Identification of functionally important residues and structural features in a bacterial lignostilbene dioxygenase

Lignostilbene-α,β-dioxygenase A (LsdA) from the bacterium Sphingomonas paucimobilis TMY1009 is a nonheme iron oxygenase that catalyzes the cleavage of lignostilbene, a compound arising in lignin transformation, to two vanillin molecules. To examine LsdA's substrate specificity, we heterologously produced the dimeric enzyme with the help of chaperones. When tested on several substituted stilbenes, LsdA exhibited the greatest specificity for lignostilbene (kcatapp = 1.00 ± 0.04 × 106 m−1 s−1). These experiments further indicated that the substrate's 4-hydroxy moiety is required for catalysis and that this moiety cannot be replaced with a methoxy group. Phenylazophenol inhibited the LsdA-catalyzed cleavage of lignostilbene in a reversible, mixed fashion (Kic = 6 ± 1 μm, Kiu = 24 ± 4 μm). An X-ray crystal structure of LsdA at 2.3 Å resolution revealed a seven-bladed β-propeller fold with an iron cofactor coordinated by four histidines, in agreement with previous observations on related carotenoid cleavage oxygenases. We noted that residues at the dimer interface are also present in LsdB, another lignostilbene dioxygenase in S. paucimobilis TMY1009, rationalizing LsdA and LsdB homo- and heterodimerization in vivo. A structure of an LsdA·phenylazophenol complex identified Phe59, Tyr101, and Lys134 as contacting the 4-hydroxyphenyl moiety of the inhibitor. Phe59 and Tyr101 substitutions with His and Phe, respectively, reduced LsdA activity (kcatapp) ∼15- and 10-fold. The K134M variant did not detectably cleave lignostilbene, indicating that Lys134 plays a key catalytic role. This study expands our mechanistic understanding of LsdA and related stilbene-cleaving dioxygenases.

Lignin is a heterogeneous aromatic polymer found in plant cell walls that contributes to the recalcitrance of biomass. It is increasingly recognized that the valorization of lignin is essential to the sustainable biorefining of lignocellulose (1). Decon-structing lignin is thus of great relevance to transforming lignocellulose to biofuels and commodity chemicals (1,2). The discovery that bacteria are able to at least partially deconstruct lignin has accelerated the study of enzymes and pathways potentially involved in lignin depolymerization and the catabolism of the resulting products (3)(4)(5). Lignin-derived aromatic compounds that are efficiently degraded by bacteria include ␤-aryl ethers, pinoresinol, 2,2Ј-dihydroxy-3,3Ј-dimethoxy-5,5Ј-dicarboxybiphenyl, diaryl propane, and phenylcoumarane (5,6).
LSDs belong to the same protein family as carotenoid cleavage oxygenases (CCOs), which typically catalyze the oxidative cleavage of a double bond in carotenoids (21,22). These enzymes are characterized by a structural fold comprising a seven-bladed ␤-propeller (21). The active site occurs at the center of this propeller and contains an Fe 2ϩ coordinated by four histidines (21). In crystal structures of LSD NOV1 and CAO1 in complex with stilbenoid substrates, the organic substrate is bound such that the scissile double bond is in close proximity to the ferrous ion (23,24).
At least two mechanisms have been proposed for LSDs. In a mechanism proposed by McAndrew et al. (23), the hydroxystilbenoid is activated via the enzyme-catalyzed deprotonation of the 4-hydroxy group, which allows electron delocalization toward an Fe 3ϩ -superoxo electrophile. In an alternate proposal by Sui et al. (24), electron density from the scissile double bond is redistributed to the iron-oxy complex to form an Fe 2ϩperoxo-substrate cation intermediate. Deprotonation of the hydroxyl moiety is critical in both mechanisms and is assisted by Lys 134 and Tyr 101 (LsdA/LSD NOV1 numbering), two activesite residues conserved among stilbene-cleaving oxygenases (23,24). However, the roles of these residues have not been investigated. Further, it is unclear whether the organic substrate gates the reactivity of the ferrous ion with O 2 to inhibit the oxidation of the metal ion, as is the case for extradiol dioxygenases (25). For example, EPR analyses have suggested that NO binds to the iron center independent of organic substrate (23). By contrast, crystallographic, X-ray absorption spectroscopy, and Mössbauer spectroscopy data indicate that a number of CCOs do not bind O 2 in the absence of the organic substrate (24).
Herein, we report the kinetic and structural characterization of LsdA from TMY1009. Steady-state kinetic studies were performed to evaluate the substrate specificity of the enzyme for a variety of stilbenes. The inhibition of the enzyme by a substrate analog was similarly described. Several X-ray crystal structures were determined, including that of LsdA bound to a substrate analog. Conserved active-site residues were substituted to evaluate their roles in substrate specificity and catalysis. The results are discussed with respect to CCOs and bacterial lignin catabolism.

Purification of LsdA
LsdA of TMY1009 was produced in Escherichia coli using a pET vector containing lsd. Most of the produced protein was insoluble, consistent with a previous account (24). However, the co-production of LsdA in a strain producing the GroEL and GroES chaperones significantly improved the level of soluble LsdA. LsdA was purified to Ͼ99% apparent homogeneity as judged by SDS-PAGE analysis at yields of ϳ10 -20 mg of purified protein per liter of cell culture. Inductively coupled plasma MS (ICP-MS) analyses revealed that purified LsdA contained ϳ1 eq of iron per protomer and insignificant amounts of cadmium, cobalt, copper, zinc, manganese, nickel, and lead. Consistent with this result, a colorimetric assay based on Ferene-S yielded a value of 1.1 Ϯ 0.2 eq of iron per LsdA protomer. Preparations of LsdA retained essentially 100% of their activities when exposed to ambient levels of O 2 for up to 16 h at room temperature.
In the oxygraph assay, neither stilbene nor 4,4Ј-dimethoxystilbene was detectably cleaved by LsdA, consistent with their lack of a 4-hydroxy substituent. Other compounds that were not cleaved include phenylazophenol and diethylstilbestrol. The identification of phenylazophenol as a nonhydrolyzable substrate analog that bears a 4-hydroxy substituent prompted us to evaluate its ability to inhibit the LsdA-catalyzed reaction. In steady-state kinetic studies, phenylazophenol inhibited the LsdA-catalyzed cleavage of lignostilbene (K ic ϭ 6 Ϯ 1 M and K iu ϭ 24 Ϯ 4 M; K ic and K iu refer to competitive and uncompetitive inhibition constant, respectively) (Fig. 3). Preincubation of LsdA with up to 50 M phenylazophenol for up to 30 min at room temperature did not significantly affect the enzymatic activity, consistent with reversible inhibition.

Structure of LsdA
LsdA crystallized in space group P3 2 21 with two LsdA protomers in the asymmetric unit. The two protomers are related by an approximately 2-fold rotational symmetry (Fig.  4A) and likely represent the dimer that LsdA forms in solution (14). The structure for apo-LsdA, solved to 2.3 Å resolution, was used to solve that of holo-LsdA) (i.e. bound to Fe 2ϩ ) to 2.6 Å ( Table 2). The apo-and holo-LsdA protomers are highly similar in structure, with an average root mean square deviation (RMSD) over all C␣ atoms of ϳ0.3 Å. The structural fold of LsdA is that of a seven-bladed ␤-propeller, typical of the CCOs (Fig. 4B). Among stilbenoid-cleaving dioxygenases of known structure, LsdA is most similar to the resveratrol-cleaving enzyme LSD NOV1 (Protein Data Bank (PDB) entry 5J53) (23), sharing an RMSD of 1.1 Å over 473 aligned C␣ atoms. LSD NOV1 is also a homodimer.
The dimer interface of LsdA has a buried surface area of 1460 Å 2 and contains many polar interactions, including 14 hydrogen bonds and 9 salt bridges, as predicted by PDB-PISA. All but Characterization of LsdA one of these interactions are mediated by a linear stretch of residues from the N terminus to Glu 31 of each subunit (Fig. 4C), which includes strand ␤1 (Glu 20 -Leu 24 ). The ␤1 strands of the two subunits are arranged anti-parallel to each other, with the Glu 20 amide from one subunit forming a hydrogen bond with the Asp 22 carbonyl of the other. This arrangement creates a 10-stranded anti-parallel ␤-sheet formed by the first propeller blade of each protomer. Other notable interactions involve the side-chain carboxylates of Asp 25 and Glu 27 , which form reciprocal salt bridges with the N terminus (Ala 2 ) and Arg 15 , respectively, of the two protomers.

Metal-binding site
As observed in other CCOs, the LsdA active site harbors a single Fe 2ϩ ion that resides at the center of the ␤-propeller. This metal ion is coordinated in a tetragonal pyramidal fashion by four conserved histidines (His 167 , His 218 , His 282 , and His 472 ) and a solvent molecule (Fig. 5). The average Fe 2ϩ -His bond length in the resting state LsdA is ϳ2.2 Å, in agreement with values reported in other CCOs (23,24). Similar to other CCOs, the sixth metal coordination site (across from His 282 ) is unoccupied and is partly occluded by Thr 121 (26). Additionally, three of the metal-coordinating histidines interact with conserved acidic residues (Glu 135 , Glu 350 , and Glu 414 ) via hydrogen bonds. Finally a cap-like structure on one face of the ␤-propeller helps shield the metal-binding site from the solvent. This cap is formed by a series of loops that otherwise connect various ␤ strands and is structurally similar to that of LSD NOV1 (RMSD of 3.6 Å over 126 superposed C␣ atoms). As with other LSDs, LsdA's cap lacks the hydrophobic patch found in CCOs that helps localize these enzymes to membranes, where their hydrophobic substrates (carotenoids or isoprenoids) are typically found.

Structure of the LsdA⅐phenylazophenol complex
To further explore substrate binding in LsdA, the enzyme was co-crystalized with phenylazophenol. Co-crystals of LsdA⅐phenyl-azophenol were yellow, similar to that of the inhibitor. Two protomers constitute the asymmetric unit, as in the inhibitor-free structures, and the complex was refined to 3.0 Å ( Table 2). The structure of LsdA in the complex is virtually indistinguishable from that of the inhibitor-free enzyme: comparison between the two yielded an RMSD over all C␣ atoms of ϳ0.3 Å. Inspection of an omit difference density map revealed positive density consistent with the presence of an inhibitor molecule adjacent to the metal at each active site (Fig. 6A). The active site of the LsdA⅐phenylazophenol complex has a lower metal occupancy, as indicated by a weaker electron density associated with the metal ion as compared with the inhibitorfree structure.
Phenylazophenol was modeled at full occupancy with an average B-factor of 76.7 Å 2 (Fig. 6B). However, the resolution of the structure precluded defining the binding orientation on the basis of density fitting alone. Instead, the binding orientation was derived from the polarity of surrounding amino acid residues and by comparison with the structures of enzyme⅐ligand complexes of homologous enzymes (Fig. 6C) (23,24). In the model, the Fe 2ϩ ion is closest to the two azo nitrogen atoms of phenylazophenol (ϳ3-4 Å). The 4-hydroxy moiety of the phenylazophenol forms hydrogen bonds with each of Tyr 101 and Lys 134 (Fig. 6B). In addition, Phe 59 forms astacking interaction with the phenolic ring of the inhibitor. Glu 350 , located within the active-site pocket distal to Tyr 101 , does not contact the inhibitor, which lacks substituents on the nonphenolic ring. These four residues are conserved throughout stilbene-cleaving dioxygenases and have been previously identified in binary complexes of LSD NOV1 and CAO1, respectively, with resveratrol (23,24). In LSD NOV1 , Ser 283 and Glu 353 form hydrogen bonds to one of the hydroxyl groups on reserveratrol's resorcinolic ring. The glutamate is conserved in LsdA (Glu 350 ) and CAO1, but the serine is replaced by glycine in both LsdA and CAO1. In LsdA, the bound phenylazophenol is covered by a constellation of phenylalanine residues (Phe 305 , Phe 307 , and Phe 308 ) from the cap. These interactions, together with an interaction with Phe 59 , help sequester the inhibitor from the solvent. As compared with the ligand-free enzyme, a rotation about 1 of the Phe 305 by Ϫ45°coupled with small changes in conformation in the main chain repositions the phenyl ring to accommodate binding of phenylazophenol. In addition, the phenyl ring of Phe 308 is repositioned by rotations of 1 and 2 by 70 and 115°, respectively, to form the ligand-binding pocket in the LsdA⅐phenylazophenol complex. Finally, no density corresponding to a metal-bound solvent or O 2 species was observed in the LsdA⅐phenylazophenol complex, in contrast to what was reported in the CAO1⅐resveratrol and LSD NOV1 ⅐resveratrol complexes, perhaps due to the low resolution.

Active-site variants
To evaluate the roles of key residues in substrate recognition and catalysis, we substituted each of three residues of LsdA identified to interact with the 4-hydroxy moiety of the phenylazophenol: Phe 59 , Tyr 101 , and Lys 134 . These residues were substituted with histidine, phenylalanine, and methionine, respectively. The variants were purified in similar yields as the WT LsdA. Further, they all contained a full complement of iron. As summarized in Table 3, all of the variants had significantly less activity compared with the WT. Indeed, the K134M variant did not detectably cleave lignostilbene. The Y101F variant had k cat app and k cat app /K M app (the superscript "app" refers to apparent parameters) values that were 10 and 20% those of the WT, whereas the corresponding values for the F59H variant were ϳ7 and 3% those of WT LsdA.

Discussion
The substrate specificity studies of LsdA are consistent with previous reports that the enzyme cleaves only 4-hydroxystilbenes. More particularly, it had previously been determined that LsdA does not cleave 2-hydroxy, 3-hydroxy, or 4-methoxy stilbenes (20). Different LSD homologs have different substrate specificities, as exemplified by the isoforms of LSD TMY1009 (14). Moreover, there are conflicting reports on whether LSD NOV1 can cleave rhapontigenin and rhaponticin, both of which are 4-methoxy stilbenes (19,23). Nevertheless, most characterized LSDs appear to require the 4-hydroxy moiety for activity (8,23,27). This is consistent with the conserved active-site lysyl and tyrosyl residues in LSDs and the strikingly similar manner in which they interact with the

Characterization of LsdA
4-hydroxyl group in each of the LSD NOV1 ⅐resveratrol, CAO1⅐resveratrol, and LsdA⅐phenylazophenol complexes (23,24). The inhibition of LsdA by phenylazophenol is consistent with previous studies in which LsdA was unable to cleave stilbenoids with substitutions at either C ␣ or C ␤ of the vinyl group (28,29). Further, both N-benzylideneaniline and N-benzylaniline potently inhibit LsdA (29). Similarly, CAO1 did not cleave fluoro-resveratrol (24). Phenylazophenol behaved as a mixed inhibitor, which suggests the presence of multiple binding sites. Whereas only a single binding site was observed in the LsdA⅐phenylazophenol structure, electron density consistent with multiple ligands was observed in a Co 2ϩ -substituted CAO1⅐resveratrol complex (24).
Although LsdA cleaves lignostilbene quite efficiently, the enzyme's physiological substrate remains unknown. Inspection of the substrate-binding site of LsdA revealed several pockets adjacent to the phenyl rings of the inhibitor, suggesting that the enzyme's physiological substrate has multiple substitutions along the aromatic groups. Interestingly, the stilbenoid 3-(4hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl)acrylate (DCA-S) has been identified as a metabolite in the catabolism of dehydrodiconiferyl alcohol in SYK-6 (12). It is presumed that DCA-S is cleaved by at least one of the eight LSD homologs harbored by SYK-6 (5). Nevertheless, LsdA's specific activity for DCA-S was 2 orders of magnitude lower than for lignostilbene (7).
In the two proposed reaction mechanism of LSDs, conserved Lys 134 and Tyr 101 have been postulated to assist in the deprotonation of the 4-hydroxyl (23,24). Our data indicate that although both residues hydrogen-bond with the substrate's hydroxyl, only Lys 134 is essential for catalysis. Although the high pK a of lysine's ⑀-amino group (ϳ10) makes it ill-suited to function as a base catalyst, it functions as such in several dehydrogenases (31)(32)(33). In these enzymes, the catalytic lysine does not act directly as the base catalyst, but instead promotes the deprotonation of the catalytic tyrosine by lowering the pK a of the latter's phenolic hydroxyl group via electrostatic polariza-tion (34). Our data are consistent with the catalytic lysine of LSDs acting directly on the substrate.
The Fe 2ϩ cofactor of LSDs and other CCOs is relatively resistant to oxidative inactivation. This is in contrast to another class of nonheme Fe 2ϩ dioxygenases, the extradiol dioxygenases, which are inactivated upon exposure to O 2 as well as during catalytic turnover (25). Whereas LSDs may use a gating mechanism similar to the extradiol dioxygenases to prevent the binding and activation of O 2 in the absence of organic substrate, this protective measure is unlikely to involve structural changes to modulate the enzyme's affinity toward O 2 . Substrate binding in CCO did not affect the protein structure or significantly change the electronic state of the metal (24). Indeed, the activesite metal appears to contribute minimally toward substrate binding and overall protein structure. However, ligand binding may have a steric or electronic effect in the formation of the ternary complex.
The residues that mediate dimerization in LsdA are conserved in LsdB. These include all of the residues involved in the predicted 14 hydrogen bonds and nine salt bridges between the two protomers of the LsdA dimer (Fig. 4C). The high degree of conservation between LsdA and LsdB rationalizes how LsdA and LsdB form homo-and heterodimers in vivo (14). Further, these residues are conserved in CAO1, consistent with its dimeric structure. LsdA and LsdB share Ͼ95% amino acid sequence identity with LSD1 and LSD2 (SLG_36640), respectively, from SYK-6. The conserved residues include all of those at the dimer interface. This suggests that the occurrence of heterodimeric LSDs is not uncommon to TMY10009, although the physiological significance of these heterodimers is unclear. The relatively small size of the dimer interface suggests that subunits might readily swap in solution. Finally, it is noted that these interfacial residues are not required for dimerization, as the third LSD of TMY10009 is dimeric despite lacking these residues (15).
In conclusion, this study presents a more in-depth look into the first characterized LSD and establishes the relative importance of Lys 134 for catalysis. Further work is required to establish the physiological role of the LSDs, particularly in bacteria containing multiple homologs. To this end, we are investigating the various LSDs of SYK-6.

Chemicals and reagents
All reagents were of analytical grade unless otherwise noted. Restriction enzymes and the Phusion PCR system used for cloning were from New England Biolabs. Water for buffers was purified using a Barnstead Nanopure Diamond TM system to a resistance of at least 18 megaohms. Lignostilbene was a gift from Prof. Victor Snieckus and Dr. Timothy E. Hurst (Queens University, Kingston, Ontario, Canada). All other stilbenes were commercially sourced.

DNA manipulation
DNA was purified, manipulated, and propagated using standard procedures (35). The lsdA gene (locus tag: 1917171A), which encodes for the ␣-isoform of LSD in TMY1009, was synthesized by back-translating the protein's amino acid sequence

Characterization of LsdA
using codon optimized for expression in E. coli (GenScript USA Inc.). The gene was subcloned into pET41b (Novagen) to yield pET41LsdA. Variants of LsdA were generated from pET41LsdA using PCR-based mutagenesis using a pair of overlapping primers. The nucleotide sequences of key constructs were confirmed by sequencing. The oligonucleotides used in this study are listed in Table S1.

Protein production and purification
LsdA was produced heterologously using E. coli BL-21 (DE3) containing pET41LsdA and pGro7 (Takara Bio Inc.). Freshly transformed cells were grown at 37°C in lysogeny broth supplemented with 30 mg/liter kanamycin, 30 mg/liter chloroamphenicol, and 1 mg/ml L-arabinose to an A 600 of ϳ0.7. Expression of lsdA was induced with 1 mM isopropyl ␤-D-thiogalactopyranoside, at which time the medium was further supplemented with 0.5 mM FeCl 3 , and the cells were incubated at 16°C for an additional 16 h. Cells were harvested by centrifugation and stored at Ϫ80°C until further processing. Cells collected from 2 liters of culture were suspended in 20 ml of 20 mM HEPPS, 2 mM DTT, 0.5 mM (NH 4 ) 2 Fe(SO 4 ) 2 , pH 8.0, and lysed at 4°C using an EmulsiFlex-C5 homogenizer (Avestin). Cellular debris was removed by centrifugation. (NH 4 ) 2 SO 4 was added to the cleared lysate to a final concentration of 1.2 M, and the supernatant was removed by centrifugation. The protein pellet was resuspended in the cell resuspension buffer supplemented with 0.8 M (NH 4 ) 2 SO 4 , and the precipitate was removed by centrifugation and filtration at 0.45 m. Subsequent purification steps were performed anaerobically by manipulating the sample inside a glovebox (Labmaster Model 100, Mbraun). Chromatography was performed using an ÄKTA Purifier interfaced to the glovebox with buffers and fraction collection inside the glovebox. Buffers used for purification were sparged with N 2 before being placed in the glovebox for equilibration overnight. The supernatant was loaded onto a Source 15 phenyl column and eluted with a linear gradient from 0.8 to 0 M (NH 4 ) 2 SO 4 in 120 ml of 20 mM HEPPS, 2 mM DTT, 0.5 mM (NH 4 ) 2 Fe(SO 4 ) 2 , pH 8.0 (ÄKTA Purifier, GE Healthcare). Fractions containing LsdA, as determined through SDS-PAGE, were pooled and dialyzed into 20 mM HEPPS, 2 mM DTT, pH 8.0. LsdA was purified further using a MonoQ 10/100 GL column (GE Healthcare). The protein was eluted with a linear gradient from 0.2 to 1 M NaCl in 120 ml of 20 mM HEPPS, 2 mM DTT, pH 8.0. Fractions containing LsdA were pooled, dialyzed into 20 mM HEPPS, 80 mM NaCl, 2 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP), pH 8.0, concentrated to ϳ10 mg/ml, flash-frozen as beads in liquid N 2 , and stored at Ϫ80°C until further use. The variants were purified similarly. Apo-LsdA was purified using a similar protocol except that the purification was performed aerobically and neither the media nor the buffers were supplemented with iron.

Protein analytical methods
Protein purity was evaluated using SDS-polyacrylamide gel stained with Coomassie Blue according to established procedures (35). Protein concentration was determined using a micro-BCA TM protein assay kit (Pierce) using BSA as a standard. Iron concentrations were determined colorimetrically using the Ferene-S assay and ferric chloride solution as a standard (36). ICP-MS was performed using a NexION 300d mass spectrometer (PerkinElmer Life Sciences) calibrated using IV-Stock-4 synthetic standard (Inorganic Ventures). To liberate metal ions, the protein samples were treated with concentrated HNO 3 and H 2 O 2 as described previously (37).
a Experiments were performed using air-saturated TAPS (I ϭ 0.1 M), pH 8.5, at 25°C. Parameters were calculated using a minimum of 20 data points at various lignostilbene concentrations. These parameters were obtained using air-saturated buffer and are thus apparent. b ND, not detected.

Steady-state kinetics
Kinetic assays were performed by monitoring the consumption of O 2 using a Clark-type polarographic O 2 electrode OXYG1 (Hansatech) connected to a circulating water bath. Assays were performed in 1 ml of air-saturated 40 mM TAPS (I ϭ 0.1 M, pH 8.5) at 25°C and initiated by adding the stilbene. Stock solutions of the stilbenes were made in dimethylformamide (DMF). The final concentration of DMF in the assay solutions was Ͻ0.5% (v/v). Reaction velocities were corrected for the background reading prior to substrate addition. The electrode was calibrated daily according to the manufacturer's instructions using air-saturated water and O 2 -depleted water via the addition of sodium hydrosulfite. Stock solutions were prepared fresh daily. Steady-state kinetic parameters were evaluated by fitting the Michaelis-Menten equation to the data using the least-squares fitting of LEONORA (38). The effect of pH on the rate of the LsdA-catalyzed reaction was evaluated using I ϭ 0.1 M solutions of citrate (pH 6.0), HEPPS (pH 7.0 -9.0), and carbonate (pH 9.6 and 10.3). The apparent steadystate kinetic parameters for O 2 were evaluated using 125 M lignostilbene and initial concentrations of O 2 from 16 to 600 M. The initial O 2 concentrations were achieved by equilibrating the reaction mixture with humidified mixtures of O 2 and N 2 gasses. Final O 2 levels were normalized to the ambient O 2 level prior to the adjustment. The electrode was equilibrated with air-saturated buffer between runs. The inhibition of LsdA by phenylazophenol was evaluated by monitoring the initial velocity using varying concentration of lignostilbene and the inhibitor. The inhibition constants were determined by fitting an equation describing mixed inhibition to the data using LEONORA (38).

Protein structure determination
Crystals of apo-LsdA were grown aerobically by sitting drop at room temperature in a 1:1 mixture of ϳ10 mg/ml apo-LsdA in 20 mM HEPPS, 80 mM NaCl, 2 mM TCEP, pH 8.0, with reservoir solution containing 0.2 M tripotassium citrate and ϳ20% PEG 3350 (v/v). Crystals were briefly soaked in reservoir buffer supplemented with ϳ30% glycerol (v/v) for cryoprotection and flash-frozen in liquid nitrogen. Diffraction data were collected at the Stanford Synchrotron Radiation Lightsource (SSRL) on beamline 7-1 and the data were processed and integrated using AUTOXDS (39). LsdA crystallized in the space group P3 2 21 with two molecules in the asymmetric unit. The structure was solved by molecular replacement using the coordinates from the structure of apocarotenoid-15,15Ј-oxygenase from Synechocystis sp. strain PCC 6803/Kazusa (26% sequence identity, PDB code 2BIW) as a search model with the program PhaserMR from the Phenix package (40, 41). The structure was manually edited using Coot, and refinement was performed with phenix.refine (42,43). The refined structure has residues 2-483 modeled, out of 485 total residues, for chain A with a gap between 381 and 384 where poor electron density was not amendable to modeling. Residues 2-482 are modeled for chain B. The model also contains 547 waters, three magnesium ions, and four glycerol molecules.
Crystals of holo-LsdA were grown aerobically by sitting drop at room temperature in a 1:1 mixture of ϳ10 mg/ml holo-LsdA in 20 mM HEPPS, 80 mM NaCl, 2 mM TCEP, pH 8.0, with reservoir solution containing 0.2 M sodium acetate and ϳ25% PEG 3350 (v/v). Crystals were briefly soaked in reservoir buffer supplemented with ϳ30% glycerol (v/v) for cryoprotection and flash-frozen in liquid nitrogen. Diffraction data were collected at the SSRL on beamline 7-1. Data were processed and integrated using Mosflm and CCP4 AIMLESS (44,45). Holo-LsdA crystallized in the space group P3 2 21 with two molecules in the asymmetric unit. The structure was solved using molecular replacement with LsdA protomer coordinates from the solved apo-structure (described above) as a search model in the program PhaserMR from the Phenix package (40, 41). The refined structure has residues 2-481 modeled for chain A. Chain B has residues 2-481 modeled with two gaps spanning residues 306 -315 and 381-386 that were not modeled due to poor electron density. The model also contains two iron molecules, one magnesium ion, three glycerols, and 351 water molecules.
A crystal structure of LsdA⅐phenylazo-phenol was obtained by co-crystallizing the enzyme and inhibitor. The crystals were prepared aerobically by sitting drop at room temperature using a 1:1 ratio of ϳ10 mg/ml LsdA in protein buffer and reservoir buffer containing 0.2 M sodium fluoride and ϳ17% PEG 3350 (v/v) supplemented with ϳ1 mM of phenylazophenol in DMF. Crystals were flash-frozen in liquid nitrogen. Diffraction data were collected at the Canadian Light Source on beamline 08ID-1 and data were processed and integrated using Mosflm and CCP4 AIMLESS (44,45). The crystal was of space group P3 2 21 with two molecules in the asymmetric unit. The structure was also solved using molecular replacement with LsdA protomer coordinates from the solved apo-structure as a search model in PhaserMR from Phenix (40, 41). The refined structure has residues 2-482 modeled for each protomer, but there was poor electron density for residues 309 -315 and 380 -386 in both protomers. A single solvent molecule was modeled in each active site.
Data collection and refinement statistics for all three structures are summarized in Table 2. The program MolProbity was used for structure validation including calculation of the fit to a Ramachandran plot (46). The coordinates and observed structure factor amplitudes have been deposited in the PDB under the accession codes 6OJR, 6OJW, and 6OJT for apo-LsdA, holo-LsdA, and LsdA⅐phenylazophenol, respectively. Structure figures were generated in PyMOL (PyMOL Molecular Graphics System, version 1.8, Schrödinger, LLC, New York). RMSD calculations between different LsdA structures were performed using the least-squared superposition tool of Coot (4). RMSD calculations between different LsdA and LSD NOV1 structures were performed using DALI (30).