Regioselective arene halogenation using the FAD-dependent halogenase RebH.
Ontology highlight
ABSTRACT:
SUBMITTER: Payne JT
PROVIDER: S-EPMC3824154 | biostudies-literature | 2013 May
REPOSITORIES: biostudies-literature
Publications
Regioselective arene halogenation using the FAD-dependent halogenase RebH.
Payne James T JT Andorfer Mary C MC Lewis Jared C JC
Angewandte Chemie (International ed. in English) 20130416 20
Similar Datasets
Project description:Halogenation of bioactive peptides via incorporation of non-natural amino acid derivatives during chemical synthesis is a common strategy to enhance functionality. Bacterial tyrptophan halogenases efficiently catalyze regiospecific halogenation of the free amino acid tryptophan, both in vitro and in vivo. Expansion of their substrate scope to peptides and proteins would facilitate highly-regulated post-synthesis/expression halogenation. Here, we demonstrate novel in vitro halogenation (chlorination and bromination) of peptides by select halogenase enzymes and identify the C-terminal (G/S)GW motif as a preferred substrate. In a first proof-of-principle experiment, we also demonstrate chemo-catalyzed derivatization of an enzymatically chlorinated peptide, albeit with low efficiency. We further rationally derive PyrH halogenase mutants showing improved halogenation of the (G/S)GW motif, both as a free peptide and when genetically fused to model proteins with efficiencies up to 90%.
| S-EPMC9775415 | biostudies-literature
Project description:The remarkable site selectivity and broad substrate scope of flavin-dependent halogenases (FDHs) has led to much interest in their potential as biocatalysts. Multiple engineering efforts have demonstrated that FDHs can be tuned for non-native substrate scope and site selectivity. FDHs have also proven useful as in vivo biocatalysts and have been successfully incorporated into biosynthetic pathways to build new chlorinated aromatic compounds in several heterologous organisms. In both cases, reduced flavin cofactor, usually supplied by a separate flavin reductase (FR), is required. Herein, we report functional synthetic, fused FDH-FR proteins containing various FDHs and FRs joined by different linkers. We show that FDH-FR fusion proteins can increase product titers compared to the individual components for in vivo biocatalysis in Escherichia coli.
| S-EPMC5898195 | biostudies-literature
Project description:A new FAD (flavin adenine dinucleotide)-dependent halogenase HalY from Streptomyces sp. JCM9888 was reported to be involved in the regioselective halogenation of adenine. HalY is a variant B FAD-dependent halogenase that is most similar to the halogenase PltA involved in pyoluteorin biosynthesis. This study reports the overexpression and purification of HalY with an N-terminal hexahistidine tag, followed by crystallization experiments and X-ray crystallographic analysis. HalY was purified as a monomer in solution and crystallized to give X-ray diffraction to a resolution of 1.7 Å. The crystal belonged to the monoclinic space group P21, with unit-cell parameters a = 41.4, b = 113.4, c = 47.6 Å, α = γ = 90, β = 107.4°, and contained one monomer of HalY in the asymmetric unit, with a calculated Matthews coefficient of 2.3 Å(3) Da(-1) and a solvent content of 46%. The structure of the halogenase CndH was used as a search model in molecular replacement to obtain the initial model of HalY. Manual model building and structure refinement of HalY are in progress.
| S-EPMC4528926 | biostudies-literature
Project description:Halogenated phenazine meroterpenoids are a structurally unusual family of marine actinobacterial natural products that exhibit antibiotic, antibiofilm, and cytotoxic bioactivities. Despite a lack of established phenazine halogenation biochemistry, genomic analysis of Streptomyces sp. CNZ-289, a prolific lavanducyanin and C2-halogenated derivative producer, suggested the involvement of vanadium-dependent haloperoxidases. We subsequently discovered lavanducyanin halogenase (LvcH), characterized it in vitro as a regioselective vanadium-dependent chloroperoxidase, and applied it in late-stage chemoenzymatic synthesis.
| S-EPMC11250029 | biostudies-literature
Project description:We previously reported that the halogenase RebH catalyzes selective halogenation of several heterocycles and carbocycles, but product yields were limited by enzyme instability. Here, we use directed evolution to engineer an RebH variant, 3-LR, with a Topt over 5 °C higher than that of wild-type, and 3-LSR, with a Tm 18 °C higher than that of wild-type. These enzymes provided significantly improved conversion (up to fourfold) for halogenation of tryptophan and several non-natural substrates. This initial evolution of RebH not only provides improved enzymes for immediate synthetic applications, but also establishes a robust protocol for further halogenase evolution.
| S-EPMC4124618 | biostudies-literature
Project description:Activating industrially important aromatic hydrocarbons by installing halogen atoms is extremely important in organic synthesis and often improves the pharmacological properties of drug molecules. To this end, tryptophan halogenase enzymes are potentially valuable tools for regioselective halogenation of arenes, including various industrially important indole derivatives and similar scaffolds. Although endogenous enzymes show reasonable substrate scope towards indole compounds, their efficacy can often be improved by engineering. Using a structure-guided semi-rational mutagenesis approach, we have developed two RebH variants with expanded biocatalytic repertoires that can efficiently halogenate several novel indole substrates and produce important pharmaceutical intermediates. Interestingly, the engineered enzymes are completely inactive towards their natural substrate tryptophan in spite of their high tolerance to various functional groups in the indole ring. Computational modelling and molecular dynamics simulations provide mechanistic insights into the role of gatekeeper residues in the substrate binding site and the dramatic switch in substrate specificity when these are mutated.
| S-EPMC8518859 | biostudies-literature
Project description:We recently characterized the substrate scope of wild-type RebH and proceeded to evolve variants of this enzyme with improved stability for biocatalysis. The substrate scopes of both RebH and the stabilized variants, however, are limited primarily to compounds similar in size to tryptophan. A substrate walking approach was used to further evolve RebH variants with expanded substrate scope. Two particularly notable variants were identified: 3-SS, which provides high conversion of tricyclic tryptoline derivatives; and 4-V, which accepts a broad range of large indoles and carbazoles. This constitutes the first reported use of directed evolution to enable the functionalization of substrates not accepted by wild-type RebH and demonstrates the utility of RebH variants for the site-selective halogenation of biologically active compounds.
| S-EPMC4506780 | biostudies-literature
Project description:RebH variants capable of chlorinating substituted indoles ortho-, meta-, and para- to the indole nitrogen were evolved by directly screening for altered selectivity on deuterium-substituted probe substrates using mass spectrometry. This systematic approach allowed for rapid accumulation of beneficial mutations using simple adaptive walks and should prove generally useful for altering and optimizing the selectivity of C-H functionalization catalysts. Analysis of the beneficial mutations showed that structure-guided selection of active site residues for targeted mutagenesis can be complicated either by activity/selectivity tradeoffs that reduce the possibility of detecting such mutations or by epistatic effects that actually eliminate the benefits of a mutation in certain contexts. As a corollary to this finding, the precise manner in which the beneficial mutations identified led to the observed changes in RebH selectivity is not clear. Docking simulations suggest that tryptamine binds to these variants as tryptophan does to native halogenases, but structural studies will be required to confirm these models and shed light on how particular mutations impact tryptamine binding. Similar directed evolution efforts on other enzymes or artificial metalloenzymes could enable a wide range of C-H functionalization reactions.
| S-EPMC4917012 | biostudies-literature
Project description:The flavin-dependent halogenase (FDH) AetF successively brominates tryptophan at C5 and C7 to generate 5,7-dibromotryptophan. In contrast to the well studied two-component tryptophan halogenases, AetF is a single-component flavoprotein monooxygenase. Here, crystal structures of AetF alone and in complex with various substrates are presented, representing the first experimental structures of a single-component FDH. Rotational pseudosymmetry and pseudomerohedral twinning complicated the phasing of one structure. AetF is structurally related to flavin-dependent monooxygenases. It contains two dinucleotide-binding domains for binding the ADP moiety with unusual sequences that deviate from the consensus sequences GXGXXG and GXGXXA. A large domain tightly binds the cofactor flavin adenine dinucleotide (FAD), while the small domain responsible for binding the nicotinamide adenine dinucleotide (NADP) is unoccupied. About half of the protein forms additional structural elements containing the tryptophan binding site. FAD and tryptophan are about 16 Å apart. A tunnel between them presumably allows diffusion of the active halogenating agent hypohalous acid from FAD to the substrate. Tryptophan and 5-bromotryptophan bind to the same site but with a different binding pose. A flip of the indole moiety identically positions C5 of tryptophan and C7 of 5-bromotryptophan next to the tunnel and to catalytic residues, providing a simple explanation for the regioselectivity of the two successive halogenations. AetF can also bind 7-bromotryptophan in the same orientation as tryptophan. This opens the way for the biocatalytic production of differentially dihalogenated tryptophan derivatives. The structural conservation of a catalytic lysine suggests a way to identify novel single-component FDHs.
| S-EPMC10306068 | biostudies-literature
Project description:This article reports an efficient CH activation process for regioselective halogenation of 1,4-benzodiazepinones. Direct halogenation with NXS (X = Br, I) affords halogenated benzodiazepinones on the central aromatic ring whereas catalyst (Pd(OAc)2) controlled CH activation furnishes regioselectively ortho halogenated benzodiazepinones on the phenyl side chain.
| S-EPMC4503988 | biostudies-literature