Crystal structures of TdsC, a dibenzothiophene monooxygenase from the thermophile Paenibacillus sp. A11-2, reveal potential for expanding its substrate selectivity

Sulfur compounds in fossil fuels are a major source of environmental pollution, and microbial desulfurization has emerged as a promising technology for removing sulfur under mild conditions. The enzyme TdsC from the thermophile Paenibacillus sp. A11-2 is a two-component flavin-dependent monooxygenase that catalyzes the oxygenation of dibenzothiophene (DBT) to its sulfoxide (DBTO) and sulfone (DBTO2) during microbial desulfurization. The crystal structures of the apo and flavin mononucleotide (FMN)-bound forms of DszC, an ortholog of TdsC, were previously determined, although the structure of the ternary substrate–FMN–enzyme complex remains unknown. Herein, we report the crystal structures of the DBT–FMN–TdsC and DBTO–FMN–TdsC complexes. These ternary structures revealed many hydrophobic and hydrogen-bonding interactions with the substrate, and the position of the substrate could reasonably explain the two-step oxygenation of DBT by TdsC. We also determined the crystal structure of the indole-bound enzyme because TdsC, but not DszC, can also oxidize indole, and we observed that indole binding did not induce global conformational changes in TdsC with or without bound FMN. We also found that the two loop regions close to the FMN-binding site are disordered in apo-TdsC and become structured upon FMN binding. Alanine substitutions of Tyr-93 and His-388, which are located close to the substrate and FMN bound to TdsC, significantly decreased benzothiophene oxygenation activity, suggesting their involvement in supplying protons to the active site. Interestingly, these substitutions increased DBT oxygenation activity by TdsC, indicating that expanding the substrate-binding site can increase the oxygenation activity of TdsC on larger sulfur-containing substrates, a property that should prove useful for future microbial desulfurization applications.

Sulfur compounds in fossil fuels are converted into sulfur oxides during combustion, are a major cause of severe environmental pollution and acid rain, and are also detrimental to human health. Hydrodesulfurization (HDS) 3 is widely applied in oil refineries to remove sulfur from crude oil-derived fuels by using hydrogen gas over a metal catalyst at high temperature and pressure. However, fossil fuels also contain heterocyclic sulfur compounds, such as dibenzothiophene (DBT) and its alkylated derivatives, that are resistant to conventional HDS. Biodesulfurization (BDS) holds promise as an alternative and complementary technology for removing these recalcitrant sulfur compounds under mild conditions and at low cost. DBT serves as a model compound for the BDS of fossil fuels, and many bacterial strains, including isolates from the genera Rhodococcus, Mycobacterium, Bacillus, Nocardia, and Paenibacillus, have been identified as DBT-desulfurizing bacteria (1)(2)(3). In particular, the Rhodococcus erythropolis strain IGTS8 has been studied extensively as a representative desulfurizing bacterium and was the first bacterium for which the genes involved in DBT desulfurization (dszABC) were characterized (4). Subsequently, the homologous genes tdsABC (5) and bdsABC (6,7) were identified from the thermophilic DBT-desulfurizing bacteria Paenibacillus sp. A11-2 (8) and Bacillus subtilis WU-S2B (9), respectively. The enzymes encoded by these genes are involved in the desulfurization of DBT to yield 2-hydroxybiphenyl (2-HBP), as shown in Fig. 1A. DBT monooxygenase (DszC/ TdsC/BdsC) oxidizes DBT to DBT sulfone (DBTO 2 ) via a twostep oxygenation reaction (10,11). DBTO 2 is then converted to 2Ј-hydroxybiphenyl 2-sulfinic acid (HBPSi) by another monooxygenase (DszA/TdsA/BdsA) (12,13). All of the enzymes that catalyze the sulfur oxidation and ring cleavage are members of the two-component flavin-dependent monooxygenase family (14 -18). Finally, HBPSi is desulfurized to 2-HBP with the release of the sulfur atom as sulfite by HBPSi desulfinase (DszB/TdsB/BdsB) (19,20). During these reactions, the carbon skeleton of DBT remains intact, and this property affords a significant advantage for the use of these microorganisms in industrial desulfurization processes because the fossil fuel is desulfurized without a decrease in its calorific value.
Recently, the crystal structures of the apo-and FMN-bound forms of a DBT monooxygenase, DszC, were determined (21)(22)(23)(24). Docking simulations of DBT into DszC were performed (21), but no crystal structure of a ternary substrate-FMNenzyme complex, which is essential for elucidating the DBT oxygenation mechanism, has been reported for any DBT monooxygenase. In this study, we obtained crystals of the DBT monooxygenase TdsC, an ortholog of DszC (amino acid sequence identity, 52%), and determined the structures of the DBT-FMN-TdsC and DBTO-FMN-TdsC complexes, as well as those of the apo-TdsC and FMN-TdsC complexes. We also determined the crystal structures of the indole-TdsC and indole-FMN-TdsC complexes, because in contrast to DszC, TdsC can also oxidize indole. These ternary complex crystal structures revealed that Tyr-93 and His-388 are located very close to the substrate and C4a of FMN, which binds molecular oxygen. We next constructed several mutant enzymes, including Y93A and H388A, and found that most of these mutations significantly reduced benzothiophene (BT) oxygenation activity. In contrast, when DBT was used as the substrate, the Y93A and H388A mutant enzymes unexpectedly exhibited higher activities than the wild-type (WT) enzyme. These results indicate that enzyme engineering of the substrate-binding pocket can increase DBT monooxygenase activity, which is required for efficient biodesulfurization.

Structure of apo-TdsC
The TdsC crystal structure comprises a homotetramer, with each chain being 414 amino acid residues long ( Fig. 2A). Analysis by size-exclusion chromatography indicated that TdsC is a tetramer in solution (data not shown). The TdsC chain is composed of an N-terminal ␣ domain (Asp-14 -Leu-124), a central ␤ domain (Gly-125-Val-222), and a C-terminal four-helixbundle domain (Tyr-223-Ser-414) (Fig. 2B). This domain architecture and quaternary structure are also seen for the acyl-CoA dehydrogenase superfamily (25), and the structure of TdsC is very similar to that of DszC (21)(22)(23)(24) and p-hydroxyphenylacetate hydroxylase C 2 (26,27). The electron densities for Ser-129 and the subsequent 7-9 residues, which are in the ␤ domain and close in space to the C terminus, were unclear due to disorder. Thr-282 and the subsequent four or five residues of the C-terminal four-helix-bundle domain in the adjacent chain are close to these disordered regions and were also disordered.

Structure of FMN-bound TdsC
FMN-bound TdsC crystals were prepared by soaking apo-TdsC crystals in a cryoprotectant solution containing FMN. The simulated annealing (SA) F o Ϫ F c omit electron density map was calculated by omitting FMN (Fig. 3A) and revealed that all four chains bind FMN. FMN binding to TdsC resulted Figure 1. A, microbial DBT desulfurization pathway. B, general catalytic mechanism of the oxygenation reaction by flavin-dependent monooxygenases and aromatic hydroxylases (16, 30 -33).

Crystal structures of ternary substrate-FMN-TdsC complexes
in no global structural changes. However, seven of the eight disordered regions in the apoenzyme, which are close to the FMN-binding site, became structured upon FMN binding, and the loops around Glu-130 -Trp-138 (loop 1) and around Thr-282-Gly-285 from the adjacent chain (loop 2) covered the active site of TdsC (Fig. 3B), as was also reported for FMNbound DszC (24). Nevertheless, these seven regions exhibited higher temperature factors compared with the other structured regions, indicating that the loop 1 and 2 regions remained flexible. An isoalloxazine moiety was inserted into the cavity between the C-terminal four-helix-bundle domain and the central ␤ domain (Fig. 3C), and its A ring formed hydrophobic interactions with Phe-158 and Trp-202 (Fig. 3D). The N and O atoms of the isoalloxazine ring formed extensive hydrogenbonding interactions with several residues, including Asn-126, Ser-129, and Ser-160, and with many main-chain atoms. The ribityl side chain of FMN was found to extend outward from the pocket and form hydrogen bonds with Tyr-413 and the mainchain carbonyl group of His-388. A water molecule formed a bridge between the O3Ј atom of the ribityl side chain and the main-chain O atom of Glu-130 and the side chain of Arg-279 of the adjacent chain through hydrogen bonds (Fig. 3E). The terminal phosphate group formed an extensive hydrogen-bond network with the guanidinium groups of Arg-279 and Arg-366 on the adjacent chain. Most of the residues involved in interactions with FMN are conserved between DszC and TdsC. Indeed, almost the same interactions with FMN were found in the crystal structure of DszC (24), and thus the FMN-binding mode is expected to be strictly conserved between these enzymes. The conformations of most of the residues involved in FMN binding remained unchanged after binding, except for the Tyr-93 side chain, which moved closer to FMN, thereby enlarging the pocket between the N-terminal ␣ domain and the central ␤ domain compared with the apoenzyme (Fig. 3B).

Indole oxidation by TdsC
Cultivation of the Escherichia coli strain overproducing TdsC resulted in the medium turning greenish, suggesting that TdsC produced indigo. This indigo was likely generated from tryptophan in the medium via indole, as shown previously for another DBT monooxygenase, BdsC from B. subtilis WU-S2B (28). We found that indigo can indeed be produced from the reaction between TdsC and indole (Table 1), and therefore we also determined the crystal structures of the indole-TdsC and indole-FMN-TdsC complexes.

Binary indole-TdsC and ternary indole-FMN-TdsC complexes
Similar to the FMN-bound form, indole binding did not induce global conformational changes in TdsC. The SA F o Ϫ F c omit electron density map (Fig. 4A) showed that three of the

Crystal structures of ternary substrate-FMN-TdsC complexes
four chains contained indole, and the hydrophobic substrate was found at almost the same position as the hydrophobic A ring of isoalloxazine in FMN-bound TdsC (Fig. 4B). The electron density map around the indole was rather unclear, and thus other orientations distinct from the model cannot be excluded.
The SA F o Ϫ F c omit electron density map of the ternary indole-FMN-TdsC complex showed that the active-site pocket of the D chain contained indole and was situated about 3 Å from the isoalloxazine ring (Fig. 4C). Although the C chain also contained indole at a similar position as in the D chain, its SA F o Ϫ F c omit electron density map was less clear. Indole binding to the FMN-TdsC complex induced no significant structural changes. Comparison of the ternary indole-FMN-TdsC structure with the binary indole-TdsC complex showed that loop 1 moved by about 3 Å upon FMN binding, making the active-site pocket larger in the ternary complex than in the binary complex (Fig. 4D). We cannot exclude other orientations of indole, because its SA F o Ϫ F c omit electron density map was rather unclear, and indole is a highly symmetric molecule. However, the indole C3 atom where the oxidation occurs can indeed face toward the isoalloxazine ring. The distance between the indole C3 atom and the FMN C4a atom was 5.1 and 5.6 Å for the C and D chains, respectively, which is similar to the distance between the substrate and the C4a atom (5.0 Å) reported for p-hydroxyphenylacetate hydroxylase C 2 (27). Thus, the binding orientation of indole and the distance between the substrate and FMN in the ternary complex are consistent with the indole oxidation activity of TdsC.

Ternary DBT-FMN-TdsC and DBTO-FMN-TdsC complexes
We have determined the first crystal structure of the ternary DBT-FMN-TdsC complex, which is important for elucidating the mechanism of DBT metabolism. The SA F o Ϫ F c omit map indicated that the B, C, and D chains hold DBT in the hydrophobic pocket of the active site ( Fig. 5A) such that DBT is surrounded by Tyr-93, Trp-138, Phe-171, Trp-247, Phe-412, and Tyr-413 (Fig. 5, B and C). The location of the two rings of DBT was found to be almost the same as that of indole in the ternary indole-FMN-TdsC complex (Fig. 5D). The sulfur atom of DBT was situated 4.6 Å from the C4a atom of FMN in the B, C, and D chains, which is also comparable with the distance between the substrate and C4a found in p-hydroxyphenylacetate hydroxylase C 2 (27) and explains the oxygenation of DBT by TdsC. The conformations of the main chain and side chains of the activesite residues in TdsC were essentially the same for the ternary indole-FMN-TdsC and DBT-FMN-TdsC complexes.
DBT is oxygenated to its sulfoxide DBTO by TdsC or its orthologs, which then undergo another monooxygenation mediated by the same enzymes to generate the sulfone DBTO 2 .

Crystal structures of ternary substrate-FMN-TdsC complexes
Consequently, DBTO is both a product and a substrate for these enzymes, and therefore the DBTO-FMN-TdsC structure should also provide important insights into the metabolism of DBT by these enzymes, which is an essential step for bacterial desulfurization. Very clear SA F o Ϫ F c omit electron density for DBTO was found in the B, C, and D chains of the ternary DBTO-FMN-TdsC complex (Fig. 6A). DBTO could be located unambiguously as a result of the electron density of the sulfoxide moiety. The ring moiety of DBTO was located at almost the same position as DBT in the ternary complexes, and the ring formed hydrophobic interactions with Trp-247 and Phe-412, as observed for the ternary indole-FMN-TdsC and DBT-FMN-TdsC complexes. The sulfoxide S atom has a lone pair of electrons and thus possesses a tetrahedral conformation similar to that of an sp 3 carbon atom. The sulfoxide oxygen atom points into the pocket comprising the residues Trp-247, Thr-251, Gln-252, His-388, and Asp-389 (Fig. 6B) and forms a hydrogen-bond network with two water molecules, Gln-252, His-388, Asp-389, and the carbonyl oxygen atom of Phe-412. This stands in contrast to the DBT-containing ternary complex, which lacks hydrogen bonds with the substrate. The hydrogen-bonding interactions of the sulfoxide oxygen result in the lone pair of electrons facing toward the isoalloxazine ring, thereby allowing the second monooxygenation by the enzyme.

Conformations and dynamics of mobile loops
Although loop 1 of apo-TdsC is disordered, the equivalent loop region in apo-DszC is structured and adopts an open conformation (21,22,24), which was also found for the indole-TdsC complex (Fig. 7A and Table 2). This disordered or "open" loop 1 is suitable for FMN incorporation and binding. The conformations of loop 1 in FMN-containing TdsCs are almost identical and are very similar to the closed conformation found for loop 1 of FMN-bound DszC (Fig. 7B) (24).
The conformation of loop 2, which interacts with the ribityl group of FMN in the adjacent chain, was almost identical for all ligand-bound TdsCs and is very similar to the closed conformation of the equivalent region of DszC (Fig. 7, A and B, Table 2) (21,22,24). For both TdsC and DszC, a pocket exists between loop 2 and FMN (Fig. 7C), where the adenine moiety is likely accommodated when FAD binds to TdsC and DszC, both of which can utilize FAD as a co-substrate (11,29). The potential adenine-loop interactions are also seen in FAD-bound acyl-CoA dehydrogenase from Mycobacterium thermoresistibile (PDB code 3NF4), suggesting that the acyl-CoA dehydrogenase-type two-component flavin-dependent monooxygenases (18) share the similar adenine-binding site for FAD.

Crystal structures of ternary substrate-FMN-TdsC complexes
The A and B chains in indole-FMN-TdsC and the A chain in DBT-FMN-TdsC did not contain substrate, and the loops 1 and 2 close to these "vacant" substrate pockets exhibited a relatively higher temperature factor compared with those close to the "occupied" substrate pocket, although interactions between the substrate and loops 1 and 2 were limited (Figs. 4D and 8). Similar correlations between the mobile loops and the ligand were also observed in other TdsC and DszC structures possessing closed loops (Fig. 8). Therefore, the flexibility of the mobile loops and the substrate binding are closely related, and the wellstructured loops 1 and 2 in the ternary substrate-FMN-TdsC complex and the "relaxed" loops in the apoenzyme would contribute toward efficient monooxygenation by these enzymes.

Roles of residues around the flavin N5 and C4a atoms
In the general catalytic mechanism of the oxygenation reactions by flavin-dependent monooxygenases and aromatic hydroxylases, the reduced flavin binds dioxygen at the C4a atom to form C4a-hydroperoxyflavin after proton addition at the terminal O atom of the peroxide (Fig. 1B) (16, 30 -33). The hydroperoxy moiety acts as an electrophile in the monooxygenation reaction, and the oxidized flavin and a water molecule are recovered. Thus, activation of molecular oxygen and substrate oxygenation are performed at and around the C4a atom of the isoalloxazine ring. Ser-160 is directly hydrogen-bonded to N5, which is adjacent to the C4a atom, and also participates in a hydrogen-bond network with His-89, His-388, and Tyr-96 (Fig.  3D). To investigate the roles of these active-site residues, we prepared mutant TdsCs and examined their oxygenation activities toward DBT, BT, and indole. The single mutations H89A and S160A substantially reduced the oxygenation activity for all of the substrates investigated. DszC contains His-92 and Ser-163 at almost the same positions as His-89 and Ser-160 in TdsC. Both mutations H92F (21) and S163A (21,34) substantially reduced the DBT oxygenation activity of DszC, as found for the TdsC mutants H89A and S160A.
The hydrogen-bonding network from the flavin N5 atom to Ser and aromatic side chains was also found in p-hydroxyphenylacetate hydroxylase C 2 . Ser-171 in this enzyme is located at a very similar position to Ser-160 in TdsC and forms a hydrogen bond with Trp-112. The mutations S171A (35) and W112A (36) drastically increased the decay rate of C4a-hydroperoxyflavin, indicating that the role of the Ser and the aromatic side chain is to stabilize this species. Recently, Vey and co-workers (34) also reported that the S163A mutation eliminated the ability of DszC to stabilize the C4a-peroxyflavin intermediate. Therefore, the substantial decreases in the oxygenation activity for the S160A and H89A mutants of TdsC suggest that Ser-160 and His-89 also stabilize the C4a-hydroperoxyflavin.
Mutations at Tyr-93 and His-388 also reduced the oxygenation activity for two-ring substrates, i.e. BT and indole. DszC

Crystal structures of ternary substrate-FMN-TdsC complexes
contains its Tyr-96 and His-391 residues at almost the same positions as Tyr-93 and His-388 in TdsC. Liu et al. (21) found that both Y96A and H391F mutations substantially reduced the oxygenation activity of DszC. p-Hydroxyphenylacetate hydroxylase C 2 also contains its His-396 at a very similar position to His-388 in TdsC. Extensive kinetic studies on this enzyme suggested that His-396 acts as a proton source for the protoncoupled electron transfer before the transition state of C4ahydroperoxide formation and that this residue is important for maintaining C4a-hydroperoxyflavin in the protonated form (35,37). In the case of TdsC, the substitution of Tyr-93 with Phe resulted in no detectable activity for BT or indole oxygenation, indicating that the presence of the phenyl group in Y93F is not sufficient for the oxygenation activity of TdsC and that the hydroxy group of Tyr-93 plays a crucial role in the oxygenation of BT and indole. Therefore, His-388 and Tyr-93 are both likely involved in proton supply to form C4a-hydroperoxyflavin. In contrast to the results for the BT and indole oxygenation activities, substitution of Tyr-93 and His-388 with Ala increased the DBT oxygenation activity. Both BT and DBT were oxygenated at the sulfur atom by TdsC, although DBT was a very poor substrate compared with BT ( Table 1). The crystal structure of p-hydroxyphenylacetate hydroxylase C 2 (27,38) indicates that the cavity above the C4a atom is required for flavin-dependent monooxygenases to allow the face-on approach of molecular oxygen with respect to the isoalloxazine ring (Fig. 1B) and that the unstable C4a-hydroperoxyflavin intermediate is stabilized in this solvent-free cavity (supplemental movie 1) (39). Re-inspection of the structure around the substrate in the ternary DBT-FMN-TdsC complex revealed that the space between the substrate and the C4a atom of FMN is very limited (Fig. 5C and supplemental movie 2), and DBT is therefore a poor substrate for TdsC. We have not yet determined the structure of the ternary BT-FMN-TdsC complex, but BT is a smaller substrate than DBT and thus likely leads to a cavity above the C4a atom, similar to that found in the indole-FMN-TdsC complex and p-hydroxyphenylacetate hydroxylase C 2 . Thus, there should be sufficient space above the C4a atom of FMN to allow the C4a-hydroperoxyflavin formation. The replacement of Tyr-93 or His-388 with Ala expands the cavity above the C4a atom, thereby promoting C4a-hydroperoxyflavin formation and thus higher DBT oxygenation activities. The cavity volume in the Y93F mutant is expected to be slightly larger than that in the WT and much smaller than that in Y93A. Indeed, the DBT oxygenation activity of Y93F was found to be higher than that of the WT and lower than that of Y93A. Similarly, introduction of the larger side chain, phenylalanine, at position 388 greatly reduced the DBT oxygenation activity. The appreciable activities of Y93A, Y93F, and H388A for DBT oxygenation implies that the proton supply system for the C4a-peroxyflavin can vary depending on the substrate.
Although H89A and S160A mutations would also be expected to create a cavity around the C4a atom, its position would not be suitable for a face-on approach of molecular oxygen to the isoalloxazine ring (Fig. 1B). Furthermore, these residues are important for stabilization of the intermediate, and thus these mutations did not increase the DBT oxygenation activity compared with the WT.
The effects of mutations of Tyr or His residues in the active sites of TdsC and DszC are starkly different; mutation substantially increased the DBT oxygenation activity of TdsC, although it destroyed the same activity of DszC. Explaining this discrepancy is not straightforward, because most of the residues in the active site and the substrate-binding pocket are conserved between TdsC and DszC. However, one residue at the entrance of the substrate-binding pocket is not conserved and could be the origin of these divergent effects. In TdsC, Tyr-413 forms a lid on the substrate-binding pocket and is replaced by Thr (Thr-416), a smaller residue, in DszC (Fig. 5E). The substrate-binding pocket in DszC is therefore larger than that in TdsC. The substrate in the ternary DBT-FMN-DszC complex can relocate farther from the C4a in FMN compared with that in the ternary DBT-FMN-TdsC complex, and the DBT-FMN-DszC complex would have a cavity above the C4a of FMN very similar to that of BT-FMN-TdsC. Therefore, mutation of DszC decreased the DBT oxygenation activity, similar to that observed for BT oxygenation by WT and mutant TdsC.
The indole oxidation activity is also quite different between TdsC and DszC; TdsC can oxidize indole to form indigo, whereas DszC cannot. The inability of DszC to perform indole oxidation would also be explained by its larger substrate-binding pocket relative to TdsC. The larger pocket is not suitable for binding indole, and/or the indole in the pocket can easily escape from the large mouth of the pocket. Detailed understanding of the substrate selectivity and the origin of these divergent mutation effects will require determination of the crystal structures of the ternary DBT-FMN-DszC and BT-FMN-TdsC complexes and molecular dynamics simulation of the substrate and mobile loops in the ternary substrate-FMN-enzyme complexes.

Conclusions
In this study, we have determined the crystal structures of ternary substrate-FMN-TdsC complexes in addition to the structures of apo-TdsC, FMN-TdsC, and indole-TdsC. The crystal structure of indole-FMN-TdsC reasonably explained the indole oxidation activity of TdsC. The structures of the ternary complexes containing DBT or DBTO revealed the key substrate interactions with the enzyme that are essential for the two-step oxygenation by TdsC and its orthologs. The active sites of TdsC and DszC are covered with the two mobile loops and these mobile loop regions are very important for substrate and co-substrate binding. Expansion of the cavity above the C4a atom, where the hydroperoxide is formed, by substitution with smaller residues enhanced the oxygenation activity for DBT, which is a poor substrate for TdsC. The DBT oxygenation activities of these mutants suggest that modification of the substrate-binding pockets of TdsC and its orthologs can be used to create engineered enzymes able to metabolize the highly alkylated DBTs that are less efficiently removed by biodesulfurization. The crystal structures of substrates complexed with TdsC can be used to guide the design of such engineered enzymes. Information regarding the dynamics of the flexible loops that are part of the substrate-binding pocket will also be important for modifying substrate selectivity.

Construction of the tdsC-overexpressing strain
The plasmid harboring tdsABC was a gift from the Japan Cooperation Center, Petroleum (Shizuoka, Japan). The tdsC gene was amplified using an Expand High Fidelity PCR System (Roche Diagnostics, Mannheim, Germany) and the buffer conditions recommended by the manufacturer. The reaction mixture was heated at 94°C for 2 min and then subjected to 30 cycles of amplification (94°C for 1 min, 56°C for 1 min, and 72°C for 2 min) with the primers 5Ј-GGGGGGGAATTCAT-GAGAACAATCCAT-3Ј (the EcoRI restriction site is underlined) and 5Ј-CCGCGGAAGCTTTCAAGAGTAAAAGCTG-3Ј (the HindIII restriction site is underlined) and the plasmid mentioned above as the template. The amplified fragments were digested with EcoRI and HindIII, and the digested fragments were separated by agarose gel electrophoresis, inserted into pKK223-3 with the tac promoter, and then used to transform E. coli JM109 cells.

Enzyme preparation
Cells of the E. coli transformant harboring the constructed plasmid pKK223-3/tdsC were grown at 37°C in 2-liter flasks containing 1 liter of LB medium supplemented with 50 mg/liter ampicillin. After incubation for 3 h, 1 mM isopropyl 1-thio-␤-D-galactopyranoside was added to the medium, and cultivation was continued for an additional 14 h. The cells were harvested from 6 liters of culture broth, suspended in basal buffer (50 mM potassium phosphate buffer (pH 7.0) containing 1 mM DTT and 10% glycerol), then sonicated, and centrifuged at 8,000 ϫ g for 20 min to remove cell debris.
The cell extract was dialyzed against basal buffer and then applied to a DEAE-Sepharose column (4.5 ϫ 53 cm) equilibrated with the same buffer. The column was washed with the same buffer, and the bound proteins were eluted with the same buffer containing 0.1 M NaCl. The resultant enzyme solution was dialyzed against basal buffer containing 1 M (NH 4 ) 2 SO 4 . After centrifugation to remove the precipitate, the supernatant was applied to a phenyl-Toyopearl column (4 ϫ 22 cm) equilibrated with the same buffer. The column was washed sequentially with basal buffer containing 1, 0.8, and 0.6 M (NH 4 ) 2 SO 4 , and then the bound proteins were eluted with basal buffer containing 0.4 M (NH 4 ) 2 SO 4 . The active fractions were dialyzed against basal buffer and then concentrated by ultrafiltration.

Mutant enzyme construction
Site-directed mutagenesis of tdsC was performed using a PrimeSTAR mutagenesis basal kit (TaKaRa Bio, Kyoto, Japan) according to the manufacturer's instructions. PCR was then conducted using pKK223-3/tdsC as a template; the products were incorporated into E. coli JM109, and the DNA sequences of the mutant genes were confirmed. The mutant strains were cultivated, and the enzyme samples were purified as described above.

Enzyme assay
Enzyme activities were determined using DBT or BT as the substrate by preparing reaction mixtures containing 100 mM potassium phosphate buffer (pH 7.0), 10 M FMN, 6 mM NADH, 5 units/ml flavin reductase (TdsD), and 0.3 mM substrate. TdsD was prepared as described previously (7). The Table 3 Crystallographic and refinement statistics Highest resolution shell is shown in parentheses. 5% of reflections were used for the calculation of R free .

Crystal structures of ternary substrate-FMN-TdsC complexes
enzyme reaction was conducted for 30 min at 50°C, and then the products were measured by HPLC as described previously (10). Enzyme reactions using indole as the substrate were conducted as described previously (28). One unit of activity was defined as the amount of TdsC necessary to produce 1 nmol/ min of DBTO 2 , BT sulfone, or indigo.

Crystallization
Crystals of apo-TdsC were obtained by the sitting-drop vapor-diffusion method at 20°C. A protein solution (12 mg/ml in 50 mM Tris-HCl buffer (pH 7.0)) was mixed with an equal volume of a reservoir solution comprising 1.3 M ammonium sulfate, 12% glycerol, and 0.1 M Tris-HCl buffer (pH 8.5). Bipyramidal crystals of native TdsC appeared within 1 day and reached their maximum size after 1 week. Crystals of the indole-TdsC complexes were obtained using similar conditions, whereas no crystals of the enzyme-FMN complex could be obtained by this method. Therefore, FMN was introduced into the protein by soaking the apo-TdsC crystals in a reservoir solution containing 2 mM FMN overnight. The crystals of the ternary substrate-FMN-TdsC complexes were obtained by soaking apo-TdsC crystals in a reservoir solution containing 1.5 mM substrate and 2 mM FMN. In the case for the ternary DBT-FMN-TdsC and DBTO-FMN-TdsC complexes, 2-hydroxypropyl-␤-cyclodextrin was added to the reservoir solution to solubilize the substrates.

Structure determination and refinement
Crystals were cryoprotected during the ligand-soaking process by gradually increasing the glycerol concentration up to 25%, followed by rapid immersion into liquid nitrogen. All of the diffraction datasets were collected at the SPring-8 beamline BL26B1 on-site or via the remote access system (40) and processed with XDS (41) or Mosflm (42) and Scala (43). When we attempted to solve the structure of apo-TdsC, no structural information of close homologs had been reported in the Protein Data Bank. Therefore, we comprehensively tested the structure of acyl-CoA dehydrogenases having low sequence identity (20 -30%) to the TdsC as molecular replacement search models using MolRep (44). The polyalanine model from M. thermoresistibile (PDB code 3NF4) showed the highest score. After application of the morph_model module (45) implemented in the Phenix suite (46), a clear electron density map of the bulky side chains was obtained, allowing us to build an initial structural model. Further refinement and model building were performed using Phenix.refine and Coot (47). All other structures were solved by molecular replacement of the apo-TdsC structure using MolRep or PHASER (48) and then refined as described above. The data collection and refinement statistics are summarized in Table 3. All of the figures showing crystal structures were prepared using PyMOL (49).