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

Open AccessPublished:July 10, 2019DOI:https://doi.org/10.1074/jbc.RA119.009428
      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.

      Introduction

      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 (
      • Ragauskas A.J.
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      Lignin valorization: improving lignin processing in the biorefinery.
      ). Deconstructing lignin is thus of great relevance to transforming lignocellulose to biofuels and commodity chemicals (
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      Lignin valorization: improving lignin processing in the biorefinery.
      ,
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      • Rahmanpour R.
      Enzymatic conversion of lignin into renewable chemicals.
      ). 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 (
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      Bacterial catabolism of lignin-derived aromatics: new findings in a recent decade: update on bacterial lignin catabolism.
      ). Lignin-derived aromatic compounds that are efficiently degraded by bacteria include β-aryl ethers, pinoresinol, 2,2′-dihydroxy-3,3′-dimethoxy-5,5′-dicarboxybiphenyl, diaryl propane, and phenylcoumarane (
      • Kamimura N.
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      Bacterial catabolism of lignin-derived aromatics: new findings in a recent decade: update on bacterial lignin catabolism.
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      Decoding how a soil bacterium extracts building blocks and metabolic energy from ligninolysis provides road map for lignin valorization.
      ).
      Lignostilbene-α,β-dioxygenase (LSD,
      The abbreviations used are: LSD
      lignostilbene-α,β-dioxygenase
      CAO1
      carotenoid oxygenase 1
      CCO
      carotenoid cleavage oxygenase
      DMF
      dimethylformamide
      HEPPS
      4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid
      ICP-MS
      inductively coupled plasma mass spectrometry
      Kic
      competitive inhibition constant
      Kiu
      uncompetitive inhibition constant
      PDB
      Protein Data Bank
      RMSD
      root mean square deviation
      TAPS
      3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid
      TCEP
      tris(2-carboxyethyl)phosphine hydrochloride
      DCA-S
      3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl)-acrylate
      SSRL
      Stanford Synchrotron Radiation Lightsource.
      EC 1.13.11.43) catalyzes the oxygenolytic fission of lignostilbene (Fig. 1A) to two molecules of vanillin, incorporating both atoms of O2 into the products (
      • Kamoda S.
      • Habu N.
      • Samejima M.
      • Yoshimoto T.
      Purification and some properties of lignostilbene-α,β-dioxygenase responsible for the Cα-Cβ cleavage of a diarylpropane type lignin model-compound from Pseudomonas sp. TMY1009.
      ,
      • Sui X.
      • Golczak M.
      • Zhang J.
      • Kleinberg K.A.
      • von Lintig J.
      • Palczewski K.
      • Kiser P.D.
      Utilization of dioxygen by carotenoid cleavage oxygenases.
      • dela Seña C.
      • Riedl K.M.
      • Narayanasamy S.
      • Curley Jr., R.W.
      • Schwartz S.J.
      • Harrison E.H.
      The human enzyme that converts dietary provitamin A carotenoids to vitamin A is a dioxygenase.
      ). Lignostilbenoids occur in a very limited number of naturally occurring lignins but are prevalent in industrial lignins due to condensation reactions (
      • Nunes S.
      • Danesi F.
      • Del Rio D.
      • Silva P.
      Resveratrol and inflammatory bowel disease: the evidence so far.
      ,
      • Crestini C.
      • Lange H.
      • Sette M.
      • Argyropoulos D.S.
      On the structure of softwood kraft lignin.
      ). Moreover, lignin-derived stilbenes are proposed to be produced in the bacterial catabolism of diaryl propane and phenylcoumarane (
      • Kamoda S.
      • Habu N.
      • Samejima M.
      • Yoshimoto T.
      Purification and some properties of lignostilbene-α,β-dioxygenase responsible for the Cα-Cβ cleavage of a diarylpropane type lignin model-compound from Pseudomonas sp. TMY1009.
      ,
      • Takahashi K.
      • Miyake K.
      • Hishiyama S.
      • Kamimura N.
      • Masai E.
      Two novel decarboxylase genes play a key role in the stereospecific catabolism of dehydrodiconiferyl alcohol in Sphingobium sp. strain SYK-6.
      ). Plants produce other stilbenes, such as resveratrol, arachidins, and astringins, as allelochemicals to protect against pathogens and physical damage (
      • Chong J.
      • Poutaraud A.
      • Hugueney P.
      Metabolism and roles of stilbenes in plants.
      ). This variety of stilbenes may explain why some bacterial strains contain multiple LSD homologs (
      • Kamimura N.
      • Takahashi K.
      • Mori K.
      • Araki T.
      • Fujita M.
      • Higuchi Y.
      • Masai E.
      Bacterial catabolism of lignin-derived aromatics: new findings in a recent decade: update on bacterial lignin catabolism.
      ,
      • Kamoda S.
      • Habu N.
      • Samejima M.
      • Yoshimoto T.
      Purification and some properties of lignostilbene-α,β-dioxygenase responsible for the Cα-Cβ cleavage of a diarylpropane type lignin model-compound from Pseudomonas sp. TMY1009.
      ,
      • Takahashi K.
      • Miyake K.
      • Hishiyama S.
      • Kamimura N.
      • Masai E.
      Two novel decarboxylase genes play a key role in the stereospecific catabolism of dehydrodiconiferyl alcohol in Sphingobium sp. strain SYK-6.
      ,
      • Kamoda S.
      • Saburi Y.
      Structural and enzymatical comparison of lignostilbene-α,β-dioxygenase isozymes, I, II, and III, from Pseudomonas paucimobilis TMY1009.
      ,
      • Kamoda S.
      • Terada T.
      • Saburi Y.
      Purification and some properties of lignostilbene-α,β-dioxygenase isozyme IV from Pseudomonas paucimobilis TMY1009.
      ). The first LSD to be described, LsdA from Sphingomonas paucimobilis TMY1009 (TMY1009 hereafter), shares 98% amino acid sequence identity with LSD1 from Sphingobium sp. SYK-6 (SYK-6 hereafter), encoded by SLG_37450. LsdA functions as a homodimer and, somewhat unexpectedly, also as a heterodimer with LsdB, a second LSD in TMY1009 with which it shares 70% identity (
      • Kamoda S.
      • Saburi Y.
      Structural and enzymatical comparison of lignostilbene-α,β-dioxygenase isozymes, I, II, and III, from Pseudomonas paucimobilis TMY1009.
      ). LsdA also shares 49% amino acid sequence identity with the pterostilbene-cleaving CCO1 from Sphingobium sp. strain JS1018 (
      • Yu R.Q.
      • Kurt Z.
      • He F.
      • Spain J.C.
      Biodegradation of the allelopathic chemical pterostilbene by a Sphingobium sp. strain from the peanut rhizosphere.
      ). Among structurally characterized enzymes, LsdA shares 56% identity with LSDNOV1 from Novosphingomonas aromaticivorans NOV1 and 40% with carotenoid oxygenase 1 (CAO1) of Neurospora crassa (
      • Kamimura N.
      • Takahashi K.
      • Mori K.
      • Araki T.
      • Fujita M.
      • Higuchi Y.
      • Masai E.
      Bacterial catabolism of lignin-derived aromatics: new findings in a recent decade: update on bacterial lignin catabolism.
      ,
      • Kamoda S.
      • Saburi Y.
      Cloning, expression, and sequence analysis of a lignostilbene-α,β-dioxygenase gene from Pseudomonas paucimobilis TMY1009.
      ,
      • Diaz-Sanchez V.
      • Estrada A.F.
      • Limón M.C.
      • Al-Babili S.
      • Avalos J.
      The oxygenase CAO-1 of Neurospora crassa is a resveratrol cleavage enzyme.
      • Marasco E.K.
      • Schmidt-Dannert C.
      Identification of bacterial carotenoid cleavage dioxygenase homologues that cleave the interphenyl α,β double bond of stilbene derivatives via a monooxygenase reaction.
      ). Among tested stilbenes, LsdA had the highest substrate specificity (kcat/KM) for lignostilbene (
      • Kamoda S.
      • Saburi Y.
      Structural and enzymatical comparison of lignostilbene-α,β-dioxygenase isozymes, I, II, and III, from Pseudomonas paucimobilis TMY1009.
      ). Moreover, the enzyme appears to act only on trans-stilbenes possessing a 4-hydroxy moiety (
      • Kamoda S.
      • Terada T.
      • Saburi Y.
      A common structure of substrate shared by lignostilbene-α,β-dioxygenase isozymes from Sphingomonas paucimobilis TMY1009.
      ).
      Figure thumbnail gr1
      Figure 1A, the LsdA-catalyzed cleavage of lignostilbene. B, compounds used in this study: lignostilbene (1), resveratrol (2), 4-hydroxystilbene (3), 4-hydroxy-4′-nitrostilbene (4), stilbene (5), 4,4′-dimethoxystilbene (6), diethylstilbestrol (7), phenylazophenol (8).
      LSDs belong to the same protein family as carotenoid cleavage oxygenases (CCOs), which typically catalyze the oxidative cleavage of a double bond in carotenoids (
      • Sui X.
      • Kiser P.D.
      • Lintig J.
      • Palczewski K.
      Structural basis of carotenoid cleavage: from bacteria to mammals.
      ,
      • Harrison P.J.
      • Bugg T.D.
      Enzymology of the carotenoid cleavage dioxygenases: reaction mechanisms, inhibition and biochemical roles.
      ). These enzymes are characterized by a structural fold comprising a seven-bladed β-propeller (
      • Sui X.
      • Kiser P.D.
      • Lintig J.
      • Palczewski K.
      Structural basis of carotenoid cleavage: from bacteria to mammals.
      ). The active site occurs at the center of this propeller and contains an Fe2+ coordinated by four histidines (
      • Sui X.
      • Kiser P.D.
      • Lintig J.
      • Palczewski K.
      Structural basis of carotenoid cleavage: from bacteria to mammals.
      ). In crystal structures of LSDNOV1 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 (
      • McAndrew R.P.
      • Sathitsuksanoh N.
      • Mbughuni M.M.
      • Heins R.A.
      • Pereira J.H.
      • George A.
      • Sale K.L.
      • Fox B.G.
      • Simmons B.A.
      • Adams P.D.
      Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase.
      ,
      • Sui X.
      • Weitz A.C.
      • Farquhar E.R.
      • Badiee M.
      • Banerjee S.
      • von Lintig J.
      • Tochtrop G.P.
      • Palczewski K.
      • Hendrich M.P.
      • Kiser P.D.
      Structure and spectroscopy of alkene-cleaving dioxygenases containing an atypically coordinated non-heme iron center.
      ).
      At least two mechanisms have been proposed for LSDs. In a mechanism proposed by McAndrew et al. (
      • McAndrew R.P.
      • Sathitsuksanoh N.
      • Mbughuni M.M.
      • Heins R.A.
      • Pereira J.H.
      • George A.
      • Sale K.L.
      • Fox B.G.
      • Simmons B.A.
      • Adams P.D.
      Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase.
      ), the hydroxystilbenoid is activated via the enzyme-catalyzed deprotonation of the 4-hydroxy group, which allows electron delocalization toward an Fe3+-superoxo electrophile. In an alternate proposal by Sui et al. (
      • Sui X.
      • Weitz A.C.
      • Farquhar E.R.
      • Badiee M.
      • Banerjee S.
      • von Lintig J.
      • Tochtrop G.P.
      • Palczewski K.
      • Hendrich M.P.
      • Kiser P.D.
      Structure and spectroscopy of alkene-cleaving dioxygenases containing an atypically coordinated non-heme iron center.
      ), π electron density from the scissile double bond is redistributed to the iron-oxy complex to form an Fe2+-peroxo-substrate cation intermediate. Deprotonation of the hydroxyl moiety is critical in both mechanisms and is assisted by Lys134 and Tyr101 (LsdA/LSDNOV1 numbering), two active-site residues conserved among stilbene-cleaving oxygenases (
      • McAndrew R.P.
      • Sathitsuksanoh N.
      • Mbughuni M.M.
      • Heins R.A.
      • Pereira J.H.
      • George A.
      • Sale K.L.
      • Fox B.G.
      • Simmons B.A.
      • Adams P.D.
      Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase.
      ,
      • Sui X.
      • Weitz A.C.
      • Farquhar E.R.
      • Badiee M.
      • Banerjee S.
      • von Lintig J.
      • Tochtrop G.P.
      • Palczewski K.
      • Hendrich M.P.
      • Kiser P.D.
      Structure and spectroscopy of alkene-cleaving dioxygenases containing an atypically coordinated non-heme iron center.
      ). 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 O2 to inhibit the oxidation of the metal ion, as is the case for extradiol dioxygenases (
      • Costas M.
      • Mehn M.P.
      • Jensen M.P.
      • Que Jr., L.
      Dioxygen activation at mononuclear nonheme iron active sites: enzymes, models, and intermediates.
      ). For example, EPR analyses have suggested that NO binds to the iron center independent of organic substrate (
      • McAndrew R.P.
      • Sathitsuksanoh N.
      • Mbughuni M.M.
      • Heins R.A.
      • Pereira J.H.
      • George A.
      • Sale K.L.
      • Fox B.G.
      • Simmons B.A.
      • Adams P.D.
      Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase.
      ). By contrast, crystallographic, X-ray absorption spectroscopy, and Mössbauer spectroscopy data indicate that a number of CCOs do not bind O2 in the absence of the organic substrate (
      • Sui X.
      • Weitz A.C.
      • Farquhar E.R.
      • Badiee M.
      • Banerjee S.
      • von Lintig J.
      • Tochtrop G.P.
      • Palczewski K.
      • Hendrich M.P.
      • Kiser P.D.
      Structure and spectroscopy of alkene-cleaving dioxygenases containing an atypically coordinated non-heme iron center.
      ).
      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.

      Results

      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 (
      • Sui X.
      • Weitz A.C.
      • Farquhar E.R.
      • Badiee M.
      • Banerjee S.
      • von Lintig J.
      • Tochtrop G.P.
      • Palczewski K.
      • Hendrich M.P.
      • Kiser P.D.
      Structure and spectroscopy of alkene-cleaving dioxygenases containing an atypically coordinated non-heme iron center.
      ). 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 >99% apparent homogeneity as judged by SDS-PAGE analysis at yields of ∼10–20 mg of purified protein per liter of cell culture. Inductively coupled plasma MS (ICP-MS) analyses revealed that purified LsdA contained ∼1 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 ± 0.2 eq of iron per LsdA protomer. Preparations of LsdA retained essentially 100% of their activities when exposed to ambient levels of O2 for up to 16 h at room temperature.

      Substrate specificity

      In an oxygraph assay, LsdA was most active at pH 8.5 (Fig. S1). Accordingly, the enzyme was subsequently characterized using air-saturated TAPS (I = 0.1 m), pH 8.5, at 25 °C. Similar activity was observed using Tris as the buffer. LsdA cleaved 4-hydroxy trans-stilbenes, such as lignostilbene, 4-hydroxystilbene, and resveratrol (Fig. 1B), but not 4-hydroxy-4′-nitro-stilbene. The LsdA-catalyzed cleavage of lignostilbene to vanillin was validated using an HPLC-based assay (Fig. S2). The initial rate of lignostilbene cleavage displayed Michaelis–Menten kinetics (Fig. 2A). As summarized in Table 1, LsdA cleaved the substrates with the following specificity: lignostilbene > 4-hydroxystilbene ≈ resveratrol. LsdA also displayed Michaelis–Menten behavior with respect to O2 concentration, with a KMO2 value of 190 ± 10 μm (Fig. 2B).
      Figure thumbnail gr2
      Figure 2Steady-state kinetic analyses of the LsdA-catalyzed reaction. A, dependence of initial velocity on lignostilbene concentration in air-saturated TAPS (I = 0.1 m, pH 8.5), 25 °C. B, dependence of initial velocity on the O2 concentration in the presence of 125 μm lignostilbene. Red lines, fits of the Michaelis–Menten equation to the data.
      Table 1Apparent steady-state kinetic parameters of LsdA for different substrates
      SubstratekcatappKMappkcatapp/KMapp
      s−1μms−1·mm−1
      Lignostilbene30 ± 131 ± 31000 ± 50
      Resveratrol0.63 ± 0.0234 ± 419 ± 2
      4-Hydroxystilbene
      Reported values based on concentration up to 30 μm, above which substrate inhibition was observed.
      0.21 ± 0.019 ± 124 ± 3
      a Reported values based on concentration up to 30 μm, above which substrate inhibition was observed.
      In the oxygraph assay, neither stilbene nor 4,4′-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 (Kic = 6 ± 1 μm and Kiu = 24 ± 4 μm; Kic and Kiu refer to competitive and uncompetitive inhibition constant, respectively) (Fig. 3). Preincubation of LsdA with up to 50 μm phenylazophenol for up to 30 min at room temperature did not significantly affect the enzymatic activity, consistent with reversible inhibition.
      Figure thumbnail gr3
      Figure 3Dixon plot of the inhibition of LsdA-catalyzed lignostilbene cleavage by phenylazophenol. Experiments were performed using TAPS (I = 0.1 m, pH 8.5), 25 °C, and 10 μm (■), 20 μm (▴), 40 μm (●), 60 μm (×), 80 μm (□), 100 μm (▵), and 120 μm (○) lignostilbene. The lines represent a best fit of an equation describing mixed inhibition to the data (Kic = 6 ± 1 μm; Kiu = 24 ± 4 μm; KM = 32 ± 3 μm; kcat = 32 ± 2 s−1).

      Structure of LsdA

      LsdA crystallized in space group P3221 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 (
      • Kamoda S.
      • Saburi Y.
      Structural and enzymatical comparison of lignostilbene-α,β-dioxygenase isozymes, I, II, and III, from Pseudomonas paucimobilis TMY1009.
      ). The structure for apo-LsdA, solved to 2.3 Å resolution, was used to solve that of holo-LsdA) (i.e. bound to Fe2+) 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 Cα atoms of ∼0.3 Å. The structural fold of LsdA is that of a seven-bladed β-propeller, typical of the CCOs (Fig. 4B). Among stilbenoid-cleaving dioxygenases of known structure, LsdA is most similar to the resveratrol-cleaving enzyme LSDNOV1 (Protein Data Bank (PDB) entry 5J53) (
      • McAndrew R.P.
      • Sathitsuksanoh N.
      • Mbughuni M.M.
      • Heins R.A.
      • Pereira J.H.
      • George A.
      • Sale K.L.
      • Fox B.G.
      • Simmons B.A.
      • Adams P.D.
      Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase.
      ), sharing an RMSD of 1.1 Å over 473 aligned Cα atoms. LSDNOV1 is also a homodimer.
      Figure thumbnail gr4
      Figure 4Crystal structure of LsdA dimer and protomer. A, ribbon and surface representation of the dimeric assembly of LsdA. The different protomers of the asymmetric unit and presumed dimer are yellow and teal, respectively. Bound Fe2+ ions are shown as black spheres. B, ribbon diagram of LsdA protomer, colored using a gradient from the N (blue) to C (red) termini. C, stick representation of residues at the dimer interface. Carbon atoms and secondary structure elements of the two subunits are yellow and teal, respectively. Intersubunit salt bridges and hydrogen-bonding interactions (≤4.0 Å) are indicated using dashed lines.
      Table 2X-ray diffraction data collection and refinement statistics for LsdA structures
      Apo-LsdAHolo-LsdALsdA·phenylazophenol
      Data collection
      Data collection values in parentheses represent the data for the highest resolution shell.
      Resolution range (Å)37.08–2.30 (2.38–2.30)60.92–2.60 (2.69–2.60)61.00–3.00 (3.11–3.00)
      Space groupP3221P3221P3221
      Unit cell dimensions (Å)a = b = 181.3, c = 94.9a = b = 181.3, c = 95.1a = b = 181.9, c = 96.4
      Unique reflections79,776 (7876)56,663 (5634)34,286 (3443)
      Completeness (%)99.9 (99.4)99.8 (99.6)92.0 (93.6)
      Redundancy13.5 (13.2)9.2 (9.9)2.0 (2.0)
      Average II20.3 (2.1)15.4 (2.2)9.9 (2.5)
      Rmerge0.146 (1.353)0.048 (0.337)0.053 (0.259)
      CC½0.998 (0.731)0.996 (0.733)0.990 (0.875)
      Wilson B-factor (Å2)35.839.055.7
      Anisotropy0.3560.1150.637
      Data refinement
      Rwork (Rfree)0.180 (0.203)0.187 (0.215)0.213 (0.241)
      Protein residues964944962
      No. of water molecules5473512
      RMSD bond length (Å)0.0030.0030.002
      RMSD bond angles (degrees)0.620.610.59
      Average B-value (Å2)40.943.056.0
      Ramachandran plot (%)
      Most favored regions97.696.394.9
      Disallowed regions0.00.20.2
      PDB code6OJR6OJW6OJT
      a Data collection values in parentheses represent the data for the highest resolution shell.
      The dimer interface of LsdA has a buried surface area of 1460 Å2 and contains many polar interactions, including 14 hydrogen bonds and 9 salt bridges, as predicted by PDB-PISA. All but one of these interactions are mediated by a linear stretch of residues from the N terminus to Glu31 of each subunit (Fig. 4C), which includes strand β1 (Glu20–Leu24). The β1 strands of the two subunits are arranged anti-parallel to each other, with the Glu20 amide from one subunit forming a hydrogen bond with the Asp22 carbonyl of the other. This arrangement creates a 10-stranded anti-parallel β-sheet formed by the first propeller blade of each protomer. Other notable interactions involve the side-chain carboxylates of Asp25 and Glu27, which form reciprocal salt bridges with the N terminus (Ala2) and Arg15, respectively, of the two protomers.

      Metal-binding site

      As observed in other CCOs, the LsdA active site harbors a single Fe2+ ion that resides at the center of the β-propeller. This metal ion is coordinated in a tetragonal pyramidal fashion by four conserved histidines (His167, His218, His282, and His472) and a solvent molecule (Fig. 5). The average Fe2+–His bond length in the resting state LsdA is ∼2.2 Å, in agreement with values reported in other CCOs (
      • McAndrew R.P.
      • Sathitsuksanoh N.
      • Mbughuni M.M.
      • Heins R.A.
      • Pereira J.H.
      • George A.
      • Sale K.L.
      • Fox B.G.
      • Simmons B.A.
      • Adams P.D.
      Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase.
      ,
      • Sui X.
      • Weitz A.C.
      • Farquhar E.R.
      • Badiee M.
      • Banerjee S.
      • von Lintig J.
      • Tochtrop G.P.
      • Palczewski K.
      • Hendrich M.P.
      • Kiser P.D.
      Structure and spectroscopy of alkene-cleaving dioxygenases containing an atypically coordinated non-heme iron center.
      ). Similar to other CCOs, the sixth metal coordination site (across from His282) is unoccupied and is partly occluded by Thr121 (
      • Sui X.
      • Zhang J.
      • Golczak M.
      • Palczewski K.
      • Kiser P.D.
      Key residues for catalytic function and metal coordination in a carotenoid cleavage dioxygenase.
      ). Additionally, three of the metal-coordinating histidines interact with conserved acidic residues (Glu135, Glu350, and Glu414) via hydrogen bonds. Finally a cap-like structure on one face of the β-propeller helps shield the metal-binding site from the solvent. This cap is formed by a series of loops that otherwise connect various β strands and is structurally similar to that of LSDNOV1 (RMSD of 3.6 Å over 126 superposed Cα 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.
      Figure thumbnail gr5
      Figure 5The metal-binding site of LsdA. Shown is a stick representation of LsdA in the resting state (PDB code 6OJW). The Fe2+ ion and solvent species are represented as orange and red spheres, respectively. Ligand bonds, as well as polar and hydrogen-bonding interactions (≤3.0 Å) are indicated using dashed lines.

      Structure of the LsdA·phenylazophenol complex

      To further explore substrate binding in LsdA, the enzyme was co-crystalized with phenylazophenol. Co-crystals of LsdA·phenyl-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 Cα atoms of ∼0.3 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 LsdA·phenylazophenol complex has a lower metal occupancy, as indicated by a weaker electron density associated with the metal ion as compared with the inhibitor-free structure.
      Figure thumbnail gr6
      Figure 6The substrate-binding site of LsdA. A, FoFc electron density omit maps (contoured at 4σ) of phenylazophenol for LsdA protomer A. B, stick representation of LsdA in complex with phenylazophenol (PDB code 6OJT). LsdA residues and phenylazophenol are drawn as sticks and colored gray and yellow, respectively. The Fe2+ ion and water molecule are represented as orange and red spheres, respectively. Ligand bonds as well as polar and hydrogen-bonding interactions (≤3.0 Å) are indicated using dashed lines. C, superposition of LsdA·phenylazophenol (yellow, 6OJT), LSDNOV1·resveratrol (gray, 5J54), and Co-CAO1·resveratrol (teal, 5U90). For clarity, only the active-site metal and key interacting residues Phe59, Tyr101, and Lys134 (LsdA numbering) are shown.
      Phenylazophenol was modeled at full occupancy with an average B-factor of 76.7 Å2 (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 enzyme·ligand complexes of homologous enzymes (Fig. 6C) (
      • McAndrew R.P.
      • Sathitsuksanoh N.
      • Mbughuni M.M.
      • Heins R.A.
      • Pereira J.H.
      • George A.
      • Sale K.L.
      • Fox B.G.
      • Simmons B.A.
      • Adams P.D.
      Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase.
      ,
      • Sui X.
      • Weitz A.C.
      • Farquhar E.R.
      • Badiee M.
      • Banerjee S.
      • von Lintig J.
      • Tochtrop G.P.
      • Palczewski K.
      • Hendrich M.P.
      • Kiser P.D.
      Structure and spectroscopy of alkene-cleaving dioxygenases containing an atypically coordinated non-heme iron center.
      ). In the model, the Fe2+ ion is closest to the two azo nitrogen atoms of phenylazophenol (∼3–4 Å). The 4-hydroxy moiety of the phenylazophenol forms hydrogen bonds with each of Tyr101 and Lys134 (Fig. 6B). In addition, Phe59 forms a π–π stacking interaction with the phenolic ring of the inhibitor. Glu350, located within the active-site pocket distal to Tyr101, 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 LSDNOV1 and CAO1, respectively, with resveratrol (
      • McAndrew R.P.
      • Sathitsuksanoh N.
      • Mbughuni M.M.
      • Heins R.A.
      • Pereira J.H.
      • George A.
      • Sale K.L.
      • Fox B.G.
      • Simmons B.A.
      • Adams P.D.
      Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase.
      ,
      • Sui X.
      • Weitz A.C.
      • Farquhar E.R.
      • Badiee M.
      • Banerjee S.
      • von Lintig J.
      • Tochtrop G.P.
      • Palczewski K.
      • Hendrich M.P.
      • Kiser P.D.
      Structure and spectroscopy of alkene-cleaving dioxygenases containing an atypically coordinated non-heme iron center.
      ). In LSDNOV1, Ser283 and Glu353 form hydrogen bonds to one of the hydroxyl groups on reserveratrol's resorcinolic ring. The glutamate is conserved in LsdA (Glu350) 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 (Phe305, Phe307, and Phe308) from the cap. These interactions, together with an interaction with Phe59, help sequester the inhibitor from the solvent. As compared with the ligand-free enzyme, a rotation about χ1 of the Phe305 by −45° 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 Phe308 is repositioned by rotations of χ1 and χ2 by 70 and 115°, respectively, to form the ligand-binding pocket in the LsdA·phenylazophenol complex. Finally, no density corresponding to a metal-bound solvent or O2 species was observed in the LsdA·phenylazophenol complex, in contrast to what was reported in the CAO1·resveratrol and LSDNOV1·resveratrol 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: Phe59, Tyr101, and Lys134. 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 kcatapp and kcatapp/KMapp (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 ∼7 and 3% those of WT LsdA.
      Table 3Apparent steady-state kinetic parameters of LsdA and select variants for lignostilbene
      EnzymekcatappKMappkcatapp/KMapp
      s−1μms−1·mm−1
      Experiments were performed using air-saturated TAPS (I = 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.
      WT30 ± 131 ± 31000 ± 50
      F59H2.0 ± 0.172 ± 528 ± 2
      Y101F3.1 ± 0.115 ± 1210 ± 20
      K134MND
      ND, not detected.
      NDND
      a Experiments were performed using air-saturated TAPS (I = 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.

      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 (
      • Kamoda S.
      • Terada T.
      • Saburi Y.
      A common structure of substrate shared by lignostilbene-α,β-dioxygenase isozymes from Sphingomonas paucimobilis TMY1009.
      ). Different LSD homologs have different substrate specificities, as exemplified by the isoforms of LSDTMY1009 (
      • Kamoda S.
      • Saburi Y.
      Structural and enzymatical comparison of lignostilbene-α,β-dioxygenase isozymes, I, II, and III, from Pseudomonas paucimobilis TMY1009.
      ). Moreover, there are conflicting reports on whether LSDNOV1 can cleave rhapontigenin and rhaponticin, both of which are 4-methoxy stilbenes (
      • Marasco E.K.
      • Schmidt-Dannert C.
      Identification of bacterial carotenoid cleavage dioxygenase homologues that cleave the interphenyl α,β double bond of stilbene derivatives via a monooxygenase reaction.
      ,
      • McAndrew R.P.
      • Sathitsuksanoh N.
      • Mbughuni M.M.
      • Heins R.A.
      • Pereira J.H.
      • George A.
      • Sale K.L.
      • Fox B.G.
      • Simmons B.A.
      • Adams P.D.
      Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase.
      ). Nevertheless, most characterized LSDs appear to require the 4-hydroxy moiety for activity (
      • Sui X.
      • Golczak M.
      • Zhang J.
      • Kleinberg K.A.
      • von Lintig J.
      • Palczewski K.
      • Kiser P.D.
      Utilization of dioxygen by carotenoid cleavage oxygenases.
      ,
      • McAndrew R.P.
      • Sathitsuksanoh N.
      • Mbughuni M.M.
      • Heins R.A.
      • Pereira J.H.
      • George A.
      • Sale K.L.
      • Fox B.G.
      • Simmons B.A.
      • Adams P.D.
      Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase.
      ,
      • Loewen P.C.
      • Switala J.
      • Wells J.P.
      • Huang F.
      • Zara A.T.
      • Allingham J.S.
      • Loewen M.C.
      Structure and function of a lignostilbene-α,β-dioxygenase orthologue from Pseudomonas brassicacearum.
      ). 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 4-hydroxyl group in each of the LSDNOV1·resveratrol, CAO1·resveratrol, and LsdA·phenylazophenol complexes (
      • McAndrew R.P.
      • Sathitsuksanoh N.
      • Mbughuni M.M.
      • Heins R.A.
      • Pereira J.H.
      • George A.
      • Sale K.L.
      • Fox B.G.
      • Simmons B.A.
      • Adams P.D.
      Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase.
      ,
      • Sui X.
      • Weitz A.C.
      • Farquhar E.R.
      • Badiee M.
      • Banerjee S.
      • von Lintig J.
      • Tochtrop G.P.
      • Palczewski K.
      • Hendrich M.P.
      • Kiser P.D.
      Structure and spectroscopy of alkene-cleaving dioxygenases containing an atypically coordinated non-heme iron center.
      ).
      The inhibition of LsdA by phenylazophenol is consistent with previous studies in which LsdA was unable to cleave stilbenoids with substitutions at either Cα or Cβ of the vinyl group (
      • Han S.
      • Inoue H.
      • Terada T.
      • Kamoda S.
      • Saburi Y.
      • Sekimata K.
      • Saito T.
      • Kobayashi M.
      • Shinozaki K.
      • Yoshida S.
      • Asami T.
      Design and synthesis of lignostilbene-α,β-dioxygenase inhibitors.
      ,
      • Han S.Y.
      • Inoue H.
      • Terada T.
      • Kamoda S.
      • Saburi Y.
      • Sekimata K.
      • Saito T.
      • Kobayashi M.
      • Shinozaki K.
      • Yoshida S.
      • Asami T.
      N-Benzylideneaniline and N-benzylaniline are potent inhibitors of lignostilbene-α,β-dioxygenase, a key enzyme in oxidative cleavage of the central double bond of lignostilbene.
      ). Further, both N-benzylideneaniline and N-benzylaniline potently inhibit LsdA (
      • Han S.Y.
      • Inoue H.
      • Terada T.
      • Kamoda S.
      • Saburi Y.
      • Sekimata K.
      • Saito T.
      • Kobayashi M.
      • Shinozaki K.
      • Yoshida S.
      • Asami T.
      N-Benzylideneaniline and N-benzylaniline are potent inhibitors of lignostilbene-α,β-dioxygenase, a key enzyme in oxidative cleavage of the central double bond of lignostilbene.
      ). Similarly, CAO1 did not cleave fluoro-resveratrol (
      • Sui X.
      • Weitz A.C.
      • Farquhar E.R.
      • Badiee M.
      • Banerjee S.
      • von Lintig J.
      • Tochtrop G.P.
      • Palczewski K.
      • Hendrich M.P.
      • Kiser P.D.
      Structure and spectroscopy of alkene-cleaving dioxygenases containing an atypically coordinated non-heme iron center.
      ). Phenylazophenol behaved as a mixed inhibitor, which suggests the presence of multiple binding sites. Whereas only a single binding site was observed in the LsdA·phenylazophenol structure, electron density consistent with multiple ligands was observed in a Co2+-substituted CAO1·resveratrol complex (
      • Sui X.
      • Weitz A.C.
      • Farquhar E.R.
      • Badiee M.
      • Banerjee S.
      • von Lintig J.
      • Tochtrop G.P.
      • Palczewski K.
      • Hendrich M.P.
      • Kiser P.D.
      Structure and spectroscopy of alkene-cleaving dioxygenases containing an atypically coordinated non-heme iron center.
      ).
      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-(4-hydroxy-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 (
      • Takahashi K.
      • Miyake K.
      • Hishiyama S.
      • Kamimura N.
      • Masai E.
      Two novel decarboxylase genes play a key role in the stereospecific catabolism of dehydrodiconiferyl alcohol in Sphingobium sp. strain SYK-6.
      ). It is presumed that DCA-S is cleaved by at least one of the eight LSD homologs harbored by SYK-6 (
      • Kamimura N.
      • Takahashi K.
      • Mori K.
      • Araki T.
      • Fujita M.
      • Higuchi Y.
      • Masai E.
      Bacterial catabolism of lignin-derived aromatics: new findings in a recent decade: update on bacterial lignin catabolism.
      ). Nevertheless, LsdA's specific activity for DCA-S was 2 orders of magnitude lower than for lignostilbene (
      • Kamoda S.
      • Habu N.
      • Samejima M.
      • Yoshimoto T.
      Purification and some properties of lignostilbene-α,β-dioxygenase responsible for the Cα-Cβ cleavage of a diarylpropane type lignin model-compound from Pseudomonas sp. TMY1009.
      ).
      In the two proposed reaction mechanism of LSDs, conserved Lys134 and Tyr101 have been postulated to assist in the deprotonation of the 4-hydroxyl (
      • McAndrew R.P.
      • Sathitsuksanoh N.
      • Mbughuni M.M.
      • Heins R.A.
      • Pereira J.H.
      • George A.
      • Sale K.L.
      • Fox B.G.
      • Simmons B.A.
      • Adams P.D.
      Structure and mechanism of NOV1, a resveratrol-cleaving dioxygenase.
      ,
      • Sui X.
      • Weitz A.C.
      • Farquhar E.R.
      • Badiee M.
      • Banerjee S.
      • von Lintig J.
      • Tochtrop G.P.
      • Palczewski K.
      • Hendrich M.P.
      • Kiser P.D.
      Structure and spectroscopy of alkene-cleaving dioxygenases containing an atypically coordinated non-heme iron center.
      ). Our data indicate that although both residues hydrogen-bond with the substrate's hydroxyl, only Lys134 is essential for catalysis. Although the high pKa of lysine's ∈-amino group (∼10) makes it ill-suited to function as a base catalyst, it functions as such in several dehydrogenases (
      • Sekimoto T.
      • Matsuyama T.
      • Fukui T.
      • Tanizawa K.
      Evidence for lysine 80 as general base catalyst of leucine dehydrogenase.
      ,
      • Zhang L.
      • Chooback L.
      • Cook P.F.
      Lysine 183 is the general base in the 6-phosphogluconate dehydrogenase-catalyzed reaction.
      • Kumar V.P.
      • Thomas L.M.
      • Bobyk K.D.
      • Andi B.
      • Cook P.F.
      • West A.H.
      Evidence in support of lysine 77 and histidine 96 as acid-base catalytic residues in saccharopine dehydrogenase from Saccharomyces cerevisiae.
      ). 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 pKa of the latter's phenolic hydroxyl group via electrostatic polarization (
      • Jörnvall H.
      • Persson B.
      • Krook M.
      • Atrian S.
      • Gonzàlez-Duarte R.
      • Jeffery J.
      • Ghosh D.
      Short-chain dehydrogenases/reductases (SDR).
      ). Our data are consistent with the catalytic lysine of LSDs acting directly on the substrate.
      The Fe2+ cofactor of LSDs and other CCOs is relatively resistant to oxidative inactivation. This is in contrast to another class of nonheme Fe2+ dioxygenases, the extradiol dioxygenases, which are inactivated upon exposure to O2 as well as during catalytic turnover (
      • Costas M.
      • Mehn M.P.
      • Jensen M.P.
      • Que Jr., L.
      Dioxygen activation at mononuclear nonheme iron active sites: enzymes, models, and intermediates.
      ). Whereas LSDs may use a gating mechanism similar to the extradiol dioxygenases to prevent the binding and activation of O2 in the absence of organic substrate, this protective measure is unlikely to involve structural changes to modulate the enzyme's affinity toward O2. Substrate binding in CCO did not affect the protein structure or significantly change the electronic state of the metal (
      • Sui X.
      • Weitz A.C.
      • Farquhar E.R.
      • Badiee M.
      • Banerjee S.
      • von Lintig J.
      • Tochtrop G.P.
      • Palczewski K.
      • Hendrich M.P.
      • Kiser P.D.
      Structure and spectroscopy of alkene-cleaving dioxygenases containing an atypically coordinated non-heme iron center.
      ). Indeed, the active-site 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 (
      • Kamoda S.
      • Saburi Y.
      Structural and enzymatical comparison of lignostilbene-α,β-dioxygenase isozymes, I, II, and III, from Pseudomonas paucimobilis TMY1009.
      ). Further, these residues are conserved in CAO1, consistent with its dimeric structure. LsdA and LsdB share >95% 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 (
      • Kamoda S.
      • Terada T.
      • Saburi Y.
      Purification and some properties of lignostilbene-α,β-dioxygenase isozyme IV from Pseudomonas paucimobilis TMY1009.
      ).
      In conclusion, this study presents a more in-depth look into the first characterized LSD and establishes the relative importance of Lys134 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.

      Experimental procedures

      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 DiamondTM 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 (
      • Ausubel F.M.
      ). The lsdA gene (locus tag: 1917171A), which encodes for the α-isoform of LSD in TMY1009, was synthesized by back-translating the protein's amino acid sequence 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 λ(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 A600 of ∼0.7. Expression of lsdA was induced with 1 mm isopropyl β-d-thiogalactopyranoside, at which time the medium was further supplemented with 0.5 mm FeCl3, and the cells were incubated at 16 °C for an additional 16 h. Cells were harvested by centrifugation and stored at −80 °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 (NH4)2Fe(SO4)2, pH 8.0, and lysed at 4 °C using an EmulsiFlex-C5 homogenizer (Avestin). Cellular debris was removed by centrifugation. (NH4)2SO4 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 (NH4)2SO4, and the precipitate was removed by centrifugation and filtration at 0.45 μm. 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 N2 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 (NH4)2SO4 in 120 ml of 20 mm HEPPS, 2 mm DTT, 0.5 mm (NH4)2Fe(SO4)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 ∼10 mg/ml, flash-frozen as beads in liquid N2, and stored at −80 °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 (
      • Ausubel F.M.
      ). Protein concentration was determined using a micro-BCATM 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 (
      • Zabinski R.
      • Münck E.
      • Champion P.M.
      • Wood J.M.
      Kinetic and Mossbauer studies on the mechanism of protocatechuic acid 4,5-oxygenase.
      ). 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 HNO3 and H2O2 as described previously (
      • Shiel A.E.
      • Weis D.
      • Orians K.J.
      Tracing cadmium, zinc and lead sources in bivalves from the coasts of western Canada and the U.S.A. using isotopes.
      ).

      Steady-state kinetics

      Kinetic assays were performed by monitoring the consumption of O2 using a Clark-type polarographic O2 electrode OXYG1 (Hansatech) connected to a circulating water bath. Assays were performed in 1 ml of air-saturated 40 mm TAPS (I = 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 <0.5% (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 O2-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 (
      • Cornish-Bowden A.
      ). The effect of pH on the rate of the LsdA-catalyzed reaction was evaluated using I = 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 steady-state kinetic parameters for O2 were evaluated using 125 μm lignostilbene and initial concentrations of O2 from 16 to 600 μm. The initial O2 concentrations were achieved by equilibrating the reaction mixture with humidified mixtures of O2 and N2 gasses. Final O2 levels were normalized to the ambient O2 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 (
      • Cornish-Bowden A.
      ).

      Protein structure determination

      Crystals of apo-LsdA were grown aerobically by sitting drop at room temperature in a 1:1 mixture of ∼10 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 ∼20% PEG 3350 (v/v). Crystals were briefly soaked in reservoir buffer supplemented with ∼30% 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 (
      • Soltis S.M.
      • Cohen A.E.
      • Deacon A.
      • Eriksson T.
      • González A.
      • McPhillips S.
      • Chui H.
      • Dunten P.
      • Hollenbeck M.
      • Mathews I.
      • Miller M.
      • Moorhead P.
      • Phizackerley R.P.
      • Smith C.
      • Song J.
      • et al.
      New paradigm for macromolecular crystallography experiments at SSRL: automated crystal screening and remote data collection.
      ). LsdA crystallized in the space group P3221 with two molecules in the asymmetric unit. The structure was solved by molecular replacement using the coordinates from the structure of apocarotenoid-15,15′-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 (
      • McCoy A.J.
      • Grosse-Kunstleve R.W.
      • Adams P.D.
      • Winn M.D.
      • Storoni L.C.
      • Read R.J.
      Phaser Crystallographic Software.
      ,
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • et al.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ). The structure was manually edited using Coot, and refinement was performed with phenix.refine (
      • Emsley P.
      • Lohkamp B.
      • Scott W.G.
      • Cowtan K.
      Features and Development of Coot.
      ,
      • Afonine P.V.
      • Grosse-Kunstleve R.W.
      • Echols N.
      • Headd J.J.
      • Moriarty N.W.
      • Mustyakimov M.
      • Terwilliger T.C.
      • Urzhumtsev A.
      • Zwart P.H.
      • Adams P.D.
      Towards automated crystallographic structure refinement with Phenix.Refine.
      ). 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 ∼10 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 ∼25% PEG 3350 (v/v). Crystals were briefly soaked in reservoir buffer supplemented with ∼30% 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 (
      • Winn M.D.
      • Ballard C.C.
      • Cowtan K.D.
      • Dodson E.J.
      • Emsley P.
      • Evans P.R.
      • Keegan R.M.
      • Krissinel E.B.
      • Leslie A.G.
      • McCoy A.
      • McNicholas S.J.
      • Murshudov G.N.
      • Pannu N.S.
      • Potterton E.A.
      • Powell H.R.
      • et al.
      Overview of the CCP4 suite and current developments.
      ,
      • Battye T.G.
      • Kontogiannis L.
      • Johnson O.
      • Powell H.R.
      • Leslie A.G.
      iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM.
      ). Holo-LsdA crystallized in the space group P3221 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 (
      • McCoy A.J.
      • Grosse-Kunstleve R.W.
      • Adams P.D.
      • Winn M.D.
      • Storoni L.C.
      • Read R.J.
      Phaser Crystallographic Software.
      ,
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • et al.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ). 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 LsdA·phenylazo-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 ∼10 mg/ml LsdA in protein buffer and reservoir buffer containing 0.2 m sodium fluoride and ∼17% PEG 3350 (v/v) supplemented with ∼1 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 (
      • Winn M.D.
      • Ballard C.C.
      • Cowtan K.D.
      • Dodson E.J.
      • Emsley P.
      • Evans P.R.
      • Keegan R.M.
      • Krissinel E.B.
      • Leslie A.G.
      • McCoy A.
      • McNicholas S.J.
      • Murshudov G.N.
      • Pannu N.S.
      • Potterton E.A.
      • Powell H.R.
      • et al.
      Overview of the CCP4 suite and current developments.
      ,
      • Battye T.G.
      • Kontogiannis L.
      • Johnson O.
      • Powell H.R.
      • Leslie A.G.
      iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM.
      ). The crystal was of space group P3221 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 (
      • McCoy A.J.
      • Grosse-Kunstleve R.W.
      • Adams P.D.
      • Winn M.D.
      • Storoni L.C.
      • Read R.J.
      Phaser Crystallographic Software.
      ,
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • et al.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ). 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 (
      • Chen V.B.
      • Arendall 3rd, W.B.
      • Headd J.J.
      • Keedy D.A.
      • Immormino R.M.
      • Kapral G.J.
      • Murray L.W.
      • Richardson J.S.
      • Richardson D.C.
      MolProbity: all-atom structure validation for macromolecular crystallography.
      ). 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 LsdA·phenylazophenol, 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 (
      • Jackson C.A.
      • Couger M.B.
      • Prabhakaran M.
      • Ramachandriya K.D.
      • Canaan P.
      • Fathepure B.Z.
      Isolation and characterization of Rhizobium sp. strain YS-1r that degrades lignin in plant biomass.
      ). RMSD calculations between different LsdA and LSDNOV1 structures were performed using DALI (
      • Holm L.
      • Sander C.
      Dali: a network tool for protein structure comparison.
      ).

      Author contributions

      E. K. and L. D. E. designed experiments and analyzed data. E. K. conducted most of the experiments. A. K. N. L. assisted in the kinetic characterization. E. K., M. M. V., M. J. K., and M. E. P. M. performed the structural refinement. E. K. and L. D. E. wrote the paper with input from M. M. V. and M. E. P. M.

      Acknowledgments

      Mariko Ikehata assisted with the ICP-MS. Use of the Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory, was supported by the United States Department of Energy, Office of Science, and Office of Basic Energy Sciences under Contract DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the Department of Energy Office of Biological and Environmental Research and by NIGMS, National Institutes of Health (including Grant P41GM103393). Research described in this paper was performed using beamline 08D1-1 at the Canadian Light Source, which is supported by the NSERC, the National Research Council Canada, the Canadian Institutes of Health Research, the Province of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan.

      Supplementary Material

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