A flavin-dependent halogenase from metagenomic analysis prefers bromination over chlorination

Neubauer, Pia R.; Widmann, Christiane; Wibberg, Daniel; Schröder, Lea; Frese, Marcel; Kottke, Tilman; Kalinowski, Jörn; Niemann, Hartmut H.; Sewald, Norbert

doi:10.1371/journal.pone.0196797

Cited by 69 publications

(117 citation statements)

References 58 publications

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“…BAL3, exhibits a high preference of indole bromination over chlorination. Conversely, Trp halogenases are reported to favor incorporation of less bulky chloride . Hence, we examined whether Thal mutagenesis also influences halide selectivity.…”

Section: Resultsmentioning

confidence: 99%

“…Conversely, Trp halogenases are reported to favor incorporation of less bulky chloride. [15,55] Hence, we examined whether Thal mutagenesis also influences halide selectivity. LC-MS analysis confirmed Thal-GWV's ability to incorporate either chloride or bromide into l-Trp with high efficiency.…”

Section: Further Characterization Of Evolved Variant Confirmed Maintamentioning

confidence: 99%

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Targeted Enzyme Engineering Unveiled Unexpected Patterns of Halogenase Stabilization

et al. 2019

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Halogenases are valuable biocatalysts for selective CÀ H activation, but despite recent efforts to broaden their application scope by means of protein engineering, improvement of thermostability and catalytic efficiency is still desired. A directed evolution campaign aimed at generating a thermostable flavindependent tryptophan 6-halogenase with reasonable activity suitable for chemoenzymatic purposes. These characteristics were tackled by combining successive rounds of epPCR along with semi-rational mutagenesis leading to a triple mutant (Thal-GLV) with substantially increased thermostability (~T M = 23.5 K) and higher activity at 25°C than the wild type enzyme. Moreover, an active-site mutation has a striking impact on thermostability but also on enantioselectivity. Our data contribute to a detailed understanding of biohalogenation and provide a profound basis for future engineering strategies to facilitate chemoenzymatic application of these attractive biocatalysts.

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Section: Resultsmentioning

confidence: 99%

Section: Further Characterization Of Evolved Variant Confirmed Maintamentioning

confidence: 99%

Targeted Enzyme Engineering Unveiled Unexpected Patterns of Halogenase Stabilization

et al. 2019

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“…Investigations on novel halogenases or engineering the existing ones, for broader substrate scope, enhanced activity, and stability have been conducted. [18,[37][38][39][40][41] Xanthomonas comprises a group of Gram negative Proteobacteria infecting over 350 monocotyledonous and dicotyledonous plant species. [42] The plant pathogen Xanthomonas campestris (Xcc) is a member of this group infecting crucifers like cabbage and cauliflower.…”

Section: Introductionmentioning

confidence: 99%

Flavin‐Dependent Halogenases from Xanthomonas campestris pv. campestris B100 Prefer Bromination over Chlorination

et al. 2019

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Flavin-dependent halogenases selectively introduce halogen substituents into (hetero-)aromatic substrates and require only molecular oxygen and halide salts for this regioselective oxidative CHfunctionalization. Genomic analysis of Xanthomonas campestris pv. campestris B100 identified three novel putative members of this enzyme class. They were shown to introduce halogen substituents into, e. g., substituted indoles, while preferring bromide over chloride.

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“…In addition, FDHs were identified that halogenate substituted tryptophan (12,13) or chlorinate tryptophan only when it is part of a substrate oligopeptide (14). FDHs acting on free substrates other than tryptophan were also described (15)(16)(17)(18), including fungal enzymes that accept large substrates (19 -21) or have rather broad substrate specificity (22,23), raising the hope that diverse compounds can be enzymatically halogenated in vitro. Moreover, FDHs have been integrated into engineered biosynthetic pathways in vivo (24,25).…”

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Structure-based switch of regioselectivity in the flavin-dependent tryptophan 6-halogenase Thal

Moritzer

Minges

Prior

et al. 2019

Journal of Biological Chemistry

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Edited by Ruma BanerjeeFlavin-dependent halogenases increasingly attract attention as biocatalysts in organic synthesis, facilitating environmentally friendly halogenation strategies that require only FADH 2 , oxygen, and halide salts. Different flavin-dependent tryptophan halogenases regioselectively chlorinate or brominate tryptophan's indole moiety at C5, C6, or C7. Here, we present the first substrate-bound structure of a tryptophan 6-halogenase, namely Thal, also known as ThdH, from the bacterium Streptomyces albogriseolus at 2.55 Å resolution. The structure revealed that the C6 of tryptophan is positioned next to the ⑀-amino group of a conserved lysine, confirming the hypothesis that proximity to the catalytic residue determines the site of electrophilic aromatic substitution. Although Thal is more similar in sequence and structure to the tryptophan 7-halogenase RebH than to the tryptophan 5-halogenase PyrH, the indole binding pose in the Thal active site more closely resembled that of PyrH than that of RebH. The difference in indole orientation between Thal and RebH appeared to be largely governed by residues positioning the Trp backbone atoms. The sequences of Thal and RebH lining the substrate binding site differ in only few residues. Therefore, we exchanged five amino acids in the Thal active site with the corresponding counterparts in RebH, generating the quintuple variant Thal-RebH5. Overall conversion of L-Trp by the Thal-RebH5 variant resembled that of WT Thal, but its regioselectivity of chlorination and bromination was almost completely switched from C6 to C7 as in RebH. We conclude that structure-based protein engineering with targeted substitution of a few residues is an efficient approach to tailoring flavin-dependent halogenases.The authors declare that they have no conflicts of interest with the contents of this article. This article contains Tables S1 and S2 and Figs. S1-S17. The atomic coordinates and structure factors (codes 6H43, 6H44, and 6IB5) have been deposited in the Protein Data Bank (http://wwpdb.org/). . 3 The abbreviations used are: FDH, flavin-dependent halogenase; aa, amino acid(s); ESI, electrospray ionization; HSQC, heteronuclear single-quantum coherence; r.m.s.d., root mean square deviation; ROESY, rotating frame nuclear Overhauser effect spectroscopy; Bicine, N,N-bis(2-hydroxyethyl)-glycine; H-bond, hydrogen bond; Ni-NTA, nickel-nitrilotriacetic acid; RR-ADH, Rhodococcus ruber alcohol dehydrogenase; PDB, Protein Data Bank.Figure 8. Steric conflicts upon placing L-Trp from Trp-RebH in Trp-Thal and vice versa. Minor clashes are indicated as small green hexagons, and more severe clashes are shown as larger red hexagons. a, L-Trp from Trp-RebH (PDB code 2E4G) was placed in Trp-Thal. Clashes mainly occur between the L-Trp indole and the side chains of conserved residues Phe-112 and Trp-466. b, L-Trp from Trp-Thal was placed in Trp-RebH (PDB code 2E4G). Clashes mainly occur between the L-Trp indole and the side chain of Asn-470 and between the L-Trp carboxylate and the side chain...

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A flavin-dependent halogenase from metagenomic analysis prefers bromination over chlorination

Cited by 69 publications

References 58 publications

Targeted Enzyme Engineering Unveiled Unexpected Patterns of Halogenase Stabilization

Targeted Enzyme Engineering Unveiled Unexpected Patterns of Halogenase Stabilization

Flavin‐Dependent Halogenases from Xanthomonas campestris pv. campestris B100 Prefer Bromination over Chlorination

Structure-based switch of regioselectivity in the flavin-dependent tryptophan 6-halogenase Thal

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