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Direct nitration and azidation of aliphatic carbons by an iron-dependent halogenase.

ABSTRACT: Iron-dependent halogenases employ cis-halo-Fe(IV)-oxo (haloferryl) complexes to functionalize unactivated aliphatic carbon centers, a capability elusive to synthetic chemists. Halogenation requires (i) coordination of a halide anion (Cl(-) or Br(-)) to the enzyme's Fe(II) cofactor, (ii) coupled activation of O2 and decarboxylation of ?-ketoglutarate to generate the haloferryl intermediate, (iii) abstraction of hydrogen (H•) from the substrate by the ferryl and (iv) transfer of the cis halogen as Cl• or Br• to the substrate radical. This enzymatic solution to an unsolved chemical challenge is potentially generalizable to installation of other functional groups, provided that the corresponding anions can support the four requisite steps. We show here that the wild-type halogenase SyrB2 can indeed direct aliphatic nitration and azidation reactions by the same chemical logic. The discovery and enhancement by mutagenesis of these previously unknown reaction types suggest unrecognized or untapped versatility in ferryl-mediated enzymatic C-H bond activation.

SUBMITTER: Matthews ML

PROVIDER: S-EPMC4076429 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Direct nitration and azidation of aliphatic carbons by an iron-dependent halogenase.

Matthews Megan L ML Chang Wei-chen WC Layne Andrew P AP Miles Linde A LA Krebs Carsten C Bollinger J Martin JM

Nature chemical biology 20140126 3

Iron-dependent halogenases employ cis-halo-Fe(IV)-oxo (haloferryl) complexes to functionalize unactivated aliphatic carbon centers, a capability elusive to synthetic chemists. Halogenation requires (i) coordination of a halide anion (Cl(-) or Br(-)) to the enzyme's Fe(II) cofactor, (ii) coupled activation of O2 and decarboxylation of α-ketoglutarate to generate the haloferryl intermediate, (iii) abstraction of hydrogen (H•) from the substrate by the ferryl and (iv) transfer of the cis halogen as ...[more]

PMID: 24463698

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Project description:Nonheme iron- and α-ketoglutarate (αKG)-dependent halogenases (NHFeHals), which catalyze the regio- and stereoselective halogenation of the unactivated C(sp3)-H bonds, exhibit tremendous potential in the challenging asymmetric halogenation. AdeV from Actinomadura sp. ATCC 39365 is the first identified carrier protein-free NHFeHal that catalyzes the chlorination of nucleotide 2'-deoxyadenosine-5'-monophosphate (2'-dAMP) to afford 2'-chloro-2'-deoxyadenosine-5'-monophosphate. Here, we determined the complex crystal structures of AdeV/FeII/Cl and AdeV/FeII/Cl/αKG at resolutions of 1.76 and 1.74 Å, respectively. AdeV possesses a typical β-sandwich topology with H194, H252, αKG, chloride, and one water molecule coordinating FeII in the active site. Molecular docking, mutagenesis, and biochemical analyses reveal that the hydrophobic interactions and hydrogen bond network between the substrate-binding pocket and the adenine, deoxyribose, and phosphate moieties of 2'-dAMP are essential for substrate recognition. Residues H111, R177, and H192 might play important roles in the second-sphere interactions that control reaction partitioning. This study provides valuable insights into the catalytic selectivity of AdeV and will facilitate the rational engineering of AdeV and other NHFeHals for synthesis of halogenated nucleotides. IMPORTANCE Halogenated nucleotides are a group of important antibiotics and are clinically used as antiviral and anticancer drugs. AdeV is the first carrier protein-independent nonheme iron- and α-ketoglutarate (αKG)-dependent halogenase (NHFeHal) that can selectively halogenate nucleotides and exhibits restricted substrate specificity toward several 2'-dAMP analogues. Here, we determined the complex crystal structures of AdeV/FeII/Cl and AdeV/FeII/Cl/αKG. Molecular docking, mutagenesis, and biochemical analyses provide important insights into the catalytic selectivity of AdeV. This study will facilitate the rational engineering of AdeV and other carrier protein-independent NHFeHals for synthesis of halogenated nucleotides.

Project description:Aliphatic halogenases activate O(2), cleave alpha-ketoglutarate (alphaKG) to CO(2) and succinate, and form haloferryl [X-Fe(IV)O; X = Cl or Br] complexes that cleave aliphatic C-H bonds to install halogens during the biosynthesis of natural products by non-ribosomal peptide synthetases (NRPSs). For the related alphaKG-dependent dioxygenases, it has been shown that reaction of the Fe(II) cofactor with O(2) to form the C-H bond-cleaving ferryl complex is "triggered" by binding of the target substrate. In this study, we have tested for and defined structural determinants of substrate triggering (ST) in the halogenase, SyrB2, from the syringomycin E biosynthetic NRPS of Pseudomonas syringae B301D. As for other halogenases, the substrate of SyrB2 is complex, consisting of l-Thr tethered via a thioester linkage to a covalently bound phosphopantetheine (PPant) cofactor of a carrier protein, SyrB1. Without an appended amino acid, SyrB1 does not trigger formation of the chloroferryl intermediate state in SyrB2, even in the presence of free l-Thr or its analogues, but SyrB1 charged either by l-Thr (l-Thr-S-SyrB1) or by any of several non-native amino acids does trigger the reaction by as much as 8000-fold (for the native substrate). Triggering efficacy is sensitive to the structures of both the amino acid and the carrier protein, being diminished by 5-24-fold when the native l-Thr is replaced with another amino acid and by approximately 40-fold when SyrB1 is replaced with the heterologous carrier protein, CytC2. The directing effect of the carrier protein and consequent tolerance for profound modifications to the target amino acid allow the chloroferryl state to be formed in the presence of substrates that perturb the ratio of its two putative coordination isomers, lack the target C-H bond (l-Ala-S-SyrB1), or contain a C-H bond of enhanced strength (l-cyclopropylglycyl-S-SyrB1). For the latter two cases, the SyrB2 chloroferryl state so formed exhibits unprecedented stability (t(1/2) = 30-110 min at 0 degree C), can be trapped at high concentration and purity by manual freezing without a cryosolvent, and represents an ideal target for structural characterization. As initial steps toward this goal, extended X-ray absorption fine structure (EXAFS) spectroscopy has been used to determine the Fe-O and Fe-Cl distances and density functional theory (DFT) calculations have been used to confirm that the measured distances are consistent with the anticipated structure of the intermediate.

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Direct nitration and azidation of aliphatic carbons by an iron-dependent halogenase.

Publications

Direct nitration and azidation of aliphatic carbons by an iron-dependent halogenase.

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