SyrB2 in syringomycin E biosynthesis is a nonheme Fe
            <sup>II</sup>
            α-ketoglutarate- and O
            <sub>2</sub>
            -dependent halogenase

Vaillancourt, Frédéric H.; Yin, Jun; Walsh, Christopher T.

doi:10.1073/pnas.0504412102

Cited by 319 publications

(376 citation statements)

References 33 publications

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“…Several other Fe II /αKG dioxygenases are purified as their apoprotein forms by inclusion of chelators (63,64). In contrast, some representatives of these enzymes have been purified anaerobically to assure that the metal remains in its reduced form (65,66).…”

Section: General Aspects Of Xana Activitymentioning

confidence: 99%

Purification and Characterization of the Fe^II- and α-Ketoglutarate-Dependent Xanthine Hydroxylase from Aspergillus nidulans

Montero-Morán

Rendón-Huerta

et al. 2007

Biochemistry

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His 6 -tagged xanthine/α-ketoglutarate (αKG) dioxygenase (XanA) of Aspergillus nidulans was purified from both the fungal mycelium and recombinant Escherichia coli cells, and the properties of the two forms of the protein were compared. Evidence was obtained for both N-and O-linked glycosylation on the fungus-derived XanA, which aggregates into an apparent dodecamer, while bacteria-derived XanA is free of glycosylation and behaves as a monomer. Immunological methods identify phosphothreonine in both forms of XanA, with phosphoserine also detected in the bacteriaderived protein. Mass spectrometric analysis confirms glycosylation and phosphorylation of the fungus-derived sample, which also undergoes extensive truncation at its amino terminus. Despite the major differences in properties of these proteins, their kinetic parameters are similar (k cat 30-70 s -1 , K m of αKG 31-50 μM, K m of xanthine ∼45 μM, and pH optima at 7.0 to 7.4). The enzyme exhibits no significant isotope effect when using 8-2 H-xanthine; however, it demonstrates a two-fold solvent deuterium isotope effect. Cu II and Zn II potently inhibit the Fe II -specific enzyme, whereas Co II , Mn II , and Ni II are weaker inhibitors. NaCl decreases the k cat and increases the K m of both αKG and xanthine. The αKG cosubstrate can be substituted by α-ketoadipate (9-fold decrease in k cat and 5-fold increase in the K m compared to the normal α-keto acid), while the αKG analogue N-oxalylglycine is a competitive inhibitor (K i 0.12 μM). No alternative purines effectively substitute for xanthine as a substrate, and only one purine analogue (6,8-dihydroxypurine) results in significant inhibition. Quenching of the endogenous fluorescence of the two enzyme forms by xanthine, αKG, and DHP † These studies were supported by the National Institutes of Health (GM063584 to R.P.H.), NSF CAREER grant 0447799 (to M.F.), Université Paris-Sud (including a Post-doctoral Fellowship to G.M.

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Section: General Aspects Of Xana Activitymentioning

confidence: 99%

Purification and Characterization of the Fe^II- and α-Ketoglutarate-Dependent Xanthine Hydroxylase from Aspergillus nidulans

Montero-Morán

Rendón-Huerta

et al. 2007

Biochemistry

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“…[44][45][46][47] Aliphatic halogenases chlorinate the terminal methyl groups of amino acids tethered via thioester linkage to the phosphopantetheine cofactor of peptidyl-carrier proteins (PCPs). They require iron, αKG, oxygen, and chloride for their activity.…”

Section: αKg-dependent Halogenasesmentioning

confidence: 99%

Non‐Heme Fe(IV)—Oxo Intermediates

et al. 2007

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High-valent non-heme iron-oxo intermediates have been proposed for decades as the key intermediates in numerous biological oxidation reactions. In the last three years, the first direct characterization of such intermediates has been provided by studies of several αKG-dependent oxygenases that catalyze either hydroxylation or halogenation of their substrates. In each case, the Fe(IV)-oxo intermediate is implicated in cleavage of the aliphatic C-H bond to initiate hydroxylation or halogenation. The observation of non-heme Fe(IV)-oxo intermediates and Fe(II)-containing product(s) complexes with almost identical spectroscopic parameters in the reactions of two distantly related αKG-dependent hydroxylases suggests that members of this sub-family follow a conserved mechanism for substrate hydroxylation. In contrast, for the αKG-dependent non-heme-iron halogenase, CytC3, two distinct Fe(IV) complexes form and decay together, suggesting that they are in rapid equilibrium. The existence of two distinct conformers of the Fe site may be the key factor accounting for the divergence of the halogenase reaction from the more usual hydroxylation pathway after C-H cleavage. Distinct transformations catalyzed by other mononuclear non-heme enzymes are likely also to involve initial C-H-cleavage by Fe(IV)-oxo complexes, followed by diverging reactivities of the resulting Fe(III)-hydroxo/substrate radical intermediates.

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“…Sequence analysis and the crystal structure of SyrB2 (14), the halogenase that supplies the 4-chloro-L-threonine fragment incorporated into syringomycin E (a phytotoxin produced by Pseudomonas syringae B301D) (12), revealed that it lacks the carboxylate ligand, having an Ala residue where the contributing Asp or Glu would normally be found (Scheme 1 A). The observation that a halide ion (X Ϫ ) coordinates to the Fe(II) cofactor at the vacated site suggested a related mechanism for the halogenase reaction involving abstraction of H • from the substrate by the oxo group of a haloferryl intermediate and rebound of the coordinated halogen radical from the resultant X-Fe(III)-OH complex to the substrate radical (green arrows).…”

mentioning

confidence: 99%

“…Before the recent discovery of the aliphatic halogenases (12), a group of ␣KG-dependent oxygenases that synthesize precursors for assembly of halogen-containing natural products by nonribosomal peptide synthetases (NRPSs), iron coordination by a conserved 2-histidine-1-carboxylate ''facial triad'' had been considered a defining feature of the family (13). Sequence analysis and the crystal structure of SyrB2 (14), the halogenase that supplies the 4-chloro-L-threonine fragment incorporated into syringomycin E (a phytotoxin produced by Pseudomonas syringae B301D) (12), revealed that it lacks the carboxylate ligand, having an Ala residue where the contributing Asp or Glu would normally be found (Scheme 1 A).…”

mentioning

confidence: 99%

Substrate positioning controls the partition between halogenation and hydroxylation in the aliphatic halogenase, SyrB2

Matthews

Neumann

Miles

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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350

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The ␣-ketoglutarate-dependent hydroxylases and halogenases employ similar reaction mechanisms involving hydrogen-abstracting Fe(IV)-oxo (ferryl) intermediates. In the halogenases, the carboxylate residue from the His 2(Asp/Glu)1''facial triad'' of iron ligands found in the hydroxylases is replaced by alanine, and a halide ion (X ؊ ) coordinates at the vacated site. Halogenation is thought to result from ''rebound'' of the halogen radical from the X-Fe ( ␣-ketoglutarate ͉ ferryl ͉ hydroxylase ͉ nonheme iron ͉ radical rebound T he ␣-ketoglutarate-dependent oxygenases activate O 2 at mononuclear Fe(II) cofactors, cleave ␣-ketoglutarate (␣KG) to CO 2 and succinate, and oxidize their substrates by two electrons (1-4). The most extensively studied members of the family are hydroxylases. A mechanism for the (pro)collagen-modifying prolyl-4-hydroxylase (P4H) originally proposed by Hanauske-Abel and Günzler (5) has accounted well for ensuing experimental data on multiple enzymes in the family. Its central tenets are the abstraction of a hydrogen atom (H • ) from the substrate by an Fe(IV)-oxo (ferryl) complex (Scheme 1A, black arrows) and the subsequent ''rebound'' of the coordinated hydroxyl radical to the substrate radical (red arrows) (6). The most compelling evidence for this mechanism was provided by the detection and spectroscopic characterization of the ferryl intermediates in taurine:␣KG dioxygenase (TauD) from Escherichia coli and a P4H from Paramecium bursaria Chlorella virus 1 (7, 8). The small Mössbauer isomer shifts (Ϸ0.3 mm/s) and S ϭ 2 electron-spin ground states of the freeze-trapped intermediates marked them as high-spin Fe(IV) complexes; the large substrate deuterium (D) kinetic isotope effects (KIEs) on their decay (k H /k D Ϸ 50) showed that they abstract hydrogen from the substrates (8, 9); and resonance Raman and X-ray absorption spectroscopic data on the TauD complex proved that it contains the ferryl unit (10, 11).Before the recent discovery of the aliphatic halogenases (12), a group of ␣KG-dependent oxygenases that synthesize precursors for assembly of halogen-containing natural products by nonribosomal peptide synthetases (NRPSs), iron coordination by a conserved 2-histidine-1-carboxylate ''facial triad'' had been considered a defining feature of the family (13). Sequence analysis and the crystal structure of SyrB2 (14), the halogenase that supplies the 4-chloro-L-threonine fragment incorporated into syringomycin E (a phytotoxin produced by Pseudomonas syringae B301D) (12), revealed that it lacks the carboxylate ligand, having an Ala residue where the contributing Asp or Glu would normally be found (Scheme 1 A). The observation that a halide ion (X Ϫ ) coordinates to the Fe(II) cofactor at the vacated site suggested a related mechanism for the halogenase reaction involving abstraction of H • from the substrate by the oxo group of a haloferryl intermediate and rebound of the coordinated halogen radical from the resultant X-Fe(III)-OH complex to the substrate radical (green arrows). Detection and ...

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SyrB2 in syringomycin E biosynthesis is a nonheme Fe ^II α-ketoglutarate- and O ₂ -dependent halogenase

Abstract: natural product biosynthesis ͉ enzymology ͉ organohalogen

Cited by 319 publications

References 33 publications

Purification and Characterization of the Fe^II- and α-Ketoglutarate-Dependent Xanthine Hydroxylase from Aspergillus nidulans

Purification and Characterization of the Fe^II- and α-Ketoglutarate-Dependent Xanthine Hydroxylase from Aspergillus nidulans

Non‐Heme Fe(IV)—Oxo Intermediates

Substrate positioning controls the partition between halogenation and hydroxylation in the aliphatic halogenase, SyrB2

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SyrB2 in syringomycin E biosynthesis is a nonheme Fe II α-ketoglutarate- and O 2 -dependent halogenase

Abstract: natural product biosynthesis ͉ enzymology ͉ organohalogen

Cited by 319 publications

References 33 publications

Purification and Characterization of the FeII- and α-Ketoglutarate-Dependent Xanthine Hydroxylase from Aspergillus nidulans

Purification and Characterization of the FeII- and α-Ketoglutarate-Dependent Xanthine Hydroxylase from Aspergillus nidulans

Non‐Heme Fe(IV)—Oxo Intermediates

Substrate positioning controls the partition between halogenation and hydroxylation in the aliphatic halogenase, SyrB2

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Resources

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SyrB2 in syringomycin E biosynthesis is a nonheme Fe ^II α-ketoglutarate- and O ₂ -dependent halogenase

Purification and Characterization of the Fe^II- and α-Ketoglutarate-Dependent Xanthine Hydroxylase from Aspergillus nidulans

Purification and Characterization of the Fe^II- and α-Ketoglutarate-Dependent Xanthine Hydroxylase from Aspergillus nidulans