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Crystal structure of halogenase PltA from the pyoluteorin biosynthetic pathway
Journal of Structural Biology xxx (2015) xxx–xxx
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Crystal structure of halogenase PltA from the pyoluteorin biosynthetic pathway Allan H. Pang, Sylvie Garneau-Tsodikova, Oleg V. Tsodikov ⇑ Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536-0596, USA
a r t i c l e
i n f o
Article history: Received 28 August 2015 Received in revised form 24 September 2015 Accepted 25 September 2015 Available online xxxx Keywords: Chlorination FAD Flavoprotein Halogenation Natural product
a b s t r a c t Pyoluteorin is an antifungal agent composed of a 4,5-dichlorinated pyrrole group linked to a resorcinol moiety. The pyoluteorin biosynthetic gene cluster in Pseudomonas fluorescens Pf-5 encodes the halogenase PltA, which has been previously demonstrated to perform both chlorinations in vitro. PltA selectively accepts as a substrate a pyrrole moiety covalently tethered to a nonribosomal peptide thiolation domain PltL (pyrrolyl-S-PltL) for FAD-dependent di-chlorination, yielding 4,5-dichloropyrrolyl-S-PltL. We report a 2.75 Å-resolution crystal structure of PltA in complex with FAD and chloride. PltA is a dimeric enzyme, containing a flavin-binding fold conserved in flavin-dependent halogenases and monooxygenases, and an additional unique helical region at the C-terminus. This C-terminal region blocks a putative substrate-binding cleft, suggesting that a conformational change involving repositioning of this region is necessary to allow binding of the pyrrolyl-S-PltL substrate for its dichlorination by PltA. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Over 4500 known natural products are halogenated (Gribble, 2003). About 95% of them are chlorinated (50%) and brominated (45%), while the rest are iodinated and fluorinated. Halogenation has been demonstrated to impart bioactive properties to several natural products. For example, the deschloro analogues of balhimycin and clorobiocin were 8-fold less active as antibiotics against Bacillus subtilis than their chlorinated counterparts (Bister et al., 2003; Eustaquio et al., 2003). The halogenated sesterterpenes neomangicol A and B displayed potent cytotoxicity against human colon cancer cells, while their non-halogenated analogues were inactive (Renner et al., 1998). Similarly, rebeccamycin analogues lacking a chlorine substituent were devoid of the antimicrobial activity of their parent compound, presumably due to the lack of cell membrane permeability (Rodrigues Pereira et al., 1996).
Abbreviations: a-KG, a-ketoglutarate; BSA, bovine serum albumin; DGGR, dige ranylgeranylglycerophospholipid reductase; FAD, flavin adenine dinucleotide; 50 FDAS, 50 -fluoro-50 -deoxyadenosine synthase; GR, glutathione reductase; HOCl, hypochlorous acid; HPO, haloperoxidase; LB, Luria–Bertani; NRPS, nonribosomal peptide synthetase; PEG, polyethylene glycol; p-HBH, p-hydroxybenzoate hydroxylase; PKS, polyketide synthase; SAM, S-adenosyl-L-methionine; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; T domain, thiolation domain. ⇑ Corresponding author. E-mail address: [email protected] (O.V. Tsodikov).
Enzymatic chlorination can occur on a wide range of carbon centers (Fig. 1a). The antibiotic chloramphenicol bears chlorinated aliphatic moieties (Podzelinska et al., 2010), while the antitumor agent C-1027 contains a chlorinated enediyne at the C3 position of the b-tyrosyl moiety (Hu et al., 1988). Aromatic chlorination can also be observed, as in the antitumor agent rebeccamycin (Bush et al., 1987) and the antibiotics chondrochloren A and B (Jansen et al., 2003). Furthermore, a heteroaromatic pyrrole ring can be chlorinated: the antifungal agents pyrrolnitrin (Kirner et al., 1998) and pyoluteorin (Nowak-Thompson et al., 1999), both produced by Pseudomonas sp., contain mono- and dichlorinated pyrrole rings, respectively. Biochemical and structural information on several halogenase enzymes indicated that they employ diverse mechanisms. There are four known classes of halogenases: haloperoxidases (HPOs), non-heme iron-dependent halogenases, SAM halogenases, and flavin-dependent halogenases. HPOs catalyze electrophilic chlorination, bromination, and iodination reactions using hydrogen peroxide as an intermediate reactive species. HPOs are diverse: they can be heme-dependent (Sundaramoorthy et al., 1995) or vanadium-dependent (Weyand et al., 1999). These enzymes have broad substrate specificity, owing to the fact that the highly reactive halogen species generated in the active site diffuses in the substratebinding pocket and even outside of the active site. Several crystal structures of different types of bacterial and eukaryotic HPOs have been determined: a heme-dependent fungal chloroperoxidase from
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Please cite this article in press as: Pang, A.H., et al. Crystal structure of halogenase PltA from the pyoluteorin biosynthetic pathway. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.013
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A.H. Pang et al. / Journal of Structural Biology xxx (2015) xxx–xxx
Fig. 1. (a) Chlorine-containing natural products with their respective proposed or observed halogenases catalyzing the chlorination at a specific carbon center. Pyoluteorin and the enzyme (PltA) responsible for chlorination of its pyrrole moiety, studied herein, are marked by a blue box. Proteins with the names in blue have been structurally characterized and will be further described in subsequent figures. The enzymes with the names in black have been biochemically, but not structurally, characterized. (b) The plt gene cluster responsible for the production of pyoluteorin in Pseudomonas sp. Light blue, gray, and dark blue arrows represent genes encoding for enzymes previously biochemically characterized, not studied, and studied herein, respectively. (c) Formation of 4,5-dichloropyrrolyl-S-PltL during pyoluteorin biosynthesis. The pathway consists of the formation of pyrrolyl-S-PltL using L-proline as a starting substrate by a set of enzymes (in black) and the dichlorination of the pyrrole moiety by PltA and associated cofactors (enclosed in a blue box), as previously reported by Walsh and colleagues. The second half of the reaction is carried out by the PKS enzymes (colored in gray), which are yet to be biochemically characterized.
Caldariomyces fumago (Sundaramoorthy et al., 1995), a vanadiumdependent bacterial iodoperoxidase Zg-VIPO1 from Zobellia galactanivorans (Fournier et al., 2014), horseradish peroxidases (Henriksen et al., 1998), and lactoperoxidases from mammals (Fenna et al., 1995; Sheikh et al., 2009; Singh et al., 2008, 2010). Non-heme iron-dependent halogenases catalyze radical halogenation reactions at aliphatic carbon sites. This group of enzymes specifically acts on carrier protein-tethered substrates and requires the use of molecular oxygen for activation of halide ions and a-ketoglutarate (a-KG). There are currently three crystal structures of Fe(II)/a-KG-dependent halogenases available: CurA (Khare et al., 2010), CytC3 (Wong et al., 2009), and SyrB2 (Blasiak et al., 2006) from the curacin A, the c,c-dichloroaminobutyrate, and the syringomycin biosynthetic pathways, respectively. SAM halogenases catalyze a nucleophilic substitution reaction, upon which a desolvated halide attacks on and displaces an L-methionine residue from SAM, with formation of 50 -fluoro (chloro)-50 -deoxyadenosine (Dong et al., 2004; Eustáquio et al., 2008). Flavin-dependent halogenases (Table 1) require reduced flavin (FADH2), which can be covalently (Podzelinska et al., 2010) or non-covalently (Bitto et al., 2008; Buedenbender et al., 2009; Dong et al., 2005; Zhu et al., 2009) bound to the enzyme. Spectroscopic studies revealed formation of the FAD-4a-OOH intermediate upon reaction of FADH2 and O2 (Yeh et al., 2006). This intermediate can react with a nearby chloride ion to yield highly reactive hypochlorous acid (HOCl). HOCl is then proposed to react with a conserved lysine residue to form a chloramine intermediate. In RebH and PrnA, the l-tryptophan
substrate is positioned for chlorination near this lysine (Yeh et al., 2007). Flavin-dependent halogenases can be classified as one of two variants based on their substrates. Variant A enzymes catalyze halogenation of small molecules, as exemplified by tryptophan halogenases (Bitto et al., 2008; Dong et al., 2005; Zhu et al., 2009). Variant B enzymes halogenate substrates that are tethered to a specific thiolation (T) domain. SgcC3 (Lin et al., 2007) and PltA (Dorrestein et al., 2005) (Table 1) are two functionally characterized Variant B enzymes. PltA is one of the first flavin-dependent halogenases to be functionally characterized, by Walsh and co-workers (Dorrestein et al., 2005). The pltA gene and the other biosynthetic genes (pltB, pltC, pltD, pltE, pltF, pltG, pltL, pltM, and pltR) are a part of the biosynthetic gene cluster for the antifungal agent pyoluteorin in Pseudomonas fluorescens Pf-5 (Fig. 1b). Even though PltM and PltD have also been originally annotated as putative halogenases based on their 25% sequence identity to each other and to PltA, only PltA was demonstrated to be an active enzyme in vitro capable of installing both chlorines present in pyoluteorin (Dorrestein et al., 2005). The PltA substrate, a thiolation domain-linked pyrrole (pyrrolyl-S-PltL), requires several enzymatic activities to be formed (Fig. 1c). This substrate gets dichlorinated by PltA with the aid of a coupled flavin reductase for subsequent use in the biosynthetic pathway. The formation of the pyrrole ring and its loading onto a T domain in other natural products was previously demonstrated (Fig. 1c) (Garneau et al., 2005; Thomas et al., 2002). Currently, only
Please cite this article in press as: Pang, A.H., et al. Crystal structure of halogenase PltA from the pyoluteorin biosynthetic pathway. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.013
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A.H. Pang et al. / Journal of Structural Biology xxx (2015) xxx–xxx Table 1 Comparison of homologous FADH2-dependent halogenases and related enzymes with known crystal structures. Enzyme
PDB code
Species
Substrate
Natural product
Oligomeric state
PltA CmlS –a CndH –b PrnA
5DBJ 3I3L 3NIX 3E1T 2PYX 2AQJ
Pseudomonas fluorescens Streptomyces venezuelae Cytophaga hutchinsonii Chondromyces crocatus Shewanella frigidimarina Pseudomonas fluorescens
Pyrrolyl-S-PltL Not known Not knowna Assumed T-bound l-Tyr Not knownb L-Trp
Pyoluteorin Chloramphenicol Unknowna Chondrochloren Unknownb Pyrrolnitrin
Dimer Monomerc Monomerc Monomer Dimer Dimer
PyrH
2WET
Streptomyces rugosporus
L-Trp
Pyrroindomycin
Dimer
RebH
2O9Z
Lechevalieria aerocolonigenes
L-Trp
Rebeccamycin
Dimer
Crystal structures solved by: a Northeast Structural Genomics Consortium. b Joint Center for Structural Genomics, no additional experiment and information available other than the structures uploaded in RCSB. c Based on analysis via PDBe PISA server.
PltA and SgcC3, a structurally uncharacterized halogenase involved in the production of an antitumor agent C-1027, have been biochemically demonstrated to discriminate between a free small molecule substrate (pyrrole-2-carboxylate for PltA and tyrosine for SgcC3) and a carrier protein-tethered substrate (pyrrolyl-SPltL for PltA and tyrosyl-S-SgcC2 for SgcC3), accepting only the carrier protein-tethered substrates for halogenation (Dorrestein et al., 2005; Lin et al., 2007). The paucity of structural information about these intriguing Variant B enzymes prompted this structural study of PltA. Here, we present a crystal structure of PltA from P. fluorescens Pf-5 in complex with its co-factors FAD and chloride. The crystal structure reveals dimeric organization and unique structural features of PltA. 2. Materials and methods 2.1. Expression and purification of PltA The pltA gene was excised from the previously reported expression construct pET28a-pltA (Dorrestein et al., 2005) by NdeI and XhoI enzymes and ligated into Int-pET19b-pps vector (Biswas and Tsodikov, 2008), which yields the protein with an N-terminal decahistidine tag cleavable with Prescission protease (GE Healthcare). This plasmid was transformed into Escherichia coli BL21 (DE3) chemically competent cells, with the transformation mixture plated on Luria–Bertani (LB) agar supplemented with 100 lg/mL ampicillin (AMP). A colony from the transformation plate was inoculated into 5 mL of LB broth containing 100 lg/mL AMP (LB/Amp) and incubated with shaking at 37 °C. This culture was then used to inoculate a 2-L LB/Amp culture, which was then incubated with shaking at 18 °C. When the culture reached an attenuance at 600 nm of 0.5, the culture was induced by the addition of isopropyl-b-D-1-thiogalactopyranoside (IPTG; final concentration of 200 lM) and kept shaking at 18 °C overnight. The cells were harvested by centrifugation at 5000g for 10 min. The cell pellet was resuspended in lysis buffer (40 mM Tris–HCl, pH 8.0, 400 mM NaCl, 10% v/v glycerol, 2 mM b-mercaptoethanol). The cells were then lysed by intermittent sonication on ice. The lysate was passed through a 5-mL HisTrap HP Ni2+ column (GE Healthcare) equilibrated in lysis buffer. The column was washed with 100 mL of lysis buffer containing 50 mM imidazole. PltA was eluted from the column with 6 mL of lysis buffer containing 500 mM imidazole. The eluant was then passed through a size-exclusion HiPrep Sephacryl S-200 HR column (GE Healthcare) equilibrated in gel filtration buffer (40 mM Tris–HCl, pH 8.0, 100 mM NaCl, 2 mM b-mercaptoethanol). Fractions containing pure PltA, as determined by SDS–PAGE gel analysis (Supplementary Fig. S1b), were pooled and concentrated using an Amicon Ultra-15 (10-kDa molecular weight cut-off) centrifugal filter device (Millipore Corporation,
MA) to 10 mg/mL. The protein was frozen by quick immersion in liquid nitrogen and stored at 80 °C. The protein preparation had a yellow color due to bound FAD (Supplementary Fig. S4a).
2.2. Crystallization of PltA Initial crystallization conditions were identified by 1536condition high-throughput crystallization screening of PltA at the Hauptman-Woodward Medical Research Institute (Luft et al., 2003) by the microbatch method in mineral oil. The crystals were grown by vapor diffusion in hanging drops containing 0.5 lL of 10 mg/mL PltA, 0.5 lL of a reservoir solution (0.1 M Bis-Tris propane, pH 7.0, 50 mM magnesium sulfate heptahydrate, 20% w/v PEG 8000) and 0.5 lL of Prescission protease in the protease dilution buffer (40 mM Tris–HCl, pH 8.0, 100 mM NaCl), at the stoichiometry of 1:100 of PltA:protease in the drop. Single yellow crystals were gradually transferred into a cryoprotecting solution (0.1 M Bis-Tris propane, pH 7, 50 mM magnesium sulfate heptahydrate, 20% w/v PEG 8000, 20% glycerol), and frozen in liquid nitrogen by quick immersion.
Table 2 X-ray diffraction data collection and refinement statistics for the PltA structure. Data collection Space group Number of monomers per asymmetric unit Unit cell dimensions a, b, c (Å) a, b, c (°) Resolution (Å) I/r Completeness (%) Redundancy Rmerge Number of unique reflections Structure refinement statistics Resolution (Å) R (%) Rfree (%) Bond length deviation (rmsd) from ideal (Å) Bond angle deviation (rmsd) from ideal (°) B factors (without/with TLS contribution, Å2) Overall Solvent Ligand Ramachandran plot statisticsb % of residues in most allowed regions % of residues in additional allowed regions % of residues in generously allowed regions % of residues in disallowed regions a b
C2 5 242.9, 95.0, 102.1 90, 91.2, 90 50.00–2.75 (2.80–2.75)a 17.56 (1.98) 95.1 (94.4) 4.6 (4.7) 0.12 (0.60) 56,814 (3976) 40.00–2.75 (2.82–2.75) 23.9 (31.3) 29.1 (34.7) 0.007 1.25 45.2/79.2 68.2/68.2 34.0/34.0 89.0 10.2 0.8 0.0 (0 residues)
Numbers in parentheses indicate the values in the highest-resolution shell. Indicates PROCHECK statistics (Laskowski et al., 1993).
Please cite this article in press as: Pang, A.H., et al. Crystal structure of halogenase PltA from the pyoluteorin biosynthetic pathway. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.013
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2.3. Data collection and crystal structure determination X-ray diffraction data were collected at beamline 22-ID of the Advanced Photon Source of the Argonne National Laboratory (Argonne, IL) at 100 K and processed with program HKL2000 (Otwinowski et al., 1997). The structure of PltA was determined by molecular replacement by using PHASER (McCoy et al., 2007), where we used as a search model the structure of a putative dehydrogenase from Cytophaga hutchinsonii (PDB accession code: 3NIX, residues 7–386), in which the C-terminal region was deleted. The structure of PltA was built by 80 iterative rounds of refinement with REFMAC (Murshudov et al., 1997) and manual building with COOT (Emsley and Cowtan, 2004). When most of the structure of each of the five monomers in the asymmetric unit was built, NCS restraints (tying all monomers) were used for several cycles, before building slightly variable structural regions. The data collection and refinement statistics are provided in Table 2. The solvent accessible surface areas were calculated by using Surface Racer program, with 1.4 Å as the solvent probe radius (Tsodikov et al., 2002). Coordinates and structure factor amplitudes have been deposited in the Worldwide Protein Data Bank site with the accession code 5DBJ.
3. Results 3.1. Dimeric organization of PltA A crystal structure of halogenase PltA from P. fluorescens Pf-5 (Fig. 2a, left panel) was determined by molecular replacement and refined at the resolution of 2.75 Å; the structure contained five PltA molecules per asymmetric unit (Table 2). The polypeptide backbone for residues 2–438 (out of 449 annotated residues) and most of the side chains were clearly defined in the electron density map with the exception of a short disordered loop (residues 408– 411) in three out of five molecules. A major part of a PltA is an FADbinding Rossmannoid fold, which starts at the N-terminus and consists of a conserved b-sheet flanked by helices. This basic fold is present in other FAD-dependent halogenases, monooxygenases, and glutathione reductase (GR) superfamily enzymes (Fig. 2). The C-terminal regions of PltA (residues 382–449) and that of other homologues are highly divergent (Fig. 2). Other structural differences include lengths, conformations, and even topologies of some b-strand connecting regions. The five copies of PltA in the asymmetric unit are organized as two nearly identical dimers, with the remaining copy forming a similar dimer with its symmetry mate. A dimer of PltA is formed by a 2-fold rotational symmetry (Fig. 3a). Size-exclusion chromatography of PltA indicated a dimeric state of PltA in solution (Supplementary Fig. S1a), consistent with the structural observation. The total of 2304 Å2 of solvent accessible surface area of both PltA monomers is buried in the monomer–monomer interface. This buried area is split approximately equally between polar (1107 Å2) and nonpolar (1197 Å2) contributions, indicating that the dimeric interface is composed of polar contacts (hydrogen bonds and salt bridges) and hydrophobic interactions (Fig. 3b). Specifically the two monomers combine their b-strands b7 (Supplementary Fig. S2) of their Rossmannoid folds in the antiparallel fashion into a conjoined b-sheet, where the main chain carbonyl oxygen of Gln143 of one monomer is hydrogen bonded to the main chain nitrogen of the same residue of the other monomer and vice versa, across the dimeric interface. Other intermolecular contacts between polar and charged residues include salt bridges between Arg128 and Glu40, Asp5 and Arg167, and a hydrogen bond between the side chain carboxyl group of Glu132 and the main chain NH of Glu40, as well as the same contacts made
Fig. 2. Comparison of PltA and structurally related homologues with the conserved FAD monooxygenase domain (pale yellow) and the unique C-terminal domain (blue). The FAD and chloride ions are shown as green sticks and orange spheres, respectively. (a) The overall crystal structure of PltA and the most similar putative halogenase (PDB ID: 3NIX), (b) non-halogenases that are structurally similar to PltA with a small C-terminal lid, examples shown here are p-hydroxybenzoate hydroxylase, p-HBH, (PDB ID: 1PBE) and digeranylgeranylglycerophospholipid reductase, DGGR, (PDB ID: 3OZ2), (c) tryptophan halogenases PrnA with co-factor FAD (PDB ID: 2AQJ) and RebH without the co-factor FAD (PDB ID: 2O9Z), and (d) non-tryptophan halogenases CndH (PDB ID: 3E1T) and CmlS (PDB ID: 3I3L). CndH is composed of a disordered C-terminal region and CmlS forms a big arch-like C-terminal lid.
symmetrically. Nonpolar interactions are between two symmetric patches of hydrophobic surface formed by side chains of Phe142, Val145, Leu164, Val169, Ile171, and the aliphatic stem of Arg167 of each monomer. A well-characterized L-Trp halogenase PrnA is also dimeric through Rossmannoid fold interactions, but its dimeric interface is different from that of PltA (Fig. 3c). 3.2. The FAD binding site Strong omit difference electron density corresponding to a bound FAD molecule was found in all five PltA monomers in the
Please cite this article in press as: Pang, A.H., et al. Crystal structure of halogenase PltA from the pyoluteorin biosynthetic pathway. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.013
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residues from several loops. The isoalloxazine ring is buried away from solvent deep inside the cleft, whereas the adenosine diphosphate portion is bound at the entrance of the cleft. The isoalloxazine ring is sandwiched between two regions of PltA: residues 44–47 and residues 320–329 (Fig. 4b). Hydrophobic interactions with the Ca of Gly45 and the side chain of Val44 on one face of the ring and with the side chains of Phe320, Pro323, and the Ca–Cb of Ser326 on the other face appear to hold the ring in place. The hydrophobic side of the ring is flanked by Ala203, Trp239, Tyr299, and the aliphatic part of Glu296. On the polar side of the FAD ring, the oxygen atom at position 2 is situated in an oxyanion hole-like environment. This oxygen is engaged in a bifurcated hydrogen bond, accepting hydrogens from a conserved buried water molecule and an amide nitrogen of Val329. The backbone amide nitrogens of the adjacent residues 326–328 interact with a bound chloride ion, which is located at the nitrogen-containing side of the isoalloxazole ring of the FAD (Fig. 4). The chloride is also held sterically by Phe320, Pro323, and Trp371 (Fig. 4b). This chloride location is conserved in FAD-dependent halogenases (Bitto et al., 2008; Dong et al., 2005; Zhu et al., 2009). A second bound chloride ion was found 25 Å away, at a site distant from the FAD. This chloride is held electrostatically by the guanidinium group of Arg277 and the carboxamide nitrogen of Asn279, as well as the main chain nitrogens of Asn279 and Glu278 (Supplementary Fig. S5). Other halogenases do not contain a bound chloride at an analogous site, consistent with the lack of conservation of Arg277 and Asn279 (Supplementary Fig. S2). However, this sec-
Fig. 3. Dimeric organization of PltA and PrnA. (a) The dimer of PltA, with the residues in the dimeric interface and the bound co-factor FAD shown as sticks. (b) A zoomed-in view of the dimeric interface of PltA. Residues of only one monomer are labeled; the residues of the other monomer are related by twofold rotational symmetry. (c) A dimer organization of PrnA (PDB ID: 2AQJ). One of the monomers (left) is oriented similarly to a monomer in PltA (left of panel a).
asymmetric unit (Figs. 4a and S3). The absorbance spectrum of the purified PltA preparation (yellow in color) indicated that FAD was indeed co-purified with the protein. The absorbance spectrum of PltA-FAD is consistent with the fully oxidized state of the bound flavin (Supplementary Fig. S4), as expected based on the purification procedure. The FAD is situated in a deep cleft, cradled by
Fig. 4. The FAD-binding site of PltA. (a) The bound FAD and chloride are well defined by the Fo Fc omit map contoured at 3r (the blue mesh), generated without the FAD and chloride. (b) A zoom-in view of the isoalloxazine ring of the bound FAD with key residues interacting with FAD explicitly shown.
Please cite this article in press as: Pang, A.H., et al. Crystal structure of halogenase PltA from the pyoluteorin biosynthetic pathway. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.013
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ondary chloride site may serve for storage of chloride, simply by increasing local concentration of chloride ions near PltA. PltA contains a so-called G-box GxGxxG (residues 12–17) that is conserved in FAD-dependent halogenases (Supplementary Fig. S2) and in the GR superfamily (Dym and Eisenberg, 2001), and is involved in binding of dinucleotides (Wierenga et al., 1983). The G-box forms a hairpin between b-strand b1 and helix a1, whose backbone amide nitrogens are engaged in hydrogen bonds with the nucleotide diphosphate moiety of the FAD and form a sterically complementary surface. The diphosphate group is also held by electrostatic interactions with Arg41, His43, and Arg123 and the positively charged N-terminal end of helix a1 (Fig. 5a). A buried conserved water molecule is bound nearby, mediating hydrogenbonding interactions between the diphosphate moiety and the protein. Both Arg residues are conserved among non-tryptophan halogenases, whereas the His residue is unique to PltA. The residues Arg41 and His43 are located in a loop, conserved in CmlS, CndH, and 3NIX. The respective region in PrnA is different in sequence and conformation (Figs. 5b and S2). This PltA loop contains a cis peptide bond between Phe39 and Glu40 (Fig. 5a). This stereochemistry appears to be important for orienting the side chain of Phe39, which serves a key structural role, as the phenyl ring of Phe39 makes van der Waals contacts with the 30 -hydroxyl group of the FAD ribose, Glu35, Arg41, and the GxGxxG motif. A number of PltA homologues (p-HBH, CndH, CmlS, 3NIX, and DGGR) contain a cis peptide bond at this position. Three of these (CndH, CmlS, and 3NIX) also contain a Phe structurally analogous to Phe39 of PltA (Supplementary Fig. S2). In DGGR, a cis peptide bond (Ser40Pro41) can be found in an analogous loop, but it is not superimposable with PltA. The tryptophan halogenase PrnA (Yeh et al., 2007)
Fig. 5. Comparison of the structural environment of the adenosine diphosphate moiety of FAD of (a) PltA and (b) PrnA. Residue Gly13 is located in the G-box motif in both enzymes. The water molecule in panel b is represented by a turquoise sphere labeled H2O.
does not contain a cis peptide in the analogous loop, but this loop is located closer to FAD (6 Å, compared to 10 Å in PltA) (Fig. 5b), apparently to provide an alternative FAD-binding mode. Additionally, the 20 - and 30 -hydroxyl groups of the ribose form hydrogen bonds donating hydrogens to the carboxyl group of Glu35, which is also positioned by the GxGxxG motif, and the 30 hydroxyl also accepts a hydrogen from the guanidinium group of Arg41 (Fig. 5a). Glu35 is nearly universally conserved in PltA and its homologues (Supplementary Fig. S2). Interestingly, despite the conservation, the analogue of Glu35 in PrnA (Glu38) does not form direct hydrogen bonds with the ribose ring of the FAD and points away from the FAD (Fig. 5b). Instead, the oxygen of the 20 -hydroxyl group of the ribose forms a hydrogen bond with the amide nitrogen of the subsequent residue (Ser39), while the 30 -hydroxyl group forms a hydrogen bond with the amide nitrogen of Gly13 (same residue number as in PltA) from the GxGxxG conserved sequence motif and with a water molecule that occupies the location of the carboxyl group of Glu35. 3.3. The unique C-terminal region of PltA A BLAST search for structurally characterized homologues yielded CmlS, 3NIX, and CndH, which are 25–27% identical to PltA (Table 1). While the FAD-binding fold of the N-terminal region is conserved among PltA and these proteins, the C-terminal regions are divergent in sequence, length (Supplementary Fig. S2), and structure (Fig. 2). The C-terminal region of PltA is composed of three helices (Fig. 2a). In contrast, the C-terminal region of CmlS is a winged helix structure, while in CndH, the C-terminal region is largely disordered (Fig. 2d). A previous proposal correlating classification between Variant A (small molecule substrate; Fig. 2c) and B (thiolation domain-tethered substrate; Fig. 2d (note that CndH is a putative Variant B enzyme)) halogenases with the degree of structural order of the C-terminal region (ordered in Variant A, disordered in Variant B) (Podzelinska et al., 2010), is directly disproved by the PltA structure: PltA is a demonstrated Variant B enzyme (Dorrestein et al., 2005) containing a mostly ordered Cterminal region (Fig. 2a). Nevertheless, the divergence of the C-terminal regions appears to be a strategy employed by halogenases to ensure substrate discrimination (Podzelinska et al., 2010). For example, the C-terminal region of PrnA forms a part of the binding pocket for the L-Trp substrate (Fig. 6b). Crystal structures of PrnA with bound L-Trp substrate (PDB: 2AQJ) and chlorotryptophan product (PDB: 2AR8) indicated that the chlorinated carbon atom of the product is in the immediate vicinity of the functionally important lysine residue, which is conserved in known FAD-dependent halogenases, including PltA (Lys73; Figs. 6a and S2). Lys73, located 10 Å away from the bound FAD, therefore, defines the putative substrate-binding site in PltA. The C-terminal region of PltA obstructs this site from the access of the large thiolation domain-tethered pyrrole substrate. More distant homologues of PltA include two non-halogenases: the hydroxylase p-HBH and the reductase DGGR (Fig. 2b). p-HBH and DGGR function differently from each other and from halogenases. p-HBH hydroxylates its substrate p-hydroxybenzoate by molecular oxygen to form 3,4-dihydroxybenzoate (Schreuder et al., 1989), whereas DGGR hydrogenates and converts unsaturated archaeols to saturated archaeols, which is an important step in the biosynthesis of archaeal membrane lipids (Xu et al., 2010). The functional difference between the two non-halogenase homologues is reflected by the structural environment of the active site tunnel. Notably, Lys73 is not conserved in p-HBH, while it is present in DGGR, but it points towards the surface of the enzyme instead of the substrate-binding tunnel. While the C-terminal regions of PltA and these two enzymes are somewhat similar, the
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respective parts from each monomer also provide a binding surface for the nucleoside moiety of the bound FAD (Fig. 3a). Therefore, dimerization of PltA may play a structural role in FAD binding, but given the significant size of the monomer–monomer interface, a likely role of the dimerization is to support the structural stability of the protein as a whole. This dimerization interface is not conserved, since not all characterized FAD-dependent halogenases (of either Variant) are dimeric (Table 1) and those that dimerize do so differently from PltA. 4.2. Mechanism of substrate binding and catalysis
Fig. 6. The view into the substrate-binding cavity from the side of the bound FAD in (a) PltA and (b) PrnA. The structures are oriented to visualize the FAD, the L-Trp substrate of PrnA, and the conserved catalytic lysine residue (Lys73 in PltA and Lys79 in PrnA).
substrate binding mechanisms must differ. The hydroxylase p-HBH contains a small opening within the FAD-binding domain for entry and exit of its small molecule substrate p-hydroxybenzoate. The reductase DGGR, due to the small size of its C-terminal region, provides unobstructed access for its large substrate, an unsaturated archaeol, to its binding site. In PltA, the respective openings are sealed off by the C-terminal region.
4. Discussion 4.1. PltA is the first structurally characterized established Variant B FAD-dependent halogenase A decade ago, PltA was demonstrated to halogenate a pyrrole moiety tethered to a thiolation (T) domain on the pathway to pyoluteorin production (Dorrestein et al., 2005). This substrate preference of an FAD-dependent halogenase defined it as a Variant B enzyme. Crystal structures of experimentally confirmed Variant A enzymes (acting on free L-Trp) have been determined (Table 1), and this study is the first structural characterization of an experimentally validated Variant B enzyme. PltA forms a dimer, where the monomer–monomer interface is distant from the catalytic center, but this interface includes a conjoined b-sheet, whose
The location of the substrate-binding site in halogenases is not trivially specified by the location of a bound FAD or a halide ion, because a reactive halogen species (such as HOCl) can diffuse through channels in the protein and react at a distance from the flavin. The halogenation site in FAD-dependent halogenases was defined by the crystal structures of tryptophan halogenases PrnA, RebH, and PyrH in complex with either the tryptophan substrate or the chlorotryptophan product (Bitto et al., 2008; Dong et al., 2005; Yeh et al., 2007; Zhu et al., 2009). Given the conservation of the fold of the catalytic domain of both Variant A and B halogenases, the bound FAD and the catalytic Lys residue, the location of the halogenation center is likely analogous in Variant A and B halogenases. In PltA, this putative substrate-binding site is obstructed by the C-terminal helical region (Figs. 6a and 7a). PltA is in this closed state, presumably, to select against binding and halogenation of small molecule substrates such as pyrrole-2-carboxylate. Given the large size of the pyrrolyl-S-PltL conjugate substrate, its entry through the FAD-binding cleft cannot occur for steric reasons, and no other channel with access to the halogenation site exists (Fig. 2a). A conformational change that most likely involves a movement of the C-terminal region to open access to the halogenation site to the carrier protein-tethered pyrrolyl moiety is required. The structure of chloramphenicol halogenase CmlS (whose exact substrate remains unknown) also contains a blocked putative substrate-binding site and is expected to undergo a conformational change to bind its substrate (Podzelinska et al., 2010). The blockage in CmlS is not as severe as in PltA and it can be relieved by a movement of only the last structured loop (Podzelinska et al., 2010) since the structure of the C-terminal region in CmlS is different. In the chondrochloren halogenase CndH, the C-terminal region is partially unstructured; therefore, the access to the halogenation site appears to be unobstructed (Buedenbender et al., 2009). With these considerations in mind, we examined whether an appropriate binding surface for a pyrrole-phosphopantetheinyl arm conjugate could be exposed upon removal of the C-terminal region of PltA from its crystal structure. Indeed, a cleft of an appropriate size gets opened as a result (Fig. 7b), abutting the putative catalytic Lys73 on one side and punctuated by a newly exposed positively-charge surface patch formed by Arg337 on the other side. The pyrrolyl and the phosphopantetheinyl arm to which it is attached fit snugly into this cleft in an extended conformation. The phospho moiety of the model substrate is in favorable electrostatic contact with Arg337. We propose that interactions with this substrate, possibly also involving the T domain, could trigger a conformational change to displace the C-terminal region of PltA for this binding interaction. The oxidation reaction cascade catalyzed by flavin-dependent monooxygenases is well understood (Huijbers et al., 2014); a similar halogenation mechanism has been proposed for tryptophan halogenases (Bitto et al., 2008; Dong et al., 2005; Zhu et al., 2009). A spectroscopic study of RebH showed that an FADH2 molecule bound to the halogenase reacted with molecular oxygen to form a highly reactive FAD-4a-OOH intermediate (Yeh et al., 2006). Since
Please cite this article in press as: Pang, A.H., et al. Crystal structure of halogenase PltA from the pyoluteorin biosynthetic pathway. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.013
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tup funds from the College of Pharmacy at the University of Kentucky (to S.G.-T. and O.V.T.). We thank the staff of sector 22 (SER-CAT) of the Advanced Photon Source at the Argonne National Laboratories for their assistance with the remote X-ray diffraction data collection. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jsb.2015.09.013. References
Fig. 7. The surface view of the putative substrate binding cleft of PltA (a) prior and (b) after the removal of the C-terminal region. The pyrrole bound to the phosphopantetheinyl arm substrate was modeled into PltA. The electrostatic potential is mapped onto the surface: white, red, and blue colors representing nonpolar, negatively, and positively charged surface, respectively. The positively charged patch due to Arg337, predicted to interact with the phospho group of substrate, is labeled.
the substrate is bound at a site remote from the FAD, a diffusible HOCl species must then form. The HOCl diffuses to the catalytic Lys (Lys73 in PltA, Lys79 in PrnA and RebH, Lys71 in CmlS, Lys76 in CndH) and modifies this Lys to an aminochloride releasing a water molecule. The aminochloride then converts the aromatic substrate to a Wheland complex. A deprotonation step is then necessary for the release of the halogenated aromatic product. The structural and mutagenesis studies of tryptophan halogenase PrnA led to the proposal that the proton abstraction is achieved by the nearby base Glu346 (Dong et al., 2005; Flecks et al., 2008). Sequence alignment of the glutamate residue reveals that it is conserved among tryptophan halogenases, but not with other homologues such as PltA, CmlS, and CndH (Supplementary Fig. S2). Instead of a Glu, these homologues contain a Phe (Phe326 in PltA, Phe304 in CmlS, and Phe307 in CndH) and no other Glu or Asp is present in PltA in the vicinity that could serve as a base. Most likely nontryptophan halogenases employ a different strategy of deprotonation; perhaps a nearby water molecule can perform this function. After the chlorination, the FAD must be reduced for the next cycle of halogenation. Previous functional study of PltA found a small population of carrier-bound monochlorinated pyrrole (Dorrestein et al., 2005), indicating that the monochlorinated product diffuses out and back in, possibly while the FAD/Cl site is being reactivated. Studies probing the substrate binding and the halogenation mechanism are aimed at testing these structural and mechanistic predictions. Acknowledgments This work was supported by an NSF Career Award MCB1149427 (to S.G.-T.), a Grant (to O.V.T.) as part of the National Center for Advancing Translational Sciences (UL1TR000117), and star-
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Crystal structure of halogenase PltA from the pyoluteorin biosynthetic pathway Allan H. Pang, Sylvie Garneau-Tsodikova, Oleg V. Tsodikov ⇑ Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536-0596, USA
a r t i c l e
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Article history: Received 28 August 2015 Received in revised form 24 September 2015 Accepted 25 September 2015 Available online xxxx Keywords: Chlorination FAD Flavoprotein Halogenation Natural product
a b s t r a c t Pyoluteorin is an antifungal agent composed of a 4,5-dichlorinated pyrrole group linked to a resorcinol moiety. The pyoluteorin biosynthetic gene cluster in Pseudomonas fluorescens Pf-5 encodes the halogenase PltA, which has been previously demonstrated to perform both chlorinations in vitro. PltA selectively accepts as a substrate a pyrrole moiety covalently tethered to a nonribosomal peptide thiolation domain PltL (pyrrolyl-S-PltL) for FAD-dependent di-chlorination, yielding 4,5-dichloropyrrolyl-S-PltL. We report a 2.75 Å-resolution crystal structure of PltA in complex with FAD and chloride. PltA is a dimeric enzyme, containing a flavin-binding fold conserved in flavin-dependent halogenases and monooxygenases, and an additional unique helical region at the C-terminus. This C-terminal region blocks a putative substrate-binding cleft, suggesting that a conformational change involving repositioning of this region is necessary to allow binding of the pyrrolyl-S-PltL substrate for its dichlorination by PltA. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Over 4500 known natural products are halogenated (Gribble, 2003). About 95% of them are chlorinated (50%) and brominated (45%), while the rest are iodinated and fluorinated. Halogenation has been demonstrated to impart bioactive properties to several natural products. For example, the deschloro analogues of balhimycin and clorobiocin were 8-fold less active as antibiotics against Bacillus subtilis than their chlorinated counterparts (Bister et al., 2003; Eustaquio et al., 2003). The halogenated sesterterpenes neomangicol A and B displayed potent cytotoxicity against human colon cancer cells, while their non-halogenated analogues were inactive (Renner et al., 1998). Similarly, rebeccamycin analogues lacking a chlorine substituent were devoid of the antimicrobial activity of their parent compound, presumably due to the lack of cell membrane permeability (Rodrigues Pereira et al., 1996).
Abbreviations: a-KG, a-ketoglutarate; BSA, bovine serum albumin; DGGR, dige ranylgeranylglycerophospholipid reductase; FAD, flavin adenine dinucleotide; 50 FDAS, 50 -fluoro-50 -deoxyadenosine synthase; GR, glutathione reductase; HOCl, hypochlorous acid; HPO, haloperoxidase; LB, Luria–Bertani; NRPS, nonribosomal peptide synthetase; PEG, polyethylene glycol; p-HBH, p-hydroxybenzoate hydroxylase; PKS, polyketide synthase; SAM, S-adenosyl-L-methionine; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; T domain, thiolation domain. ⇑ Corresponding author. E-mail address: [email protected] (O.V. Tsodikov).
Enzymatic chlorination can occur on a wide range of carbon centers (Fig. 1a). The antibiotic chloramphenicol bears chlorinated aliphatic moieties (Podzelinska et al., 2010), while the antitumor agent C-1027 contains a chlorinated enediyne at the C3 position of the b-tyrosyl moiety (Hu et al., 1988). Aromatic chlorination can also be observed, as in the antitumor agent rebeccamycin (Bush et al., 1987) and the antibiotics chondrochloren A and B (Jansen et al., 2003). Furthermore, a heteroaromatic pyrrole ring can be chlorinated: the antifungal agents pyrrolnitrin (Kirner et al., 1998) and pyoluteorin (Nowak-Thompson et al., 1999), both produced by Pseudomonas sp., contain mono- and dichlorinated pyrrole rings, respectively. Biochemical and structural information on several halogenase enzymes indicated that they employ diverse mechanisms. There are four known classes of halogenases: haloperoxidases (HPOs), non-heme iron-dependent halogenases, SAM halogenases, and flavin-dependent halogenases. HPOs catalyze electrophilic chlorination, bromination, and iodination reactions using hydrogen peroxide as an intermediate reactive species. HPOs are diverse: they can be heme-dependent (Sundaramoorthy et al., 1995) or vanadium-dependent (Weyand et al., 1999). These enzymes have broad substrate specificity, owing to the fact that the highly reactive halogen species generated in the active site diffuses in the substratebinding pocket and even outside of the active site. Several crystal structures of different types of bacterial and eukaryotic HPOs have been determined: a heme-dependent fungal chloroperoxidase from
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Please cite this article in press as: Pang, A.H., et al. Crystal structure of halogenase PltA from the pyoluteorin biosynthetic pathway. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.013
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Fig. 1. (a) Chlorine-containing natural products with their respective proposed or observed halogenases catalyzing the chlorination at a specific carbon center. Pyoluteorin and the enzyme (PltA) responsible for chlorination of its pyrrole moiety, studied herein, are marked by a blue box. Proteins with the names in blue have been structurally characterized and will be further described in subsequent figures. The enzymes with the names in black have been biochemically, but not structurally, characterized. (b) The plt gene cluster responsible for the production of pyoluteorin in Pseudomonas sp. Light blue, gray, and dark blue arrows represent genes encoding for enzymes previously biochemically characterized, not studied, and studied herein, respectively. (c) Formation of 4,5-dichloropyrrolyl-S-PltL during pyoluteorin biosynthesis. The pathway consists of the formation of pyrrolyl-S-PltL using L-proline as a starting substrate by a set of enzymes (in black) and the dichlorination of the pyrrole moiety by PltA and associated cofactors (enclosed in a blue box), as previously reported by Walsh and colleagues. The second half of the reaction is carried out by the PKS enzymes (colored in gray), which are yet to be biochemically characterized.
Caldariomyces fumago (Sundaramoorthy et al., 1995), a vanadiumdependent bacterial iodoperoxidase Zg-VIPO1 from Zobellia galactanivorans (Fournier et al., 2014), horseradish peroxidases (Henriksen et al., 1998), and lactoperoxidases from mammals (Fenna et al., 1995; Sheikh et al., 2009; Singh et al., 2008, 2010). Non-heme iron-dependent halogenases catalyze radical halogenation reactions at aliphatic carbon sites. This group of enzymes specifically acts on carrier protein-tethered substrates and requires the use of molecular oxygen for activation of halide ions and a-ketoglutarate (a-KG). There are currently three crystal structures of Fe(II)/a-KG-dependent halogenases available: CurA (Khare et al., 2010), CytC3 (Wong et al., 2009), and SyrB2 (Blasiak et al., 2006) from the curacin A, the c,c-dichloroaminobutyrate, and the syringomycin biosynthetic pathways, respectively. SAM halogenases catalyze a nucleophilic substitution reaction, upon which a desolvated halide attacks on and displaces an L-methionine residue from SAM, with formation of 50 -fluoro (chloro)-50 -deoxyadenosine (Dong et al., 2004; Eustáquio et al., 2008). Flavin-dependent halogenases (Table 1) require reduced flavin (FADH2), which can be covalently (Podzelinska et al., 2010) or non-covalently (Bitto et al., 2008; Buedenbender et al., 2009; Dong et al., 2005; Zhu et al., 2009) bound to the enzyme. Spectroscopic studies revealed formation of the FAD-4a-OOH intermediate upon reaction of FADH2 and O2 (Yeh et al., 2006). This intermediate can react with a nearby chloride ion to yield highly reactive hypochlorous acid (HOCl). HOCl is then proposed to react with a conserved lysine residue to form a chloramine intermediate. In RebH and PrnA, the l-tryptophan
substrate is positioned for chlorination near this lysine (Yeh et al., 2007). Flavin-dependent halogenases can be classified as one of two variants based on their substrates. Variant A enzymes catalyze halogenation of small molecules, as exemplified by tryptophan halogenases (Bitto et al., 2008; Dong et al., 2005; Zhu et al., 2009). Variant B enzymes halogenate substrates that are tethered to a specific thiolation (T) domain. SgcC3 (Lin et al., 2007) and PltA (Dorrestein et al., 2005) (Table 1) are two functionally characterized Variant B enzymes. PltA is one of the first flavin-dependent halogenases to be functionally characterized, by Walsh and co-workers (Dorrestein et al., 2005). The pltA gene and the other biosynthetic genes (pltB, pltC, pltD, pltE, pltF, pltG, pltL, pltM, and pltR) are a part of the biosynthetic gene cluster for the antifungal agent pyoluteorin in Pseudomonas fluorescens Pf-5 (Fig. 1b). Even though PltM and PltD have also been originally annotated as putative halogenases based on their 25% sequence identity to each other and to PltA, only PltA was demonstrated to be an active enzyme in vitro capable of installing both chlorines present in pyoluteorin (Dorrestein et al., 2005). The PltA substrate, a thiolation domain-linked pyrrole (pyrrolyl-S-PltL), requires several enzymatic activities to be formed (Fig. 1c). This substrate gets dichlorinated by PltA with the aid of a coupled flavin reductase for subsequent use in the biosynthetic pathway. The formation of the pyrrole ring and its loading onto a T domain in other natural products was previously demonstrated (Fig. 1c) (Garneau et al., 2005; Thomas et al., 2002). Currently, only
Please cite this article in press as: Pang, A.H., et al. Crystal structure of halogenase PltA from the pyoluteorin biosynthetic pathway. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.013
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A.H. Pang et al. / Journal of Structural Biology xxx (2015) xxx–xxx Table 1 Comparison of homologous FADH2-dependent halogenases and related enzymes with known crystal structures. Enzyme
PDB code
Species
Substrate
Natural product
Oligomeric state
PltA CmlS –a CndH –b PrnA
5DBJ 3I3L 3NIX 3E1T 2PYX 2AQJ
Pseudomonas fluorescens Streptomyces venezuelae Cytophaga hutchinsonii Chondromyces crocatus Shewanella frigidimarina Pseudomonas fluorescens
Pyrrolyl-S-PltL Not known Not knowna Assumed T-bound l-Tyr Not knownb L-Trp
Pyoluteorin Chloramphenicol Unknowna Chondrochloren Unknownb Pyrrolnitrin
Dimer Monomerc Monomerc Monomer Dimer Dimer
PyrH
2WET
Streptomyces rugosporus
L-Trp
Pyrroindomycin
Dimer
RebH
2O9Z
Lechevalieria aerocolonigenes
L-Trp
Rebeccamycin
Dimer
Crystal structures solved by: a Northeast Structural Genomics Consortium. b Joint Center for Structural Genomics, no additional experiment and information available other than the structures uploaded in RCSB. c Based on analysis via PDBe PISA server.
PltA and SgcC3, a structurally uncharacterized halogenase involved in the production of an antitumor agent C-1027, have been biochemically demonstrated to discriminate between a free small molecule substrate (pyrrole-2-carboxylate for PltA and tyrosine for SgcC3) and a carrier protein-tethered substrate (pyrrolyl-SPltL for PltA and tyrosyl-S-SgcC2 for SgcC3), accepting only the carrier protein-tethered substrates for halogenation (Dorrestein et al., 2005; Lin et al., 2007). The paucity of structural information about these intriguing Variant B enzymes prompted this structural study of PltA. Here, we present a crystal structure of PltA from P. fluorescens Pf-5 in complex with its co-factors FAD and chloride. The crystal structure reveals dimeric organization and unique structural features of PltA. 2. Materials and methods 2.1. Expression and purification of PltA The pltA gene was excised from the previously reported expression construct pET28a-pltA (Dorrestein et al., 2005) by NdeI and XhoI enzymes and ligated into Int-pET19b-pps vector (Biswas and Tsodikov, 2008), which yields the protein with an N-terminal decahistidine tag cleavable with Prescission protease (GE Healthcare). This plasmid was transformed into Escherichia coli BL21 (DE3) chemically competent cells, with the transformation mixture plated on Luria–Bertani (LB) agar supplemented with 100 lg/mL ampicillin (AMP). A colony from the transformation plate was inoculated into 5 mL of LB broth containing 100 lg/mL AMP (LB/Amp) and incubated with shaking at 37 °C. This culture was then used to inoculate a 2-L LB/Amp culture, which was then incubated with shaking at 18 °C. When the culture reached an attenuance at 600 nm of 0.5, the culture was induced by the addition of isopropyl-b-D-1-thiogalactopyranoside (IPTG; final concentration of 200 lM) and kept shaking at 18 °C overnight. The cells were harvested by centrifugation at 5000g for 10 min. The cell pellet was resuspended in lysis buffer (40 mM Tris–HCl, pH 8.0, 400 mM NaCl, 10% v/v glycerol, 2 mM b-mercaptoethanol). The cells were then lysed by intermittent sonication on ice. The lysate was passed through a 5-mL HisTrap HP Ni2+ column (GE Healthcare) equilibrated in lysis buffer. The column was washed with 100 mL of lysis buffer containing 50 mM imidazole. PltA was eluted from the column with 6 mL of lysis buffer containing 500 mM imidazole. The eluant was then passed through a size-exclusion HiPrep Sephacryl S-200 HR column (GE Healthcare) equilibrated in gel filtration buffer (40 mM Tris–HCl, pH 8.0, 100 mM NaCl, 2 mM b-mercaptoethanol). Fractions containing pure PltA, as determined by SDS–PAGE gel analysis (Supplementary Fig. S1b), were pooled and concentrated using an Amicon Ultra-15 (10-kDa molecular weight cut-off) centrifugal filter device (Millipore Corporation,
MA) to 10 mg/mL. The protein was frozen by quick immersion in liquid nitrogen and stored at 80 °C. The protein preparation had a yellow color due to bound FAD (Supplementary Fig. S4a).
2.2. Crystallization of PltA Initial crystallization conditions were identified by 1536condition high-throughput crystallization screening of PltA at the Hauptman-Woodward Medical Research Institute (Luft et al., 2003) by the microbatch method in mineral oil. The crystals were grown by vapor diffusion in hanging drops containing 0.5 lL of 10 mg/mL PltA, 0.5 lL of a reservoir solution (0.1 M Bis-Tris propane, pH 7.0, 50 mM magnesium sulfate heptahydrate, 20% w/v PEG 8000) and 0.5 lL of Prescission protease in the protease dilution buffer (40 mM Tris–HCl, pH 8.0, 100 mM NaCl), at the stoichiometry of 1:100 of PltA:protease in the drop. Single yellow crystals were gradually transferred into a cryoprotecting solution (0.1 M Bis-Tris propane, pH 7, 50 mM magnesium sulfate heptahydrate, 20% w/v PEG 8000, 20% glycerol), and frozen in liquid nitrogen by quick immersion.
Table 2 X-ray diffraction data collection and refinement statistics for the PltA structure. Data collection Space group Number of monomers per asymmetric unit Unit cell dimensions a, b, c (Å) a, b, c (°) Resolution (Å) I/r Completeness (%) Redundancy Rmerge Number of unique reflections Structure refinement statistics Resolution (Å) R (%) Rfree (%) Bond length deviation (rmsd) from ideal (Å) Bond angle deviation (rmsd) from ideal (°) B factors (without/with TLS contribution, Å2) Overall Solvent Ligand Ramachandran plot statisticsb % of residues in most allowed regions % of residues in additional allowed regions % of residues in generously allowed regions % of residues in disallowed regions a b
C2 5 242.9, 95.0, 102.1 90, 91.2, 90 50.00–2.75 (2.80–2.75)a 17.56 (1.98) 95.1 (94.4) 4.6 (4.7) 0.12 (0.60) 56,814 (3976) 40.00–2.75 (2.82–2.75) 23.9 (31.3) 29.1 (34.7) 0.007 1.25 45.2/79.2 68.2/68.2 34.0/34.0 89.0 10.2 0.8 0.0 (0 residues)
Numbers in parentheses indicate the values in the highest-resolution shell. Indicates PROCHECK statistics (Laskowski et al., 1993).
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2.3. Data collection and crystal structure determination X-ray diffraction data were collected at beamline 22-ID of the Advanced Photon Source of the Argonne National Laboratory (Argonne, IL) at 100 K and processed with program HKL2000 (Otwinowski et al., 1997). The structure of PltA was determined by molecular replacement by using PHASER (McCoy et al., 2007), where we used as a search model the structure of a putative dehydrogenase from Cytophaga hutchinsonii (PDB accession code: 3NIX, residues 7–386), in which the C-terminal region was deleted. The structure of PltA was built by 80 iterative rounds of refinement with REFMAC (Murshudov et al., 1997) and manual building with COOT (Emsley and Cowtan, 2004). When most of the structure of each of the five monomers in the asymmetric unit was built, NCS restraints (tying all monomers) were used for several cycles, before building slightly variable structural regions. The data collection and refinement statistics are provided in Table 2. The solvent accessible surface areas were calculated by using Surface Racer program, with 1.4 Å as the solvent probe radius (Tsodikov et al., 2002). Coordinates and structure factor amplitudes have been deposited in the Worldwide Protein Data Bank site with the accession code 5DBJ.
3. Results 3.1. Dimeric organization of PltA A crystal structure of halogenase PltA from P. fluorescens Pf-5 (Fig. 2a, left panel) was determined by molecular replacement and refined at the resolution of 2.75 Å; the structure contained five PltA molecules per asymmetric unit (Table 2). The polypeptide backbone for residues 2–438 (out of 449 annotated residues) and most of the side chains were clearly defined in the electron density map with the exception of a short disordered loop (residues 408– 411) in three out of five molecules. A major part of a PltA is an FADbinding Rossmannoid fold, which starts at the N-terminus and consists of a conserved b-sheet flanked by helices. This basic fold is present in other FAD-dependent halogenases, monooxygenases, and glutathione reductase (GR) superfamily enzymes (Fig. 2). The C-terminal regions of PltA (residues 382–449) and that of other homologues are highly divergent (Fig. 2). Other structural differences include lengths, conformations, and even topologies of some b-strand connecting regions. The five copies of PltA in the asymmetric unit are organized as two nearly identical dimers, with the remaining copy forming a similar dimer with its symmetry mate. A dimer of PltA is formed by a 2-fold rotational symmetry (Fig. 3a). Size-exclusion chromatography of PltA indicated a dimeric state of PltA in solution (Supplementary Fig. S1a), consistent with the structural observation. The total of 2304 Å2 of solvent accessible surface area of both PltA monomers is buried in the monomer–monomer interface. This buried area is split approximately equally between polar (1107 Å2) and nonpolar (1197 Å2) contributions, indicating that the dimeric interface is composed of polar contacts (hydrogen bonds and salt bridges) and hydrophobic interactions (Fig. 3b). Specifically the two monomers combine their b-strands b7 (Supplementary Fig. S2) of their Rossmannoid folds in the antiparallel fashion into a conjoined b-sheet, where the main chain carbonyl oxygen of Gln143 of one monomer is hydrogen bonded to the main chain nitrogen of the same residue of the other monomer and vice versa, across the dimeric interface. Other intermolecular contacts between polar and charged residues include salt bridges between Arg128 and Glu40, Asp5 and Arg167, and a hydrogen bond between the side chain carboxyl group of Glu132 and the main chain NH of Glu40, as well as the same contacts made
Fig. 2. Comparison of PltA and structurally related homologues with the conserved FAD monooxygenase domain (pale yellow) and the unique C-terminal domain (blue). The FAD and chloride ions are shown as green sticks and orange spheres, respectively. (a) The overall crystal structure of PltA and the most similar putative halogenase (PDB ID: 3NIX), (b) non-halogenases that are structurally similar to PltA with a small C-terminal lid, examples shown here are p-hydroxybenzoate hydroxylase, p-HBH, (PDB ID: 1PBE) and digeranylgeranylglycerophospholipid reductase, DGGR, (PDB ID: 3OZ2), (c) tryptophan halogenases PrnA with co-factor FAD (PDB ID: 2AQJ) and RebH without the co-factor FAD (PDB ID: 2O9Z), and (d) non-tryptophan halogenases CndH (PDB ID: 3E1T) and CmlS (PDB ID: 3I3L). CndH is composed of a disordered C-terminal region and CmlS forms a big arch-like C-terminal lid.
symmetrically. Nonpolar interactions are between two symmetric patches of hydrophobic surface formed by side chains of Phe142, Val145, Leu164, Val169, Ile171, and the aliphatic stem of Arg167 of each monomer. A well-characterized L-Trp halogenase PrnA is also dimeric through Rossmannoid fold interactions, but its dimeric interface is different from that of PltA (Fig. 3c). 3.2. The FAD binding site Strong omit difference electron density corresponding to a bound FAD molecule was found in all five PltA monomers in the
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residues from several loops. The isoalloxazine ring is buried away from solvent deep inside the cleft, whereas the adenosine diphosphate portion is bound at the entrance of the cleft. The isoalloxazine ring is sandwiched between two regions of PltA: residues 44–47 and residues 320–329 (Fig. 4b). Hydrophobic interactions with the Ca of Gly45 and the side chain of Val44 on one face of the ring and with the side chains of Phe320, Pro323, and the Ca–Cb of Ser326 on the other face appear to hold the ring in place. The hydrophobic side of the ring is flanked by Ala203, Trp239, Tyr299, and the aliphatic part of Glu296. On the polar side of the FAD ring, the oxygen atom at position 2 is situated in an oxyanion hole-like environment. This oxygen is engaged in a bifurcated hydrogen bond, accepting hydrogens from a conserved buried water molecule and an amide nitrogen of Val329. The backbone amide nitrogens of the adjacent residues 326–328 interact with a bound chloride ion, which is located at the nitrogen-containing side of the isoalloxazole ring of the FAD (Fig. 4). The chloride is also held sterically by Phe320, Pro323, and Trp371 (Fig. 4b). This chloride location is conserved in FAD-dependent halogenases (Bitto et al., 2008; Dong et al., 2005; Zhu et al., 2009). A second bound chloride ion was found 25 Å away, at a site distant from the FAD. This chloride is held electrostatically by the guanidinium group of Arg277 and the carboxamide nitrogen of Asn279, as well as the main chain nitrogens of Asn279 and Glu278 (Supplementary Fig. S5). Other halogenases do not contain a bound chloride at an analogous site, consistent with the lack of conservation of Arg277 and Asn279 (Supplementary Fig. S2). However, this sec-
Fig. 3. Dimeric organization of PltA and PrnA. (a) The dimer of PltA, with the residues in the dimeric interface and the bound co-factor FAD shown as sticks. (b) A zoomed-in view of the dimeric interface of PltA. Residues of only one monomer are labeled; the residues of the other monomer are related by twofold rotational symmetry. (c) A dimer organization of PrnA (PDB ID: 2AQJ). One of the monomers (left) is oriented similarly to a monomer in PltA (left of panel a).
asymmetric unit (Figs. 4a and S3). The absorbance spectrum of the purified PltA preparation (yellow in color) indicated that FAD was indeed co-purified with the protein. The absorbance spectrum of PltA-FAD is consistent with the fully oxidized state of the bound flavin (Supplementary Fig. S4), as expected based on the purification procedure. The FAD is situated in a deep cleft, cradled by
Fig. 4. The FAD-binding site of PltA. (a) The bound FAD and chloride are well defined by the Fo Fc omit map contoured at 3r (the blue mesh), generated without the FAD and chloride. (b) A zoom-in view of the isoalloxazine ring of the bound FAD with key residues interacting with FAD explicitly shown.
Please cite this article in press as: Pang, A.H., et al. Crystal structure of halogenase PltA from the pyoluteorin biosynthetic pathway. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.013
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ondary chloride site may serve for storage of chloride, simply by increasing local concentration of chloride ions near PltA. PltA contains a so-called G-box GxGxxG (residues 12–17) that is conserved in FAD-dependent halogenases (Supplementary Fig. S2) and in the GR superfamily (Dym and Eisenberg, 2001), and is involved in binding of dinucleotides (Wierenga et al., 1983). The G-box forms a hairpin between b-strand b1 and helix a1, whose backbone amide nitrogens are engaged in hydrogen bonds with the nucleotide diphosphate moiety of the FAD and form a sterically complementary surface. The diphosphate group is also held by electrostatic interactions with Arg41, His43, and Arg123 and the positively charged N-terminal end of helix a1 (Fig. 5a). A buried conserved water molecule is bound nearby, mediating hydrogenbonding interactions between the diphosphate moiety and the protein. Both Arg residues are conserved among non-tryptophan halogenases, whereas the His residue is unique to PltA. The residues Arg41 and His43 are located in a loop, conserved in CmlS, CndH, and 3NIX. The respective region in PrnA is different in sequence and conformation (Figs. 5b and S2). This PltA loop contains a cis peptide bond between Phe39 and Glu40 (Fig. 5a). This stereochemistry appears to be important for orienting the side chain of Phe39, which serves a key structural role, as the phenyl ring of Phe39 makes van der Waals contacts with the 30 -hydroxyl group of the FAD ribose, Glu35, Arg41, and the GxGxxG motif. A number of PltA homologues (p-HBH, CndH, CmlS, 3NIX, and DGGR) contain a cis peptide bond at this position. Three of these (CndH, CmlS, and 3NIX) also contain a Phe structurally analogous to Phe39 of PltA (Supplementary Fig. S2). In DGGR, a cis peptide bond (Ser40Pro41) can be found in an analogous loop, but it is not superimposable with PltA. The tryptophan halogenase PrnA (Yeh et al., 2007)
Fig. 5. Comparison of the structural environment of the adenosine diphosphate moiety of FAD of (a) PltA and (b) PrnA. Residue Gly13 is located in the G-box motif in both enzymes. The water molecule in panel b is represented by a turquoise sphere labeled H2O.
does not contain a cis peptide in the analogous loop, but this loop is located closer to FAD (6 Å, compared to 10 Å in PltA) (Fig. 5b), apparently to provide an alternative FAD-binding mode. Additionally, the 20 - and 30 -hydroxyl groups of the ribose form hydrogen bonds donating hydrogens to the carboxyl group of Glu35, which is also positioned by the GxGxxG motif, and the 30 hydroxyl also accepts a hydrogen from the guanidinium group of Arg41 (Fig. 5a). Glu35 is nearly universally conserved in PltA and its homologues (Supplementary Fig. S2). Interestingly, despite the conservation, the analogue of Glu35 in PrnA (Glu38) does not form direct hydrogen bonds with the ribose ring of the FAD and points away from the FAD (Fig. 5b). Instead, the oxygen of the 20 -hydroxyl group of the ribose forms a hydrogen bond with the amide nitrogen of the subsequent residue (Ser39), while the 30 -hydroxyl group forms a hydrogen bond with the amide nitrogen of Gly13 (same residue number as in PltA) from the GxGxxG conserved sequence motif and with a water molecule that occupies the location of the carboxyl group of Glu35. 3.3. The unique C-terminal region of PltA A BLAST search for structurally characterized homologues yielded CmlS, 3NIX, and CndH, which are 25–27% identical to PltA (Table 1). While the FAD-binding fold of the N-terminal region is conserved among PltA and these proteins, the C-terminal regions are divergent in sequence, length (Supplementary Fig. S2), and structure (Fig. 2). The C-terminal region of PltA is composed of three helices (Fig. 2a). In contrast, the C-terminal region of CmlS is a winged helix structure, while in CndH, the C-terminal region is largely disordered (Fig. 2d). A previous proposal correlating classification between Variant A (small molecule substrate; Fig. 2c) and B (thiolation domain-tethered substrate; Fig. 2d (note that CndH is a putative Variant B enzyme)) halogenases with the degree of structural order of the C-terminal region (ordered in Variant A, disordered in Variant B) (Podzelinska et al., 2010), is directly disproved by the PltA structure: PltA is a demonstrated Variant B enzyme (Dorrestein et al., 2005) containing a mostly ordered Cterminal region (Fig. 2a). Nevertheless, the divergence of the C-terminal regions appears to be a strategy employed by halogenases to ensure substrate discrimination (Podzelinska et al., 2010). For example, the C-terminal region of PrnA forms a part of the binding pocket for the L-Trp substrate (Fig. 6b). Crystal structures of PrnA with bound L-Trp substrate (PDB: 2AQJ) and chlorotryptophan product (PDB: 2AR8) indicated that the chlorinated carbon atom of the product is in the immediate vicinity of the functionally important lysine residue, which is conserved in known FAD-dependent halogenases, including PltA (Lys73; Figs. 6a and S2). Lys73, located 10 Å away from the bound FAD, therefore, defines the putative substrate-binding site in PltA. The C-terminal region of PltA obstructs this site from the access of the large thiolation domain-tethered pyrrole substrate. More distant homologues of PltA include two non-halogenases: the hydroxylase p-HBH and the reductase DGGR (Fig. 2b). p-HBH and DGGR function differently from each other and from halogenases. p-HBH hydroxylates its substrate p-hydroxybenzoate by molecular oxygen to form 3,4-dihydroxybenzoate (Schreuder et al., 1989), whereas DGGR hydrogenates and converts unsaturated archaeols to saturated archaeols, which is an important step in the biosynthesis of archaeal membrane lipids (Xu et al., 2010). The functional difference between the two non-halogenase homologues is reflected by the structural environment of the active site tunnel. Notably, Lys73 is not conserved in p-HBH, while it is present in DGGR, but it points towards the surface of the enzyme instead of the substrate-binding tunnel. While the C-terminal regions of PltA and these two enzymes are somewhat similar, the
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respective parts from each monomer also provide a binding surface for the nucleoside moiety of the bound FAD (Fig. 3a). Therefore, dimerization of PltA may play a structural role in FAD binding, but given the significant size of the monomer–monomer interface, a likely role of the dimerization is to support the structural stability of the protein as a whole. This dimerization interface is not conserved, since not all characterized FAD-dependent halogenases (of either Variant) are dimeric (Table 1) and those that dimerize do so differently from PltA. 4.2. Mechanism of substrate binding and catalysis
Fig. 6. The view into the substrate-binding cavity from the side of the bound FAD in (a) PltA and (b) PrnA. The structures are oriented to visualize the FAD, the L-Trp substrate of PrnA, and the conserved catalytic lysine residue (Lys73 in PltA and Lys79 in PrnA).
substrate binding mechanisms must differ. The hydroxylase p-HBH contains a small opening within the FAD-binding domain for entry and exit of its small molecule substrate p-hydroxybenzoate. The reductase DGGR, due to the small size of its C-terminal region, provides unobstructed access for its large substrate, an unsaturated archaeol, to its binding site. In PltA, the respective openings are sealed off by the C-terminal region.
4. Discussion 4.1. PltA is the first structurally characterized established Variant B FAD-dependent halogenase A decade ago, PltA was demonstrated to halogenate a pyrrole moiety tethered to a thiolation (T) domain on the pathway to pyoluteorin production (Dorrestein et al., 2005). This substrate preference of an FAD-dependent halogenase defined it as a Variant B enzyme. Crystal structures of experimentally confirmed Variant A enzymes (acting on free L-Trp) have been determined (Table 1), and this study is the first structural characterization of an experimentally validated Variant B enzyme. PltA forms a dimer, where the monomer–monomer interface is distant from the catalytic center, but this interface includes a conjoined b-sheet, whose
The location of the substrate-binding site in halogenases is not trivially specified by the location of a bound FAD or a halide ion, because a reactive halogen species (such as HOCl) can diffuse through channels in the protein and react at a distance from the flavin. The halogenation site in FAD-dependent halogenases was defined by the crystal structures of tryptophan halogenases PrnA, RebH, and PyrH in complex with either the tryptophan substrate or the chlorotryptophan product (Bitto et al., 2008; Dong et al., 2005; Yeh et al., 2007; Zhu et al., 2009). Given the conservation of the fold of the catalytic domain of both Variant A and B halogenases, the bound FAD and the catalytic Lys residue, the location of the halogenation center is likely analogous in Variant A and B halogenases. In PltA, this putative substrate-binding site is obstructed by the C-terminal helical region (Figs. 6a and 7a). PltA is in this closed state, presumably, to select against binding and halogenation of small molecule substrates such as pyrrole-2-carboxylate. Given the large size of the pyrrolyl-S-PltL conjugate substrate, its entry through the FAD-binding cleft cannot occur for steric reasons, and no other channel with access to the halogenation site exists (Fig. 2a). A conformational change that most likely involves a movement of the C-terminal region to open access to the halogenation site to the carrier protein-tethered pyrrolyl moiety is required. The structure of chloramphenicol halogenase CmlS (whose exact substrate remains unknown) also contains a blocked putative substrate-binding site and is expected to undergo a conformational change to bind its substrate (Podzelinska et al., 2010). The blockage in CmlS is not as severe as in PltA and it can be relieved by a movement of only the last structured loop (Podzelinska et al., 2010) since the structure of the C-terminal region in CmlS is different. In the chondrochloren halogenase CndH, the C-terminal region is partially unstructured; therefore, the access to the halogenation site appears to be unobstructed (Buedenbender et al., 2009). With these considerations in mind, we examined whether an appropriate binding surface for a pyrrole-phosphopantetheinyl arm conjugate could be exposed upon removal of the C-terminal region of PltA from its crystal structure. Indeed, a cleft of an appropriate size gets opened as a result (Fig. 7b), abutting the putative catalytic Lys73 on one side and punctuated by a newly exposed positively-charge surface patch formed by Arg337 on the other side. The pyrrolyl and the phosphopantetheinyl arm to which it is attached fit snugly into this cleft in an extended conformation. The phospho moiety of the model substrate is in favorable electrostatic contact with Arg337. We propose that interactions with this substrate, possibly also involving the T domain, could trigger a conformational change to displace the C-terminal region of PltA for this binding interaction. The oxidation reaction cascade catalyzed by flavin-dependent monooxygenases is well understood (Huijbers et al., 2014); a similar halogenation mechanism has been proposed for tryptophan halogenases (Bitto et al., 2008; Dong et al., 2005; Zhu et al., 2009). A spectroscopic study of RebH showed that an FADH2 molecule bound to the halogenase reacted with molecular oxygen to form a highly reactive FAD-4a-OOH intermediate (Yeh et al., 2006). Since
Please cite this article in press as: Pang, A.H., et al. Crystal structure of halogenase PltA from the pyoluteorin biosynthetic pathway. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.013
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tup funds from the College of Pharmacy at the University of Kentucky (to S.G.-T. and O.V.T.). We thank the staff of sector 22 (SER-CAT) of the Advanced Photon Source at the Argonne National Laboratories for their assistance with the remote X-ray diffraction data collection. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jsb.2015.09.013. References
Fig. 7. The surface view of the putative substrate binding cleft of PltA (a) prior and (b) after the removal of the C-terminal region. The pyrrole bound to the phosphopantetheinyl arm substrate was modeled into PltA. The electrostatic potential is mapped onto the surface: white, red, and blue colors representing nonpolar, negatively, and positively charged surface, respectively. The positively charged patch due to Arg337, predicted to interact with the phospho group of substrate, is labeled.
the substrate is bound at a site remote from the FAD, a diffusible HOCl species must then form. The HOCl diffuses to the catalytic Lys (Lys73 in PltA, Lys79 in PrnA and RebH, Lys71 in CmlS, Lys76 in CndH) and modifies this Lys to an aminochloride releasing a water molecule. The aminochloride then converts the aromatic substrate to a Wheland complex. A deprotonation step is then necessary for the release of the halogenated aromatic product. The structural and mutagenesis studies of tryptophan halogenase PrnA led to the proposal that the proton abstraction is achieved by the nearby base Glu346 (Dong et al., 2005; Flecks et al., 2008). Sequence alignment of the glutamate residue reveals that it is conserved among tryptophan halogenases, but not with other homologues such as PltA, CmlS, and CndH (Supplementary Fig. S2). Instead of a Glu, these homologues contain a Phe (Phe326 in PltA, Phe304 in CmlS, and Phe307 in CndH) and no other Glu or Asp is present in PltA in the vicinity that could serve as a base. Most likely nontryptophan halogenases employ a different strategy of deprotonation; perhaps a nearby water molecule can perform this function. After the chlorination, the FAD must be reduced for the next cycle of halogenation. Previous functional study of PltA found a small population of carrier-bound monochlorinated pyrrole (Dorrestein et al., 2005), indicating that the monochlorinated product diffuses out and back in, possibly while the FAD/Cl site is being reactivated. Studies probing the substrate binding and the halogenation mechanism are aimed at testing these structural and mechanistic predictions. Acknowledgments This work was supported by an NSF Career Award MCB1149427 (to S.G.-T.), a Grant (to O.V.T.) as part of the National Center for Advancing Translational Sciences (UL1TR000117), and star-
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Please cite this article in press as: Pang, A.H., et al. Crystal structure of halogenase PltA from the pyoluteorin biosynthetic pathway. J. Struct. Biol. (2015), http://dx.doi.org/10.1016/j.jsb.2015.09.013