An Unusual Flavin-Dependent Halogenase from the Metagenome of the Marine Sponge <i>Theonella swinhoei</i> WA

Smith, Duncan R. M.; Uria, Agustinus Robert; Helfrich, Eric J. N.; Milbredt, Daniela; Pée, Karl‐Heinz van; Piel, Jörn; Goss, Rebecca J. M.

doi:10.1021/acschembio.6b01115

Cited by 53 publications

(70 citation statements)

References 38 publications

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“…Like RebH (33), several other FDHs, including the Trp-FDHs SttH (56), Thal (33), PrnA (82), PyrH (82), and KrmI (83) as well as the phenol FDHs Rdc2 and GsfI (33, 59, 84), have demonstrated broad substrate scope and interesting site-selectivities (Fig. 4B, 14–20 ).…”

Section: Flavin-dependent Halogenases (Fdhs)mentioning

confidence: 99%

Understanding and Improving the Activity of Flavin-Dependent Halogenases via Random and Targeted Mutagenesis

Andorfer

Lewis

2018

Annu. Rev. Biochem.

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Flavin dependent halogenases (FDHs) catalyze the halogenation of organic substrates by coordinating reactions of reduced flavin, molecular oxygen, and chloride. Targeted and random mutagenesis of these enzymes has been used to both understand and alter their reactivity. These studies have led to insights into residues essential for catalysis and FDH variants with improved stability, expanded substrate scope, and altered site selectivity. Mutations throughout FDH structures have contributed to all of these advances. More recent studies have sought to rationalize the impact of these mutations on FDH function and to identify new FDHs to deepen our understanding of this enzyme class and to expand their utility for biocatalytic applications.

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Section: Flavin-dependent Halogenases (Fdhs)mentioning

confidence: 99%

Understanding and Improving the Activity of Flavin-Dependent Halogenases via Random and Targeted Mutagenesis

Andorfer

Lewis

2018

Annu. Rev. Biochem.

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“…Bacterial tryptophan halogenases were the first FDHs to be described (9 -11). 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.…”

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confidence: 99%

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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“…No iodinated tryptophan products were detected following incubation with iodide.T of urther investigate its biocatalytic halogenation potential, Tar14 was interrogated with al ibrary of Tr pd erivatives ( Table 1, Figures S16-S21, Table S7). Tar14 was capable of generating aw ide variety of dihalogenated Tr ps pecies by accepting monohalogenated substrates.P reviously,t he kutzneride FDH KtzR was shown to have l-7-Cl-Trp as its preferred substrate,y ielding l-6,7-diCl-Trp, [24] while FDH KrmI from asponge metagenome was able to chlorinate l-7-F-Trp, [25] albeit at trace level.…”

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confidence: 99%

Biosynthesis of l‐4‐Chlorokynurenine, an Antidepressant Prodrug and a Non‐Proteinogenic Amino Acid Found in Lipopeptide Antibiotics

et al. 2019

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l-4-Chlorokynurenine (l-4-Cl-Kyn) is aneuropharmaceutical drug candidate that is in development for the treatment of major depressive disorder.R ecently,t his amino acid was naturally found as ar esidue in the lipopeptide antibiotic taromycin. Herein, we report the unprecedented conversion of l-tryptophan into l-4-Cl-Kyn catalyzedbyfour enzymes in the taromycin biosynthetic pathway from the marine bacterium Saccharomonospora sp.CNQ-490. We used genetic, biochemical, structural, and analytical techniques to establish l-4-Cl-Kyn biosynthesis,w hichi si nitiated by the flavin-dependent tryptophan chlorinaseT ar14 and its flavin reductase partner Tar15. This work revealed the first tryptophan 2,3-dioxygenase (Tar13) and kynurenine formamidase (Tar16) enzymes that are selective for chlorinated substrates. The substrate scope of Tar13, Tar14, and Tar16 was examined and revealed intriguing promiscuity,therebyopening doors for the targeted engineering of these enzymes as useful biocatalysts.Supportinginformation (including experimental information) and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

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An Unusual Flavin-Dependent Halogenase from the Metagenome of the Marine Sponge Theonella swinhoei WA

Abstract: Uncultured bacteria from sponges have been demonstrated to be responsible for the generation of many potent, bioactive natural products including halogenated metabolites.

Cited by 53 publications

References 38 publications

Understanding and Improving the Activity of Flavin-Dependent Halogenases via Random and Targeted Mutagenesis

Understanding and Improving the Activity of Flavin-Dependent Halogenases via Random and Targeted Mutagenesis

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

Biosynthesis of l‐4‐Chlorokynurenine, an Antidepressant Prodrug and a Non‐Proteinogenic Amino Acid Found in Lipopeptide Antibiotics

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