cycles of alternate flushing with 95% N 2 , 5% H 2 and vacuum evacuating. Reaction mixtures were prepared in an anaerobic chamber and reactions were started by enzyme addition. An assay in which NADH was omitted was used as a blank. One unit was defined as the amount of enzyme catalyzing the reduction of 1 mol of flavin substrate per minute at 30°C under anaerobic conditions. Activity was calculated from the slope of the absorbance decrease at 375 nm using ⑀ 375 ϭ 9.3 mM Ϫ1 cm Ϫ1 for FAD and ⑀ 375 ϭ 10.4 mM Ϫ1 cm Ϫ1 for FMN. The obtained values were corrected for residual NADH absorption at 375 nm using ⑀ 375 ϭ 2.0 mM Ϫ1 cm Ϫ1 for NADH.
Alkanesulfonate Monooxygenase Assay-SsuD activity was routinely assayed with Ellman's reagent (5,5Ј-dithiobis(2-nitrobenzoic acid)) which produces a bright yellow color upon reaction with sulfite (13,14). The assay mixture (1.5 ml final volume) contained 500 M NADPH, 3 M FMN, 500 M sulfonated substrate, SsuE and SsuD to a SsuE/SsuD molar ratio of 3.0, in 10 mM Tris-HCl, pH 9.1, and was stirred on a magnetic stirrer at 30°C. Reactions were started by addition of SsuD to the reaction mixture and stopped by the addition of 200-l samples to a spectrophotometric cuvette containing 100 l of 5,5Ј-dithiobis(2-nitrobenzoic acid) (1 mg/ml in 100 mM sodium phosphate buffer, pH 7.0) to which 700 l of deionized water was finally added. The colorimetric reaction was allowed to develop at room temperature for 2 min. Sulfite determination with Ellman's reagent was linear up to 250 M sulfite. One unit of activity was defined as the amount of enzyme forming 1 mol of sulfite per minute at 30°C under standard assay conditions, calculated from the slope of absorbance increase at 430 nm with reference to sulfite calibration curves. These were prepared daily from fresh solutions of sodium sulfite in deionized water. When assayed under anaerobic conditions, all reagents were made anaerobic by at least 10 cycles of alternate flushing with 95% N 2 , 5% H 2 and vacuum pulling, reaction mixtures were prepared in an anaerobic chamber and sulfite detection was carried out as described above, but using anaerobized 5,5Ј-dithiobis(2-nitrobenzoic acid).
Analysis of Enzyme Reaction Products-The presence of pentaldehyde in reaction mixtures containing pentanesulfonate as substrate for SsuD was detected by gas chromatography analysis using co-chromatography with authentic pentaldehyde (Fluka). A Perkin-Elmer 8700 gas chromatograph equipped with a flame ionization detector was used. 10-l samples of reaction mixture were injected directly and chromatographed on a 180 ϫ 0.2-cm Porapak P (80/100-mesh) column (Supelco) run with nitrogen at 175°C. The flow rate was 30 ml/min and the detector temperature was 250°C.
SDS-Polyacrylamide Gel Electrophoresis-SDS-PAGE was performed on a Mini-PROTEAN II system (Bio-Rad) with 12% polyacrylamide gels under denaturing conditions (8). Protein concentrations were measured using the method of Bradford (15) with Bio-Rad reagent dye concentrate, following the manufacturer's instructions. Bovine serum albumin was used as a standard.

Purification of NAD(P)H:FMN Oxidoreductase-
The wild type SsuE enzyme produced from the overexpression plasmid pME4146 lost its NAD(P)H-dependent FMN reduction activity within 2-3 h in crude extracts, when kept on ice. When partially purified by ammonium sulfate fractionation (30 -40% cut) and stored at Ϫ20°C with 20% glycerol, activity could be maintained for a few days. However, cation or anion exchange chromatography on Fractogel EMD SO 3 Ϫ (S) and EMD TMAE (S) (Merck) or SP-and Q-Sepharose (Amersham Pharmacia Biotech), respectively, or dye-ligand affinity chromatography on Green A-agarose (Millipore AG) led to significant protein inactivation. Affinity chromatography with FMN-agarose (Sig-ma) did not lead to any purification of SsuE. Purification of SsuE as an N-terminal histidine-tagged fusion protein expressed from plasmid pME4287 turned out to be an efficient alternative to conventional chromatography techniques for obtaining pure, active and stable enzyme. The SsuE enzyme was purified in one step with 40% yield (Fig. 1A) by metal chelate affinity chromatography. The purified histidine-tagged SsuE had a specific activity of 32.4 units/mg (NADPH oxidation) and was stable for several months when stored at Ϫ20°C in elution buffer.
Characterization of NAD(P)H:FMN Oxidoreductase-Densitometric scanning of lanes 2 and 3 of the SDS-PAGE gel (Fig.  1A) showed that wild type SsuE represented around 18%, whereas the histidine-tagged SsuE amounted to approximately 13% of the total soluble protein in crude extracts. The relative specific activities calculated as NAD(P)H oxidation rates of both extracts were identical, indicating that the histidine tag had no influence on SsuE activity. Enzyme characterization was therefore done with the histidine-tagged SsuE fusion protein.
The effect of pH on NAD(P)H:FMN oxidoreductase activity was investigated over a range from 4.5 to 10.8 using appropriate buffer systems (16). SsuE activity was maximal in 20 mM Tris-HCl buffer, pH 7.9, supplemented with 20 mM imidazole and 50 mM NaCl. In all other buffer systems used, including acetate, bis-Tris, and glycine-NaOH, SsuE specific activity was less than 5% of the maximal activity. This was also the case when the enzyme was dialyzed or subjected to buffer exchange using PD10 gel filtration columns (Amersham Pharmacia Biotech). It is thus likely that a cofactor essential for activity was lost upon dialysis or gel filtration. The pure SsuE protein showed no typical absorption spectrum of flavin-containing flavoproteins, suggesting that the enzyme does not contain any bound flavin cofactor. Gel filtration chromatography on Superdex 75 was used to estimate a M r of 58,400 Ϯ 100 for the native enzyme. The subunit molecular mass calculated from the histidine-tagged SsuE amino acid sequence of 23.7 kDa was estimated by SDS-PAGE analysis as 25.4 Ϯ 0.7 kDa. These results therefore suggest a homodimeric structure for the SsuE enzyme.
Substrate Range and Kinetic Constants of NAD(P)H:FMN Oxidoreductase-The SsuE enzyme showed a Michaelis-Menten type saturation curve in response to increasing substrate concentrations. NADPH was the best pyrimidinic substrate for SsuE, which was also able to oxidize NADH (Table I). The preference for NADPH was also found in crude extracts of both wild type and histidine-tagged SsuE proteins.
FMN was the preferred flavin substrate of SsuE, but FAD and riboflavin were also reduced at significant rates, whereas lumiflavin was not (Fig. 2). When NADH was the pyrimidinic substrate, a distinct activity maximum was obtained at an FMN concentration of 0.5 M, whereas concentrations higher than 2.5 M led to more than 60% decrease in specific activity. On the contrary, the SsuE specific activity increased with increasing FAD concentrations and reached saturation at 10 to 25 M FAD. When NADPH was supplied as pyrimidinic substrate, maximal reductase activity was obtained with 2.5-10 M FMN, while higher FMN concentration led to 15% decrease in SsuE activity. These results showed the necessity of performing the determination of kinetic constants for NADH in reaction mixtures containing only 0.5 M FMN, whereas 3 M FMN was used when the kinetic constants for NADPH were investigated. When affinities for FMN or FAD were explored, measurements were done under anaerobic conditions using NADH because a strong abiotic FMN reduction was observed when NADPH was given as pyrimidinic substrate. Table I   Purification of SsuD-The SsuD protein initially produced from an overexpression construct in which the ssuD gene originating from plasmid pME4180 (4) was under the control of the T7 RNA polymerase promoter, was not active. Sequencing of the ssuD gene of plasmid pME4180 revealed a single point mutation leading to an arginine to cysteine exchange at position 298 of the SsuD sequence, which was responsible for the complete loss of activity of the enzyme. The SsuD protein was therefore produced from the overexpression plasmid pME4282, which contains the ssuD gene sequence amplified from E. coli EC1250 genomic DNA. Using the three-step procedure summarized in Table II, the ssuD gene product was purified to near homogeneity with a yield of 35% from E. coli BL21(DE3)-(pME4282) as shown in Fig. 1B. The band corresponding to a 32-kDa protein which co-purified with SsuD and which represented less than 2% of the phenyl-Sepharose purified protein preparation could not be removed by any other chromatography technique tested. After an additional purification step on hydroxyapatite, the SsuD protein amounted to only 90% of total protein, and a net increase in intensity of the 32-kDa protein band was observed at the expense of SsuD. After storage for 24 h at Ϫ20°C of the hydroxyapatite-purified protein, SsuD was only 70% of the total protein, the remaining 30% being represented by the 32-kDa protein. We therefore presume that this 32-kDa protein is a degradation product of the native SsuD protein. For this reason, we chose to perform SsuD characterization with the enzyme preparation obtained after phenyl-Sepharose chromatography, in which no further degradation of SsuD was observed. Densitometric scanning indicated that SsuD was over 98% pure (Fig. 1B, lane 5).
Characterization of SsuD-The purified enzyme was stable upon storage at Ϫ20°C in buffer with 15% glycerol and it had a specific activity of 2.5 units/mg. The activity increased slightly during the first 2 to 3 weeks of storage.
The effect of the pH on enzyme activity was examined over a range from 4.5 to 10.8 using appropriate buffer systems (16). SsuD showed a distinct activity maximum in 10 mM Tris-HCl, pH 9.1.
When SsuD activity was assayed in crude extracts of E. coli BL21(DE3)(pME4282) cells that overproduced SsuD, desulfonation was observed even without addition of SsuE, but it was dependent on NAD(P)H and enhanced by addition of FMN. On the contrary, alkanesulfonate desulfonation by pure SsuD was absolutely dependent on the presence of SsuE, NAD(P)H, and FMN. Maximal activity was obtained with 3 M FMN and 500 M NADPH and was not inhibited by higher NADPH concentrations. Since the ssuE gene product is present at very low concentration in cells grown under sulfate replete conditions, these results suggest that in crude extracts SsuE activity was provided by other NAD(P)H-dependent flavin reductases.
When SsuD was assayed under anaerobic conditions, no sulfite production could be detected. However, when anaerobic reaction mixtures were exposed to air and stirred, sulfite production was observed, showing that alkanesulfonate desulfonation by SsuD was absolutely dependent on oxygen.
Gel filtration chromatography on Superose 6 and Superose 12 H/R was used to estimate a M r of 181,000 Ϯ 11,000 for the native enzyme. The calculated subunit molecular mass from the ssuD gene sequence of 41.73 kDa was estimated by SDS-PAGE analysis as 41.2 Ϯ 0.7 kDa. The results therefore suggest a homotetrameric structure for the SsuD enzyme.
Optimization of the Reconstitution of Alkanesulfonate Monooxygenase Activity-Since the activity of pure SsuD was dependent on the FMN reducing activity of SsuE, the influence of the concentration of SsuE on alkanesulfonate desulfonation at a constant concentration of SsuD was investigated. An increase in SsuD activity with increasing SsuE concentration was observed (Fig. 3). Maximal activity was obtained for SsuE/SsuD  molar ratios between 2.1 and 4.2, whereas for higher ratios up to 6.3, SsuD activity decreased to around 2/3 of the activity reached at saturation. Therefore, the characterization of SsuD activity was performed at a SsuE/SsuD molar ratio of 3.0. Maximal desulfonation activity was obtained only when the reaction mixtures were vigorously stirred on a magnetic stirrer. Under these conditions, the enzyme showed a Michaelis-Menten type saturation curve in response to increasing alkanesulfonate concentrations when activity was assayed at 30°C in reaction mixtures containing 3 M FMN, 500 M NADPH, SsuE and SsuD to a molar ratio of 3.0, in 10 mM Tris-HCl, pH 9.1.
Substrate Range and Kinetic Constants-Among 26 potential sulfonated substrates tested, activity was observed neither with taurine, methanesulfonic acid, L-cysteic acid, and ethanedisulfonic acid, nor with the aromatic sulfonates toluene-4-sulfonic acid, p-sulfobenzoic acid, benzenesulfonic acid, and 4-hydroxybenzenesulfonic acid. SsuD was able to desulfonate C-2 to C-10 unsubstituted alkanesulfonates, substituted ethanesulfonic acids as well as N-phenyltaurine, 4-phenyl-1butanesulfonic acid, and the sulfonated buffers tested (Table  III). Based on catalytic efficiencies, the best substrates for SsuD were decanesulfonic acid, octanesulfonic acid, and 1,3dioxo-2-isoindolineethanesulfonic acid. Significant activity was supported by the compounds listed in Table IV, which shows that SsuD is a broad substrate range alkanesulfonate monooxygenase. Among unsubstituted 1-alkanesulfonates, the catalytic efficiency increased with increasing chain length up to decanesulfonic acid. V max values with regard to 1-alkanesulfonates were quite low compared with those obtained with 1,3-dioxo-2-isoindolineethanesulfonic acid, 2-(4-pyridyl)ethanesulfonic acid, and N-phenyltaurine. The affinity of SsuD for FMNH 2 as reduced flavin substrate was strict since no desulfonation activity was observed when FAD was added at various concentrations to the reaction mixtures as flavin substrate for SsuE.
Product of Alkanesulfonate Desulfonation by SsuD-Reaction mixtures containing pentanesulfonic acid as sulfonated substrate for SsuD were analyzed with gas chromatography as described under "Experimental Procedures." A distinct peak was obtained, which had exactly the same retention time as the peak observed when authentic pentaldehyde was added to the reaction mixture. This peak was absent when reaction mixtures were analyzed from which pentanesulfonic acid had been omitted. Furthermore, the amount of pentaldehyde increased with increasing incubation time, concomitantly with the amount of sulfite produced. Accordingly, we postulate that SsuD is an FMNH 2 -dependent monooxygenase catalyzing the conversion of alkanesulfonates to aldehyde and sulfite according to the overall reaction shown in Fig. 4.

DISCUSSION
Several reports on the utilization of primary aliphatic sulfonates by bacteria as carbon or sulfur sources have appeared (2,(17)(18)(19). Apart from the previously characterized ␣-ketoglutarate-dependent taurine dioxygenase TauD from E. coli (3), the enzymology of the desulfonation of these compounds remains largely unexplored. Crude extracts of 1-octanesulfonate-grown cells of a Pseudomonas strain were reported to desulfonate 1-alkanesulfonates by monooxygenation, giving a putative 1-hydroxyalkane sulfonate which spontaneously rearranged to give the corresponding aldehyde and sulfite (18). An inducible, NADH-specific methanesulfonic acid monooxygenase was detected in a methylotrophic bacterium growing with methanesulfonic acid as a sole source of carbon and energy. This threecomponent oxygenase was partially purified and shown to FIG. 4. Overall reaction catalyzed by the SsuD/SsuE two-component alkanesulfonate monooxygenase system from E. coli.

TABLE III
Substrate ranges of alkanesulfonate monooxygenase SsuD and ␣-KG-dependent taurine dioxygenase TauD SsuD desulfonation activity was measured in 10 mM Tris-HCl, pH 9.1, containing 500 M sulfonated substrate, 500 M NADPH, 3 M FMN, with SsuE to SsuD at a molar ratio of 3.0. Activity measurements with TauD were performed in 10 mM imidazole, pH 6.9, containing 500 M sulfonated substrate, 600 M ␣-KG, 100 M Fe(II)SO 4 , 200 M sodium ascorbate. Activity was calculated as sulfite released after incubation for 3 min at 30°C. A relative specific activity of 100 corresponds to 4.1 units/mg with both enzymes and with regard to their preferred substrate.  desulfonate unsubstituted C-1 to C-3 1-alkanesulfonates but not substituted short-chain 1-alkanesulfonates such as isethionic acid, taurine, and L-cysteic acid (19). The SsuDE two-component alkanesulfonate monooxygenase characterized in this study is, to our knowledge, the first purified enzymatic system reported that is capable of oxygenolytic cleavage of the C-S bond of 1-alkanesulfonates by monooxygenation. In contrast to the monooxygenase systems for alkanesulfonate desulfonation mentioned above, it does not play a role in carbon metabolism, and its synthesis is regulated by the sulfur supply to the cell (4). Analogous systems involved in the utilization of sulfur from aliphatic sulfonates in Bacillus subtilis (20) and from methanesulfonic acid in Pseudomonas aeruginosa (21) were recently identified in our laboratory, but are not characterized as purified enzymatic systems.
Based on their biochemical properties, the SsuD and SsuE proteins represent a two-component system that can be assigned to the group of FMNH 2 -dependent monooxygenases. Besides luciferase, the most extensively studied flavin-monooxygenase, several two-component monooxygenase systems belonging to this group have already been studied. These are involved in nitrilotriacetate hydroxylation (6), EDTA oxidation (22), pristinamycin II A biosynthesis (7), and dibenzothiophene desulfurization (23). These two-component systems consist of an oxidoreductase providing FMNH 2 for the oxygenation reaction catalyzed by the second component.
The SsuE enzyme is different from the flavin reductases characterized in the systems mentioned above. SsuE is an NAD(P)H:FMN oxidoreductase whose preferred pyrimidinic substrate is NADPH, for which it showed a 12 times higher affinity and a 25 times higher turnover rate than for NADH. Furthermore, the SsuE enzyme does not seem to be a flavopro-tein since the visible spectrum of the purified enzyme did not show absorption at wavelengths typical for flavoproteins. These results set the enzyme clearly apart from the characterized NADH:FMN oxidoreductases of the nitrilotriacetate and EDTA monooxygenases as well as from the two-enzyme system involved in pristinamycin II A synthesis. Their substrate specificity for NADH was strict, and they were shown to be flavoproteins. At the amino acid sequence level, no significant homology of the SsuE protein was found with either of these flavoproteins, or to the Fre-type enzymes, which are flavin reductases that do not contain any light-absorbing prosthetic group and utilize flavin only as a substrate and not a coenzyme (24). However, SsuE showed amino acid sequence identity to an iron-sulfur flavoprotein of Methanosarcina thermophila (25). A sequence alignment (Fig. 5) shows a small conserved motif at the N terminus and over 40% identity within a 45-amino acid internal region. This conserved domain could be involved in flavin binding. The archaeal flavoproteins contain a 4 cysteine motif forming an iron-sulfur cluster. Since this motif is not present in SsuE, electron transfer from the pyrimidinic substrate to the flavin substrate in the SsuE-catalyzed reaction does not occur via an iron-sulfur cluster, but perhaps, as has been shown for the Fre protein, directly from NAD(P)H to FMN by an ordered mechanism. In this case NADPH binds first to the active site, followed by the flavin. After electron transfer, the first product released is the reduced flavin, followed by NADP ϩ (24).
The homotetrameric structure for SsuD is rather uncommon among flavin-dependent monooxygenases. Bacterial luciferase and pristinamycin II A synthase were shown to be ␣␤ heterodimers of M r 79,000 and 85,000, respectively (7), whereas component A of the nitrilotriacetate monooxygenase was shown to be a homodimeric enzyme of M r 99,000 (6). The only other tetrameric flavin-dependent monooxygenase reported as homotetrameric enzyme is component A of the EDTA oxidizing system of bacterial strain DSM 9103 with a M r of 210,000 (22). Whatever their quaternary structures, around 25% amino acid identity was found between the pristinamycin II A synthase subunit A (SnaA), component A of the nitrilotriacetate monooxygenase (NtaA), the dibenzothiophene desulfurization enzyme SoxA (DszA) (26), and SsuD (20) (not shown). The sequence of SsuD was also similar to those of LuxA and LuxB subunits, which are components of heterodimeric FMNH 2 -dependent bacterial luciferases (27), and to the product of the mer gene of Methanobacterium thermoautotrophicum, which encodes coenzyme F 420 -dependent N 5 ,N 10 -methylenetetrahydromethanopterin reductase (28). Although there were only few residues completely conserved in these sequences (Fig. 6), calculation of the secondary structure elements from SsuD and from Mer indicated that these proteins might fold similarly as bacterial luciferase, whose three-dimensional structure has been determined (27). Arginine 298, which is conserved in SnaA, NtaA, and SoxA but not in LuxA, LuxB, or Mer, was essential for SsuD catalysis.
Comparison of the substrate range of the previously characterized ␣-ketoglutarate-dependent taurine dioxygenase TauD with the substrate range of alkanesulfonate monooxygenase SsuD shows that TauD is specific for taurine, whereas SsuD, whose preferred substrate was decanesulfonic acid, desulfonated nearly all substrates tested with the exception of taurine, methanesulfonic acid, aromatic sulfonates, and L-cysteic acid (Table III). Thus, SsuD and TauD are complementary. Taken together, the substrate ranges of the two alkanesulfonate desulfonation systems are in accordance with the range of alkanesulfonates, that are used as sulfur sources by E. coli (4). However, they do not explain the reported ability of E. coli to utilize  (TrEMBL accession number Q50744), and LuxB and LuxA from Vibrio harveyi (Swiss-Prot accession numbers P07739 and P07740). Only the alignment between SsuD, Mer, and LuxB is shown. Identical residues in all three sequences are in white on a black background, identical residues occurring in two sequences are in white on a gray background. The secondary structure elements of LuxB (27) are shown below the sequence as arrows (␣-helices) or thick lines (␤-sheets). The predicted secondary structure elements of SsuD and Mer were calculated with the program PredictProtein (31) and are indicated by h for ␣-helices, e for ␤-sheets, and l for loops. Dots represent lack of defined secondary structure. methanesulfonic acid and L-cysteic acid as sulfur sources (17). Characterization of the two systems has given insight into the completely different biochemical mechanisms responsible for sulfite liberation in E. coli from taurine on the one hand and from a wide range of aliphatic sulfonates on the other. Sulfite liberated from sulfonate-sulfur has been shown to enter the sulfate reduction pathway to cysteine (29) and thereby enables growth in the absence of the preferred sulfur sources sulfate and cysteine. In line with their function in metabolism, the proteins encoded by the tau and ssu gene clusters have a lower than average content of sulfur-containing amino acids. There is one cysteine residue in SsuD and none are present in SsuE and TauD. Economizing on sulfur-containing amino acids is metabolically sensible since these proteins are specifically synthesized when growth is limited by the biosynthesis of cysteine.