Structure of T4moF, the Toluene 4‑Monooxygenase Ferredoxin Oxidoreductase

The 1.6 Å crystal structure of toluene 4-monooxygenase reductase T4moF is reported. The structure includes ferredoxin, flavin, and NADH binding domains. The position of the ferredoxin domain relative to the other two domains represents a new configuration for the iron–sulfur flavoprotein family. Close contacts between the C8 methyl group of FAD and [2Fe-2S] ligand Cys36-O represent a plausible pathway for electron transfer between the redox cofactors. Energy-minimized docking of NADH and calculation of hingelike motions between domains suggest how simple coordinated shifts of residues at the C-terminus of the enzyme could expose the N5 position of FAD for productive interaction with the nicotinamide ring. The domain configuration revealed by the T4moF structure provides an excellent steric and electrostatic match to the obligate electron acceptor, Rieske-type [2Fe-2S] ferredoxin T4moC. Protein–protein docking and energy minimization of the T4moFC complex indicate that T4moF [2Fe-2S] ligand Cys41 and T4moC [2Fe-2S] ligand His67, along with other electrostatic interactions between the protein partners, form the functional electron transfer interface

Spectroscopic and Computational Investigation of the H155A Variant of Cysteine Dioxygenase: Geometric and Electronic Consequences of a Third-Sphere Amino Acid Substitution

Cysteine dioxygenase (CDO) is a mononuclear, non-heme iron­(II)-dependent enzyme that utilizes molecular oxygen to catalyze the oxidation of l-cysteine (Cys) to cysteinesulfinic acid. Although the kinetic consequences of various outer-sphere amino acid substitutions have previously been assessed, the effects of these substitutions on the geometric and electronic structures of the active site remained largely unexplored. In this work, we have performed a spectroscopic and computational characterization of the H155A CDO variant, which was previously shown to display a rate of Cys oxidation ∼100-fold decreased relative to that of wild-type (WT) CDO. Magnetic circular dichroism and electron paramagnetic resonance spectroscopic data indicate that the His155 → Ala substitution has a significant effect on the electronic structure of the Cys-bound Fe­(II)­CDO active site. An analysis of these data within the framework of density functional theory calculations reveals that Cys-bound H155A Fe­(II)­CDO possesses a six-coordinate Fe­(II) center, differing from the analogous WT CDO species in the presence of an additional water ligand. The enhanced affinity of the Cys-bound Fe­(II) center for a sixth ligand in the H155A CDO variant likely stems from the increased level of conformational freedom of the cysteine–tyrosine cross-link in the absence of the H155 imidazole ring. Notably, the nitrosyl adduct of Cys-bound Fe­(II)­CDO [which mimics the (O₂/Cys)–CDO intermediate] is essentially unaffected by the H155A substitution, suggesting that the primary role played by the H155 side chain in CDO catalysis is to discourage the binding of a water molecule to the Cys-bound Fe­(II)­CDO active site

Spectroscopic and Computational Investigation of Iron(III) Cysteine Dioxygenase: Implications for the Nature of the Putative Superoxo-Fe(III) Intermediate

Cysteine dioxygenase (CDO) is a mono­nuclear, non-heme iron-dependent enzyme that converts exogenous cysteine (Cys) to cysteine sulfinic acid using molecular oxygen. Although the complete catalytic mechanism is not yet known, several recent reports presented evidence for an Fe­(III)-superoxo reaction intermediate. In this work, we have utilized spectroscopic and computational methods to investigate the as-isolated forms of CDO, as well as Cys-bound Fe­(III)­CDO, both in the absence and presence of azide (a mimic of superoxide). An analysis of our electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance data of the azide-treated as-isolated forms of CDO within the framework of density functional theory (DFT) computations reveals that azide coordinates directly to the Fe­(III), but not the Fe­(II) center. An analogous analysis carried out for Cys-Fe­(III)­CDO provides compelling evidence that at physiological pH, the iron center is six coordinate, with hydroxide occupying the sixth coordination site. Upon incubation of this species with azide, the majority of the active sites retain hydroxide at the iron center. Nonetheless, a modest perturbation of the electronic structure of the Fe­(III) center is observed, indicating that azide ions bind near the active site. Additionally, for a small fraction of active sites, azide displaces hydroxide and coordinates directly to the Cys-bound Fe­(III) center to generate a low-spin (<i>S</i> = ¹/₂) Fe­(III) complex. In the DFT-optimized structure of this complex, the central nitrogen atom of the azide moiety lies within 3.12 Å of the cysteine sulfur. A similar orientation of the superoxide ligand in the putative Fe­(III)-superoxo reaction intermediate would promote the attack of the distal oxygen atom on the sulfur of substrate Cys

Spectroscopic and Computational Characterization of the NO Adduct of Substrate-Bound Fe(II) Cysteine Dioxygenase: Insights into the Mechanism of O₂ Activation

Cysteine dioxygenase (CDO) is a mononuclear nonheme iron­(II)-dependent enzyme critical for maintaining appropriate cysteine (Cys) and taurine levels in eukaryotic systems. Because CDO possesses both an unusual 3-His facial ligation sphere to the iron center and a rare Cys–Tyr cross-link near the active site, the mechanism by which it converts Cys and molecular oxygen to cysteine sulfinic acid is of broad interest. However, as of yet, direct experimental support for any of the proposed mechanisms is still lacking. In this study, we have used NO as a substrate analogue for O₂ to prepare a species that mimics the geometric and electronic structures of an early reaction intermediate. The resultant unusual <i>S</i> = ¹/₂ {FeNO}⁷ species was characterized by magnetic circular dichroism, electron paramagnetic resonance, and electronic absorption spectroscopies as well as computational methods including density functional theory and semiempirical calculations. The NO adducts of Cys- and selenocysteine (Sec)-bound Fe­(II)­CDO exhibit virtually identical electronic properties; yet, CDO is unable to oxidize Sec. To explore the differences in reactivity between Cys- and Sec-bound CDO, the geometries and energies of viable O₂-bound intermediates were evaluated computationally, and it was found that a low-energy quintet-spin intermediate on the Cys reaction pathway adopts a different geometry for the Sec-bound adduct. The absence of a low-energy O₂ adduct for Sec-bound CDO is consistent with our experimental data and may explain why Sec is not oxidized by CDO

Crystallographic Analysis of Active Site Contributions to Regiospecificity in the Diiron Enzyme Toluene 4-Monooxygenase

Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric μ-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors <i>p</i>-NH₂-benzoate and <i>p</i>-Br-benzoate showed a μ-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 and A107, while F196 positioned the aromatic ring against this surface via a π-stacking interaction. The proximity of G103 and F176 to the <i>para</i> substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (<1 Å) were produced in F176, F196, and other interior residues of the active site. Likewise, products bound to the diiron center in either the presence or absence of effector protein did not significantly shift the position of the interior residues, suggesting that positioning of the cognate substrates will not be strongly influenced by effector protein binding. Thus, changes in product distributions in the absence of the effector protein are proposed to arise from differences in rates of chemical steps of the reaction relative to motion of substrates within the active site channel of the uncomplexed, less efficient enzyme, while structural changes in diiron ligand geometry associated with cycling between diferrous and diferric states are discussed for their potential contribution to product release

Mutations in FLS2 Ser-938 Dissect Signaling Activation in FLS2-Mediated Arabidopsis Immunity

<div><p>FLAGELLIN-SENSING 2 (FLS2) is a leucine-rich repeat/transmembrane domain/protein kinase (LRR-RLK) that is the plant receptor for bacterial flagellin or the flagellin-derived flg22 peptide. Previous work has shown that after flg22 binding, FLS2 releases BIK1 kinase and homologs and associates with BAK1 kinase, and that FLS2 kinase activity is critical for FLS2 function. However, the detailed mechanisms for activation of FLS2 signaling remain unclear. The present study initially identified multiple FLS2 in vitro phosphorylation sites and found that Serine-938 is important for FLS2 function in vivo. FLS2-mediated immune responses are abolished in transgenic plants expressing <i>FLS2_S938A</i>, while the acidic phosphomimic mutants FLS2_S938D and FLS2_S938E conferred responses similar to wild-type FLS2. FLS2-BAK1 association and FLS2-BIK1 disassociation after flg22 exposure still occur with FLS2_S938A, demonstrating that flg22-induced BIK1 release and BAK1 binding are not sufficient for FLS2 activity, and that Ser-938 controls other aspects of FLS2 activity. Purified BIK1 still phosphorylated purified FLS2_S938A and FLS2_S938D mutant kinase domains in vitro. Phosphorylation of BIK1 and homologs after flg22 exposure was disrupted in transgenic <i>Arabidopsis thaliana</i> plants expressing <i>FLS2_S938A</i> or <i>FLS2_D997A</i> (a kinase catalytic site mutant), but was normally induced in FLS2_S938D plants. BIK1 association with FLS2 required a kinase-active FLS2, but FLS2-BAK1 association did not. Hence FLS2-BIK1 dissociation and FLS2-BAK1 association are not sufficient for FLS2-mediated defense activation, but the proposed FLS2 phosphorylation site Ser-938 and FLS2 kinase activity are needed both for overall defense activation and for appropriate flg22-stimulated phosphorylation of BIK1 and homologs.</p> </div

Induced phosphorylation of BIK1 and its homologous proteins under flg22 treatment.

<p>cMyc tagged BIK1, PBS1, PBL1, and PBL2 were transiently expressed (under control of 35S promoters) in protoplasts made from Arabidopsis <i>fls2-101</i> lines stably transgenic for full-length <i>FLS2_WT</i>, <i>FLS2_S938A</i>, <i>FLS2_S938D</i>, and <i>FLS2</i>_D997A (under control of <i>FLS2</i> promoters; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003313#ppat.1003313.s002" target="_blank">Figure S2A</a>). Protoplasts were treated with (+) or without (−) 1 µM flg22 for 15 min prior to harvest. Total protein extracts were separated by SDS-PAGE and immunoblots were probed with anti-cMyc to detect protein size shift attributable to phosphorylation. Lower panel of each pair shows Ponceau S staining of same immunoblot to assess similarity of total protein levels.</p

Interaction of BAK1 or BIK1 with variants of FLS2.

<p>HA-tagged full-length FLS2 wild type (WT), S938A, S938D or D997A (as labeled), and cMyc-tagged full length BAK1 or BIK1, were coexpressed under control of <i>35S</i> promoters in Arabidopsis <i>fls2-101</i> protoplasts. Protoplasts were harvested prior to (−) or 15 min. after (+) treatment with 1 µM flg22. Coimmunoprecipitation was carried out using anti-cMyc antibody. Input blots are from SDS-PAGE of total protein extracts; each lane was loaded with equivalent volumes of total protoplasts. WB: antibody used to probe immunoblot. <b>A.</b> FLS2_WT, FLS2_S938A and FLS2_S938D interact with BAK1 upon flg22 treatment. <b>B.</b> BIK1 dissociates from FLS2_WT, FLS2_S938A and FLS2_S938D after flg22 treatment. <b>C.</b> FLS2-FLS2 association before and after flg22 exposure is not reduced when both FLS2 partners carry the <i>FLS2_S938A</i> mutation. FLS2-BAK1 interaction from the same experiment is shown as a control (all six lanes in C from same protoplast batch, gel, blot and immunodetection). <b>D.</b> FLS2_D997A interacts with BAK_D416A upon flg22 treatment. FLS2_D997A does not interact as well as FLS2_WT with BIK1_D202A before flg22 treatment, and flg22-elicited release of BIK1 is not detected. <b>E.</b> FLS2_S938D, and separately, FLS2_D997A, form FLS2-FLS2 associations before and after flg22 treatment.</p

Identification of Ser-938 as a candidate autophosphorylation site of FLS2 <i>in vitro</i> and <i>in vivo</i>.

<p><b>A</b> and <b>B.</b> Intact (A) and antarctic phosphatase-treated (B) intracellular domains of FLS2 (aa #840-1172) were analyzed by Mass Spectrometry (MS). M: predicted molecular weight; 1P: predicted peptide with one phosphate group; 2P: predicted peptide with two phosphate groups. <b>C.</b> Peptides containing phosphorylated amino acids, identified by mass spectrometry (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003313#ppat.1003313.s001" target="_blank">Figure S1</a>). <b>D–F.</b> Functional test of three serine sites identified by MS. Reactive oxygen species were measured in leaf discs from transgenic Arabidopsis <i>fls2-101</i> plants for 30 min. after treatment with 1 µM flg22. Stable transgenic plants carried <i>FLS2</i> serine mutant alleles as specified, with expression driven by native <i>FLS2</i> promoter. Data shown are mean ± SE for four to six independent T1 plants per construct. RLU: relative luminescence units; wt: wild-type Col-0 FLS2; S938A: FLS2_S938A; other <i>FLS2</i> alleles similarly labeled.</p

Kinetic constants determined for SACTE_2347 variants.

a<p>Pure β-1,4 d-mannan.</p>b<p>Acetylated glucomannan contain mannan (60%) and glucose (40%).</p>c<p>Locust bean gum is a natural galactomannan with composition of ∼3.5 mannose per galactose.</p>d<p>IL-pine has the following composition: 34% glucose; 9% xylose; 8% mannose; 4% arabinose, and 8% galactose.</p><p>SACTE_2347 did not hydrolyze cellulose, xylan and other polysaccharides described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094166#s4" target="_blank">Materials and Methods</a>, and likewise did not react with fluorogenic small molecule analogs.</p