Outer-membrane transport of aromatic hydrocarbons as a first step in biodegradation

Abstract

Bacterial biodegradation of hydrocarbons, an important process for environmental remediation, requires the passage of hydrophobic substrates across the cell membrane. Here, we report crystal structures of two outer membrane proteins, Pseudomonas putida TodX and Ralstonia pickettii TbuX, which have been implicated in hydrocarbon transport and are part of a subfamily of the FadL fatty acid transporter family. The structures of TodX and TbuX show significant differences with those previously determined for Escherichia coli FadL, which may provide an explanation for the substrate-specific transport of TodX and TbuX observed with in vivo transport assays. The TodX and TbuX structures revealed 14-stranded β-barrels with an N-terminal hatch domain blocking the barrel interior. A hydrophobic channel with bound detergent molecules extends from the extracellular surface and is contiguous with a passageway through the hatch domain, lined by both hydrophobic and polar or charged residues. The TodX and TbuX structures support a mechanism for transport of hydrophobic substrates from the extracellular environment to the periplasm via a channel through the hatch domain.

The monoaromatic hydrocarbons benzene, toluene, ethylbenzene, and xylene, referred to as BTEX, pose a significant human health concern. Human exposure to BTEX can cause liver and kidney damage, nervous disorders, and reproductive and developmental effects (1–4). Moreover, benzene is classified as a human carcinogen (5). Despite their negative impact on human health, BTEX compounds continue to be produced for use in the petroleum industry, in plastic and polymer manufacturing, and as solvents in paints and household cleaners (1–4). In 2005, industrial disposal practices in the United States resulted in >1.5 million lb of BTEX released to surface waters and land (6). BTEX-contaminated water and soil environments are remediated primarily through biodegradation by natural microbial populations (7, 8). In particular, Gram-negative bacteria are known for their biodegradative abilities (8).

In Gram-negative bacteria, five aerobic pathways for BTEX biodegradation have been characterized in great detail, both chemically and genetically (9): the toluene o-monooxygenation (tom) (10), toluene m-monooxygenation (tbu) (11), toluene p-monooxygenation (tmo) (12), xylene monooxygenase (xyl) (13), and toluene dioxygenation (tod) pathways (14). Although much is known about the intracellular degradation of BTEX, virtually nothing is known about how such compounds are taken up by the cell, a prerequisite for their biodegradation. In addition to the enzymatic genes, the BTEX degradation pathways include genes encoding outer membrane proteins, which may be required for hydrocarbon uptake across the outer membrane (OM). Because the Gram-negative bacterial OM is surrounded by a hydrophilic lipopolysaccharide layer (15), membrane proteins are required to facilitate transport of hydrophobic compounds across the OM. The involvement of OM proteins in BTEX uptake was demonstrated in vivo by the enhanced toluene metabolism upon expression of the Pseudomonas putida F1 TodX (16) and Ralstonia pickettii PKO1 TbuX (17) proteins. These proteins and the P. putida XylN (18) and Pseudomonas mendocina TmoX proteins (19) show 40–80% identity to each other and low (15–25%) sequence identity to the Escherichia coli long-chain fatty acid transporter FadL [supporting information (SI) Fig. S1]. The TodX-type proteins form a subfamily of the FadL transporter family (20), lending support to their proposed function in hydrocarbon transport.

The E. coli FadL protein is the only well characterized OM protein known to facilitate the uptake of hydrophobic compounds (21). Recently, two FadL crystal structures were solved (21), revealing a monomeric 14-stranded β-barrel whose interior is blocked by an N-terminal hatch domain. An inward-pointing kink formed by strand S3 in the β-barrel wall constrains the N terminus located within the interior of the barrel. From the FadL structures, a transport model was proposed that involves the formation of a channel through the hatch domain. However, no such channel was observed in either FadL structure. Here, we present the crystal structures of P. putida TodX and R. pickettii TbuX, the first structures of any transporters involved in xenobiotics uptake. Both structures show a continuous passageway leading from the extracellular surface through the hatch domain, all the way to the periplasmic surface. We discuss the implications of these structures on the mechanism of hydrocarbon transport of TodX family proteins.

Results and Discussion

Overall Structures.

Because the yield of protein expressed within the OM was low, P. putida TodX was purified from inclusion bodies and refolded in detergent. X-ray crystal structures of refolded TodX were obtained in two space groups: orthorhombic (I222; 3.2-Å resolution, one molecule in the asymmetric unit) and triclinic [P1; 2.6-Å resolution, two molecules in the asymmetric unit (Table S1)]. Neither molecule in the triclinic structure shows visible electron density for the N-terminal five residues, whereas the orthorhombic structure has a complete N terminus. Both structures are missing electron density for residues 405–414 of extracellular loop L7 and smaller regions of loops L1 (residues 72–77), L3 (residues 206–209), and L6 (residues 359–364). The orthorhombic and triclinic forms of TodX are very similar (Fig. 1A), with an average rms deviation for the C_α atoms of 0.9 Å.

Fig. 1.

Structural features of TodX and TbuX. (A) Comparison of triclinic TodX (Left), TbuX (Center) and monoclinic FadL (Right), shown as ribbon representations. The extracellular loops are shown in purple (L3), light blue (L4), and dark blue (L5). Although the electron density for some of the extracellular loops in the TbuX structure was weak and could not be modeled accurately, the conformations of the loops are similar to TodX. The region in β-strand S3 associated with the kink is colored pink. Detergent molecules are indicated in red (C₈E₄) and blue (LDAO). The N and C termini are indicated, and the hatch domain is colored yellow. The approximate position of the OM is indicated by horizontal lines with the extracellular side (E) at the top and the periplasm (P) at the bottom. (B) Ribbon representations of the N-terminal hatch domains, colored by B-factor, of triclinic and orthorhombic TodX (superimposed, Left), TbuX (Center), and monoclinic and hexagonal FadL (superimposed, Right). All figures were made with PyMOL (www.pymol.org).

R. pickettii TbuX was purified from the membrane fraction and crystallized in its native form. TbuX crystals were obtained in the orthorhombic (F222) space group at a resolution of 2.8 Å, with two molecules in the asymmetric unit (Table S1). Electron density is weak or absent in parts of extracellular loops L1 (residues 61–66), L3 (residues 206–212), L4 (residues 258–261), and in the majority of loops L6 (residues 362–376) and L7 (residues 406–418), which could not be modeled. Despite the incompleteness of the extracellular loops, the overall features of the native TbuX structure are similar to those of refolded TodX (average C_α rms deviation of 4 Å; Fig. 1A), indicating that the TodX structure is not likely to be an artifact of refolding.

TodX and TbuX have a 14-stranded β-barrel similar to that observed in the fatty acid transporter FadL (21) (Fig. 1A). Like FadL, TodX and TbuX have an N-terminal hatch domain consisting of three short helices that fold within the barrel interior. The N-terminal 10–12 residues in the TodX and TbuX structures have significantly higher B-factors relative to the rest of the hatch domain (Fig. 1B), suggesting that the N terminus is flexible. Moreover, the first five residues in the triclinic TodX structure are not visible in the electron-density maps, presumably because of high mobility. The mobility of the N terminus is reminiscent of the situation in FadL, where two otherwise identical crystal structures showed different conformations for the N-terminal seven residues (21).

Substrate Binding by Aromatic Hydrocarbon Transporters.

Within the extracellular domains of both TodX and TbuX, several tubes of electron density are visible, which were assigned as C₈E₄ detergent molecules used during the later stages of the purification process (Methods). Two C₈E₄ molecules, designated as upper and lower, were observed at slightly different positions in both the orthorhombic and triclinic TodX structures. A single C₈E₄ molecule was identified in TbuX, in approximately the same position as the lower C₈E₄ molecule in TodX (Fig. 1). Inspection of the TodX and TbuX structures reveals a continuous hydrophobic channel extending from the extracellular surface of the protein to the N terminus (Fig. 2). On the extracellular surface of the protein, the space between the two antiparallel α-helices of loop L3 forms a hydrophobic cleft, which contains the upper C₈E₄ molecule (Fig. 2B). This detergent molecule is in close proximity (<4.5 Å) to residues Ser-171, Val-173, Thr-177, Leu-182, Val-187, Ala-191, Val-194, and Ala-199 from L3 (Fig. 2). The lower C₈E₄ molecule is close to residue Leu-134 in loop L2; residues Leu-162, Leu-164, Leu-166, Leu-168, Gln-172, and Phe-202 in loop L3; residues Ile-270, Val-272, and Phe-275 in loop L4; residues Val-313, Val-324, and Leu-326 in loop L5; and residues Leu-367 and Ile-370 in loop L6 (Fig. 2A). Together, these residues form the lining of a pronounced hydrophobic channel that extends all the way to the N terminus in the interior of the β-barrel. The hydrophobic nature of the channel is conserved among the homologues implicated in hydrocarbon transport (Fig. S1). We propose that the hydrophobic channel forms a conduit for the initial binding and transport of aromatic hydrocarbon substrates by members of the TodX subfamily. Interestingly, inspection of the structures of FadL with those of TodX/TbuX reveals that the detergent-binding sites are located at similar positions in the structures, with the upper C₈E₄ molecule in TodX at a position analogous to the C₈E₄ molecule in the low-affinity binding site of FadL, and the lower TodX C₈E₄ molecule at a position analogous to that occupied by an LDAO molecule in the high-affinity binding site of FadL (ref. 21; Fig. 1).

Fig. 2.

Detergent binding to TodX. (A) Stereo diagram view showing the locations of bound C₈E₄ detergent molecules (green, with oxygens in red), designated as “upper” and “lower.” 2F_o−F_c density, contoured at 1.5σ, is shown as an orange mesh. Loop L3 is colored yellow, and the segment of loop L5 that interacts with L3 is colored magenta. Residues that are located within 4.5-Å distance of the C₈E₄ molecules are shown as stick models (carbons gray, oxygens red, and nitrogens blue). (B) Surface view from the extracellular side, rotated ≈45° relative to A, showing the upper C₈E₄ molecule bound within the hydrophobic cleft formed by loop L3. Polar residues are colored salmon, and hydrophobic residues are colored gray.

FadL Family Members Are Substrate-Specific.

Because transport assays involving volatile substrates like BTEX are technically challenging, we tested the ability of TbuX and TodX to transport long-chain fatty acids, using an in vivo assay with radiolabeled oleic acid (22). Although E. coli cells expressing FadL showed a high level of oleic acid uptake, cells expressing TodX and TbuX were unable to transport oleic acid, despite the observation that the (outer) membrane levels of TodX and TbuX are higher than those of FadL (Fig. 3). These data demonstrate that members of the FadL family are substrate-specific transporters. Substrate specificity provides an explanation for the fact that many bacteria have more than one FadL homolog (for example, Pseudomonas aeruginosa and Vibrio cholerae each have three). The substrate specificity of FadL homologues is different from that of multidrug efflux pumps, which display a broad specificity for various hydrophobic compounds (23).

Fig. 3.

TodX and TbuX are substrate specific. Upper shows activity for oleic acid uptake, normalized to the cell density (OD₆₀₀), as measured in whole cells expressing the various proteins. Scale bars represent the average of at least three independent measurements, and error bars represent the standard deviation. Representative Western immunoblots indicating expression levels of the various proteins within the OM (see Methods) are shown in Bottom.

Which structural features underlie the different substrate specificities of FadL proteins? The answer to this question is not yet clear, but one of the most obvious possibilities would be differences within the substrate-binding sites. In FadL, two positively charged residues (Arg-157 and Lys-317) that likely interact with the fatty acid carboxylate group (21) are present in the otherwise completely hydrophobic high-affinity binding pocket. Sequence and structural alignments of FadL with TodX and TbuX show that the residues corresponding to Arg-157 and Lys-317 in FadL are hydrophobic residues in the aromatic hydrocarbon transporters (Leu-166 and Leu-326 in TodX; Fig. S1), which is in accord with the uncharged nature of BTEX substrates. Thus, as a first step in elucidating the basis for substrate specificity in FadL family members, we mutated both Leu-166 and -326 in TodX to arginine and lysine, respectively (L166R/L326K). Subsequent oleate transport assays showed very little, if any, transport for the TodX double mutant (Fig. 3), indicating that these residues alone are not responsible for substrate specificity. Future more extensive mutagenesis experiments will be required to uncover the structural features governing the substrate specificity of FadL family members.

Structural Differences Between FadL and Aromatic Hydrocarbon Transporters.

One of the most striking differences between TodX/TbuX and FadL is the conformation of the extracellular loops L3 and L4 (Fig. 1). In TodX (and TbuX), loop L3 consists of two antiparallel α-helices that lie flat on the top of the barrel (Figs. 1A and 2A), forming a hydrophobic cleft that is occupied by a detergent molecule. In E. coli FadL, loop L3 also consists of two antiparallel α-helices, but these protrude into the extracellular environment (Fig. 1A). Between L3 and L4 in FadL, a hydrophobic groove is present that serves as an initial low-affinity-binding site for fatty acids (21). Thus, while the first interaction between FadL and substrate occurs in a hydrophobic groove between loops L3 and L4, in TodX and TbuX the hydrophobic cleft is formed solely by loop L3. The reason for the differences in the arrangement of the L3 and L4 loops between FadL and TodX/TbuX is unclear but is likely functionally significant for several reasons. First, very similar conformations of L3 and L4 are observed in all three aromatic hydrocarbon transporter structures. Second, the tip of loop L3 (residues Gly-321–Val-324) forms extensive interactions with loop L5 (residues Asn-181–Gly-184; Fig. 2A), suggesting that the conformation of L3 is not likely to be transient. Finally, the structures of several E. coli FadL mutant proteins all show L3 and L4 loops that are very similar in conformation to that of wild-type FadL (data not shown), indicating that crystal-packing effects are not likely to be responsible for the structural differences of L3 and L4 between FadL and TodX/TbuX.

A second structural difference between TodX/TbuX and FadL that is likely to be functionally important is the conformation of β-strand S3. E. coli FadL has an unusual inward-pointing kink in strand S3 of the barrel wall (ref. 21; Fig. 1A). The kink makes a number of interactions with the N terminus, thereby likely constraining the mobility and conformational space accessible to the N terminus (21). In TodX and TbuX, the kink is much less pronounced (Figs. 1A and 4). This is most evident in TodX, where the interstrand hydrogen-bonding pattern between S3 and the neighboring strands S2 and S4 is largely intact (Fig. 1A). The S3 kink region in TodX and TbuX may be flexible, as suggested by the relatively high B-factors for the kink residues (100–104 in TodX and 102–106 in TbuX) relative to the neighboring residues in S3 (Fig. S2). The absence of a pronounced kink in TodX/TbuX suggests there may be fewer interactions of this segment with the N terminus; as a result, we speculate that the N terminus is less constrained in the aromatic hydrocarbon transporters compared with the N terminus in E. coli FadL. This is borne out by the fact that the N-terminal ≈10 residues in TodX and TbuX have high B-factors or do not show electron density at all (Fig. 1B).

Fig. 4.

TodX and TbuX have a channel through the hatch domain. (A) Stereo diagrams viewed from the extracellular side, showing the conformational differences in the hatch domains (most notably the N terminus and helix 1) and the S3 kink region for TodX (P1 crystal form, red), TbuX (blue), and both crystal forms of FadL (white). The superposition (showing the barrels of only FadL and TodX for clarity) was made in Coot (34). The position of the hatch channel is indicated by the asterisk. (B) Stereo cutaway surface view from the side, showing the TodX hatch channel as a continuous black tube. The upper and lower C₈E₄ detergent molecules are shown as orange stick models (with oxygens in red). The residues at the narrowest constriction of the channel (Tyr-9, Gln-83, and Phe-100) are shown in green as stick models (oxygens, red and nitrogens, blue). The hatch domain is shown in pink and the signature NPA sequence in yellow.

Mechanism of Substrate Transport.

Using the two crystal structures of E. coli FadL (21), a transport model was proposed in which hydrophobic substrates are captured from the extracellular medium by a low-affinity-binding groove, followed by diffusion into an adjacent high-affinity-binding site. Subsequently, spontaneous conformational changes of the N terminus, postulated from the observation of different conformations of the N terminus in both FadL structures, result in substrate release from the high-affinity-binding site. As the final and most crucial step of the transport model, further hypothetical conformational changes in the hatch and possibly the S3 kink were proposed that would open a channel for substrate diffusion through the hatch domain into the periplasm (21).

The hatch domains, consisting of three short helices connected by short loops, have very similar folds in FadL and TodX/TbuX (Fig. 4A). However, the positions of the hatch domains within the barrel are clearly different between FadL and TodX/TbuX, with shifts occurring for several of the helices and loops, most notably for the N terminus and helix 1 (residues 11–15; Fig. 4A). These shifts, together with the presence of a much less prominent kink in strand S3, result in the presence of a channel through the hatch in TodX and TbuX. The presence of the hatch channel generates a continuous substrate passageway that leads from the extracellular substrate-binding site, via the hydrophobic channel (Fig. 2), to the periplasm (Fig. 4B). The hatch channel is located at identical positions in TodX and TbuX, on the opposite side from the absolutely conserved NPA sequence (Fig. S2). The channel is lined by (TodX) residues 5, 8–12, 14 (N terminus, hatch helix 1), 24–27 (hatch helix 2), 45, 47, 51 (strand S1), 83 (strand S2), 100, 102–105 (S3 kink), 132, 134, 136 (strand S4), 388, 389, 391 (strand S13), and 426, 428, 430, 432 (strand S14; C terminus; Fig. S3). The narrowest point (≈4.5–5-Å diameter; atom-center to atom-center distance) of the channel is close to residues Gln-83, Phe-100, and Tyr-9 (Fig. 4B). The constriction site observed in the TodX and TbuX structures may be large enough to allow passage of substrate molecules; manual fitting of benzene or toluene within the constriction is possible without having any interatom distances <2.5 Å (atom-center to atom-center). In marked contrast with the exclusively hydrophobic character of the initial substrate passageway (Fig. 2), the hatch channel in TodX/TbuX is lined by both hydrophobic and polar and charged residues (Fig. S4), some of which are not highly conserved among xenobiotic transporters (Fig. S1). We speculate that the more polar character of the hatch channel provides substrate-binding sites of lower affinity compared with those in the hydrophobic channel, which prevent the substrate from stalling in the channel during diffusion toward the periplasm. Thus, the TodX and TbuX structures support the transport model derived from the E. coli FadL structures, with spontaneous conformational changes within the hatch domain and the S3 kink opening a channel for substrate diffusion through the hatch. This hypothesis is supported by the fact that removal of the N-terminal 12 residues from the E. coli FadL structures results in a continuous channel through the FadL hatch domain (Fig. S5).

The presence of a continuous channel through TodX and TbuX might be expected to generate a channel for conductance of small ions in planar lipid bilayer experiments, analogous to what has been observed for many other OM channels. Preliminary experiments with both TbuX and FadL, however, did not show any conductance (data not shown). We speculate that the absence of measurable ion conductance in FadL family members may be due to the hydrophobic character of the initial substrate passageway (Fig. 2), which may preclude the passage of ions.

Based on the new crystal structures of TodX and TbuX, we propose the following general model for transport of BTEX hydrocarbons across the OM, representing the essential first step in the biodegradation of these compounds: (i) substrate capture from the extracellular environment by the hydrophobic cleft in extracellular loop L3; (ii) movement of the substrate through the hydrophobic channel to a position close to the N terminus; (iii) conformational changes in the S3 kink and the hatch domain, resulting in the opening of a continuous channel through the hatch; and (iv) diffusion through the hatch channel and substrate release into the periplasm. Upon emergence from the hatch channel, the hydrocarbons must diffuse through the periplasm and cross the inner membrane to be accessible to the cytosolic mono- or dioxygenase enzymes. Because BTEX hydrocarbons are relatively water-soluble (the aqueous solubility of toluene is ≈6 mM; ref. 24), they are likely to enter and diffuse through the periplasm without the aid of periplasmic-binding proteins. Crossing the inner membrane is likely to occur by spontaneous diffusion, and, like OM transport, is driven by biodegradation occurring in the cytoplasm, providing a sink.

The putative substrate diffusion channel observed in TodX and TbuX is unique among OM proteins for several reasons. First, the distance from the initial substrate-binding site on the extracellular surface (Fig. 2) to the periplasmic exit of the hatch channel is >50 Å. Along much of this length, the channel diameter does not exceed ≈10 Å. This long and narrow channel is very different from those present in other OM proteins, such as porins (OmpC, OmpF) and substrate-specific channels like LamB, which typically have short constrictions (often called “eyelets”) with lengths of ≈5–10 Å (15). Second, the TodX/TbuX channels consist of an initial, very hydrophobic part (Fig. 2) and a more polar part contributed by the hatch channel (Fig. S4). All other known OM channels have entirely polar passageways, in accordance with the polar nature of their substrates.

Besides the FadL family, the only other OM proteins in which the barrel lumen is occluded by hatch domains are the TonB-dependent receptors (TBDRs), which transport large (>800 Da) hydrophilic molecules such as vitamin B12 and iron–siderophore complexes (25). TBDRs have much larger barrels (22 β-strands) than FadL family members (14 β-strands) and consequently much larger hatch domains comprising ≈150–200 residues. TBDRs bind their substrates with high (nanomolar) affinities, require exogenous energy input for transport provided by the proton motive force, and require the inner membrane protein TonB. TonB interacts with the hatch domain of TBDRs, likely resulting in local unfolding of the hatch, generating a transient wide passageway through the hatch for transport of the large substrates (26). This elaborate mechanism may have evolved because a permanently open pore of a size required for passage of TBDR substrates would likely be harmful for the cell. By contrast, the substrates of FadL family members are small, removing the need for formation of a large channel through the hatch. Moreover, substrates likely bind to FadL proteins with relatively low affinities (27), allowing small and spontaneous conformational changes to suffice for transport via passive diffusion.

The current structures of TodX and TbuX support and extend the general transport model for hydrophobic molecules that was proposed previously, based on structures of E. coli FadL (21). The structural data provide a solid framework to test the proposed transport mechanism by site-directed mutagenesis experiments, in combination with in vivo and in vitro substrate-binding and transport assays. Such experiments will also shed light on the structural determinants of substrate specificity of FadL family members. A detailed understanding of how FadL family members transport their hydrophobic substrates not only is of fundamental importance but could also lead to the design of novel hydrophobic drugs and more efficient biodegrading bacterial strains.

Methods

Vector Construction.

Signal sequence cleavage sites were predicted to occur between residues 20 and 21 for P. putida F1 TodX and residues 23 and 24 for R. pickettii PKO1 by using the SignalP program (28). For expression and OM localization of TodX and TbuX, the mature todX and tbuX genes (lacking the endogenous signal sequences and the first two residues, and modified with a C-terminal hexahistidine tag) were amplified by PCR from P. putida F1 (American Type Culture Collection 700007) genomic DNA and from R. pickettii PKO1. The mature gene fragments were cloned in-frame with the E. coli FadL signal sequence (plus the first two residues of the mature FadL sequence) into the pBAD22 vector (29). The resulting plasmids were designated pTodX(native) and pTbuX(native). For expression of TodX in inclusion bodies, plasmid pTodX(ib) was constructed by cloning the mature TodX gene sequence (corresponding to residues 22–453), with a C-terminal hexahistidine tag, downstream of the arabinose-inducible promoter in pBAD22 (29).

Protein Expression and Purification.

Proteins were overexpressed in E. coli C43(DE3) (30) by induction with 0.2% (wt/vol) arabinose in 2×YT medium for 3 h at 37°C. Selenomethionine-substituted proteins were produced in E. coli C43(DE3) by inhibition of the methionine biosynthesis pathway (31). For native TodX and TbuX proteins, membrane protein purification was performed as described (21). TodX protein expressed from plasmid pTodX(ib) was purified from inclusion bodies and refolded as follows. Cells were harvested, resuspended in TSB buffer [20 mM Tris, 300 mM NaCl, 10% (vol/vol) glycerol, pH 8] and ruptured by passage at 15,000–20,000 psi through a microfluidizer (Avestin). Inclusion bodies and cell membranes were collected by centrifugation at 17,000 × g, washed in 10 mM Tris (pH 8), 50 mM NaCl, and resuspended in 10 mM Tris (pH 8), 50 mM NaCl containing 8 M urea. The suspension was stirred for 2 h at room temperature. After centrifugation (46,000 × g), the supernatant containing urea-denatured protein was slowly diluted 10-fold in 1% (wt/vol) N-lauroylsarkosine (sarkosyl) in TSB. Protein folding was achieved by stirring the sarkosyl solution at 4°C for 18 h. Folded TodX protein was purified by nickel affinity and gel filtration chromatography as described for FadL (21). Success of the sarkosyl folding was confirmed by observing the heat-modifiability on SDS/PAGE gels (32); before boiling, TodX migrates on SDS/PAGE gels with an apparent molecular mass of ≈28 kDa, and after boiling, the migration shifts to ≈46 kDa (data not shown).

Crystallization.

Crystallization trials were set up by using the hanging-drop technique with commercially available crystallization screens (The Classics and MB Class II screens, Qiagen). For TodX, the best diffracting crystals (native and selenomethionine) were obtained at 22°C in 25% (wt/vol) PEG4000, 0.2 M ammonium sulfate, 0.1 M sodium acetate (pH 4.6). TbuX crystallized at 22°C in 20% (wt/vol) PEG4000, 0.1 M magnesium acetate, 0.05 M sodium acetate (pH 4.5). Crystals were flash-frozen (100 K) in reservoir solution containing C₈E₄ and 20% (vol/vol) glycerol by plunging in liquid nitrogen.

Data Collection and Structure Determination.

Datasets for the TodX and TbuX crystals were collected on beamline X6A at the NSLS (Brookhaven National Laboratory, Upton, NY) and processed using HKL2000 (33). For selenomethionine-substituted TodX, diffraction data were collected at the selenium peak wavelength. Selenium sites were identified using SOLVE (34) and refined using SHARP (35). This structure was used to solve the structure of the native TodX crystal by molecular replacement using PHASER (36). For all structures, model building was done manually using Coot (37), and refinement was done with CNS (38).

For TbuX, an initial model was obtained from a dataset collected from a selenomethionine-substituted TbuX crystal that diffracted to 3.2 Å at the selenium peak wavelength. A partial model was built and used to solve the structure of a native TbuX crystal (space group F222) by molecular replacement using PHASER (36). Model building was done manually using Coot (37), and refinement was done with CNS (38).

TodX and TbuX Activity Assays Toward Fatty Acid.

Plasmids pBAD22, pFadL (21), pTodX(native), pTbuX(native), and pTsx (39) were transformed into E. coli LS6164 (ΔfadR ΔfadL) cells (40). The TodX L166R/L326K mutant was constructed using the QuikChange Site-directed Mutagenesis kit (Stratagene). Transport assays with [³H-9,10]-oleic acid (specific activity 40 Ci mmol⁻¹; Sigma) were performed in E. coli LS6164 (ΔfadR ΔfadL) cells as described by Kumar and Black (22), except cells were induced with 0.001% (wt/vol) arabinose for 1 h at 37°C, and chloramphenicol was omitted from the buffers. To determine the presence of FadL, TodX, TbuX, or Tsx proteins in the OM, cells were incubated with BugBuster (Novagen) while shaking for 20 min at 25°C and centrifuged for 10 min at 10,000 × g to separate solubilized proteins and inclusion bodies. The soluble and membrane proteins were subjected to western immunoblotting using an anti-His antibody (Qiagen).