- Targeted Enzyme Engineering Unveiled Unexpected Patterns of Halogenase Stabilization
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Halogenases are valuable biocatalysts for selective C?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 evoluti
- Minges, Hannah,Schnepel, Christian,B?ttcher, Dominique,Wei?, Martin S.,Spro?, Jens,Bornscheuer, Uwe T.,Sewald, Norbert
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p. 818 - 831
(2019/12/24)
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- Structure-based switch of regioselectivity in the flavin-dependent tryptophan 6-halogenase Thal
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Flavin-dependent halogenases increasingly attract attention as biocatalysts in organic synthesis, facilitating environmentally friendly halogenation strategies that require only FADH2, 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 ? 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 electro-philic 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.
- Moritzer, Ann-Christin,Minges, Hannah,Prior, Tina,Frese, Marcel,Sewald, Norbert,Niemann, Hartmut H.
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p. 2529 - 2542
(2019/03/12)
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- Biosynthesis of l-4-Chlorokynurenine, an Antidepressant Prodrug and a Non-Proteinogenic Amino Acid Found in Lipopeptide Antibiotics
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l-4-Chlorokynurenine (l-4-Cl-Kyn) is a neuropharmaceutical drug candidate that is in development for the treatment of major depressive disorder. Recently, this amino acid was naturally found as a residue in the lipopeptide antibiotic taromycin. Herein, we report the unprecedented conversion of l-tryptophan into l-4-Cl-Kyn catalyzed by four 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, which is initiated by the flavin-dependent tryptophan chlorinase Tar14 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, thereby opening doors for the targeted engineering of these enzymes as useful biocatalysts.
- Luhavaya, Hanna,Sigrist, Renata,Chekan, Jonathan R.,McKinnie, Shaun M. K.,Moore, Bradley S.
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supporting information
p. 8394 - 8399
(2019/05/21)
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- Unlocking Reactivity of TrpB: A General Biocatalytic Platform for Synthesis of Tryptophan Analogues
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Derivatives of the amino acid tryptophan (Trp) serve as precursors for the chemical and biological synthesis of complex molecules with a wide range of biological properties. Trp analogues are also valuable as building blocks for medicinal chemistry and as tools for chemical biology. While the enantioselective synthesis of Trp analogues is often lengthy and requires the use of protecting groups, enzymes have the potential to synthesize such products in fewer steps and with the pristine chemo- and stereoselectivity that is a hallmark of biocatalysis. The enzyme TrpB is especially attractive because it can form Trp analogues directly from serine (Ser) and the corresponding indole analogue. However, many potentially useful substrates, including bulky or electron-deficient indoles, are poorly accepted. We have applied directed evolution to TrpB from Pyrococcus furiosus and Thermotoga maritima to generate a suite of catalysts for the synthesis of previously intractable Trp analogues. For the most challenging substrates, such as nitroindoles, the key to improving activity lay in the mutation of a universally conserved and mechanistically important residue, E104. The new catalysts express at high levels (>200 mg/L of Escherichia coli culture) and can be purified by heat treatment; they can operate up to 75 °C (where solubility is enhanced) and can synthesize enantiopure Trp analogues substituted at the 4-, 5-, 6-, and 7-positions, using Ser and readily available indole analogues as starting materials. Spectroscopic analysis shows that many of the activating mutations suppress the decomposition of the active electrophilic intermediate, an amino-acrylate, which AIDS in unlocking the synthetic potential of TrpB.
- Romney, David K.,Murciano-Calles, Javier,Wehrmüller, J?ri E.,Arnold, Frances H.
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supporting information
p. 10769 - 10776
(2017/08/15)
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- An Unusual Flavin-Dependent Halogenase from the Metagenome of the Marine Sponge Theonella swinhoei WA
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Uncultured bacteria from sponges have been demonstrated to be responsible for the generation of many potent, bioactive natural products including halogenated metabolites.1 The identification of gene clusters from the metagenomes of such bacterial communities enables the discovery of enzymes that mediate new and useful chemistries and allows insight to be gained into the biogenesis of potentially pharmacologically important natural products. Here we report a new pathway to the keramamides (krm); the first functional evidence for the existence of a distinct producer in the Theonella swinhoei WA chemotype is revealed, and a key enzyme on the pathway, a unique flavin-dependent halogenase with a broad substrate specificity, with potential as a useful new biocatalytic tool, is described.
- Smith, Duncan R. M.,Uria, Agustinus R.,Helfrich, Eric J. N.,Milbredt, Daniela,Van Pée, Karl-Heinz,Piel, J?rn,Goss, Rebecca J. M.
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p. 1281 - 1287
(2017/05/29)
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- A Structure-Guided Switch in the Regioselectivity of a Tryptophan Halogenase
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Flavin-dependent halogenases are potentially useful biocatalysts for the regioselective halogenation of aromatic compounds. Haloaromatic compounds can be utilised in the synthesis and biosynthesis of pharmaceuticals and other valuable products. Here we report the first X-ray crystal structure of a tryptophan 6-halogenase (SttH), which enabled key residues that contribute to the regioselectivity in tryptophan halogenases to be identified. Structure-guided mutagenesis resulted in a triple mutant (L460F/P461E/P462T) that exhibited a complete switch in regioselectivity; with the substrate 3-indolepropionate 75 % 5-chlorination was observed with the mutant in comparison to 90 % 6-chlorination for the wild-type SttH. This is the first clear example of how regiocomplementary halogenases can be created from a single parent enzyme. The biocatalytic repertoire of SttH was also expanded to include a range of indolic and non-indolic substrates.
- Shepherd, Sarah A.,Menon, Binuraj R. K.,Fisk, Heidi,Struck, Anna-Winona,Levy, Colin,Leys, David,Micklefield, Jason
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p. 821 - 824
(2016/05/19)
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- Structure and biocatalytic scope of thermophilic flavin-dependent halogenase and flavin reductase enzymes
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Flavin-dependent halogenase (Fl-Hal) enzymes have been shown to halogenate a range of synthetic as well as natural aromatic compounds. The exquisite regioselectively of Fl-Hal enzymes can provide halogenated building blocks which are inaccessible using standard halogenation chemistries. Consequently, Fl-Hal are potentially useful biocatalysts for the chemoenzymatic synthesis of pharmaceuticals and other valuable products, which are derived from haloaromatic precursors. However, the application of Fl-Hal enzymes, in vitro, has been hampered by their poor catalytic activity and lack of stability. To overcome these issues, we identified a thermophilic tryptophan halogenase (Th-Hal), which has significantly improved catalytic activity and stability, compared with other Fl-Hal characterised to date. When used in combination with a thermostable flavin reductase, Th-Hal can efficiently halogenate a number of aromatic substrates. X-ray crystal structures of Th-Hal, and the reductase partner (Th-Fre), provide insights into the factors that contribute to enzyme stability, which could guide the discovery and engineering of more robust and productive halogenase biocatalysts.
- Menon, Binuraj R. K.,Latham, Jonathan,Dunstan, Mark S.,Brandenburger, Eileen,Klemstein, Ulrike,Leys, David,Karthikeyan, Chinnan,Greaney, Michael F.,Shepherd, Sarah A.,Micklefield, Jason
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supporting information
p. 9354 - 9361
(2016/10/13)
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- MACROCYCLIC PEPTIDOMIMETICS FOR ALPHA-HELIX MIMICRY
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Methods and compositions are provided for generating macrocyclic peptides constrained by side-chain-to-C-terminus non-peptidic tethers for use as functional and structural mimics of α-helical motifs, including in therapeutic applications. These methods can be used to produce libraries of conformationally constrained peptidomimetics to identify compounds with desired activity properties.
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Paragraph 00256
(2015/11/02)
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- Synthesis of tripeptides containing d-Trp substituted at the indole ring, assessment of opioid receptor binding and in vivo central antinociception
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The noncationizable tripeptide Ac-d-Trp-Phe-GlyNH2 was recently proposed as a novel minimal recognition motif for μ-opioid receptor. The introduction of different substituents (methyl, halogens, nitro, etc.) at the indole of d-Trp significantly influenced receptor affinities and resulted in serum stability and in a measurable effect on central antinociception in mice after ip administration.
- De Marco, Rossella,Bedini, Andrea,Spampinato, Santi,Gentilucci, Luca
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supporting information
p. 6861 - 6866
(2014/10/15)
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- An expression system for the efficient incorporation of an expanded set of tryptophan analogues
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Biosynthetic incorporation of tryptophan (Trp) analogues in recombinant proteins using an E. coli Trp auxotroph expression host is limited to analogues modified with a small substituent like a fluoro atom or a hydroxyl or amine group. We report here the e
- Petrovic, Dejan M.,Leenhouts, Kees,Van Roosmalen, Maarten L.,Broos, Jaap
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p. 1329 - 1336
(2013/06/27)
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- Total synthesis of chloptosin: A dimeric cyclohexapeptide
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Here we describe in full our investigations into the synthesis of the dimeric cyclohexapeptide chloptosin in 17 linear steps. Particularly, this work features an organocatalytic tandem process for the synthesis of the embedded piperazic acids, in which a differentially protected azodicarboxylate is used together with pyrrolidinyl tetrazole as the catalyst. The central biaryl bond is being formed by Stille coupling of two sterically demanding ortho-chloropyrroloindole fragments. The inherent flexibility of the synthetic strategy proved beneficial as the route could be adjusted smoothly during the progression of the synthesis programme. Copyright
- Oelke, Alexander J.,Antonietti, Francesca,Bertone, Leonardo,Cranwell, Philippa B.,France, David J.,Goss, Rebecca J. M.,Hofmann, Tatjana,Knauer, Stephan,Moss, Steven J.,Skelton, Paul C.,Turner, Richard M.,Wuitschik, Georg,Ley, Steven V.
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supporting information; experimental part
p. 4183 - 4194
(2011/06/17)
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- β-Peptides with improved affinity for hDM2 and hDMX
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We previously described a series of 314-helical β-peptides that bind the hDM2 protein and inhibit its interaction with a p53-derived peptide in vitro. Here we present a detailed characterization of the interaction of these peptides with hDM2 an
- Harker, Elizabeth A.,Daniels, Douglas S.,Guarracino, Danielle A.,Schepartz, Alanna
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experimental part
p. 2038 - 2046
(2009/06/06)
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- Tandem action of the O2- and FADH2-dependent halogenases KtzQ and KtzR produce 6,7-dichlorotryptophan for kutzneride assembly
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Kutznerides are actinomycete-derived antifungal nonribosomal hexadepsipeptides which are assembled from five unsual nonproteinogenic amino acids and one hydroxy acid. Conserved in all structurally characterized kutznerides is a dichlorinated tricyclic hexahydropyrroloindole postulated to be derived from 6,7-dichlorotryptophan. In this Communication, we identify KtzQ and KtzR as tandem acting FADH2-dependent halogenases that work sequentially on free l-tryptophan to generate 6,7-dichloro-l-tryptophan. Kinetic characterization of these two enzymes has shown that KtzQ (along with the flavinreductase KtzS) acts first to chlorinate at the 7-position of l-tryptophan. KtzR, with a ～120 fold preference for 7-chloro-l-tryptophan over l-tryptophan, then installs the second chlorine at the 6-position of 7-chloro-l-tryptophan to generate 6,7-dichloro-l-tryptophan. These findings provide further insights into the enzymatic logic of carbon-chloride bond formation during the biosynthesis of halogenated secondary metabolites. Copyright
- Heemstra Jr., John R.,Walsh, Christopher T.
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supporting information; body text
p. 14024 - 14025
(2009/03/11)
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- The facile synthesis of a series of tryptophan derivatives
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This study reports a facile method for the synthesis of a variety of 5- and 6-substituted tryptophan derivatives that are difficult to prepare using alternative enzymatic approaches. Acylation of an activated amino acid, derived from serine in situ, is coupled with an enzymatic resolution step to furnish enantiopure analogues bearing a range of electron withdrawing and releasing substituents. Isolation of a dehydroalanine derivative as a by-product from some reactions provides some insights into the likely mechanism of the reaction.
- Blaser, Georg,Sanderson, John M.,Batsanov, Andrei S.,Howard, Judith A.K.
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p. 2795 - 2798
(2008/09/19)
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- Synthesis of optically active ring-A substituted tryptophans as IDO inhibitors
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The first synthesis of optically active 7-methoxy-d-tryptophan as well as other ring-A substituted tryptophans is described.
- Li, Xiaoyan,Yin, Wenyuan,Sarma, P.V.V. Srirama,Zhou, Hao,Jun Ma,Cook, James M.
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p. 8569 - 8573
(2007/10/03)
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- Synthesis of L-6-chloropyrroloindoline of chloptosin cyclohexapeptide
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Chloptosin is an apoptosis-inducing dimeric cyclohexapeptide. Enantio-selective synthesis of L-6-chloropyrroloindoline, as the key chiral synthon of the cyclohexapeptide of chloptosin, was successfully achieved starting from 3-chloroaniline by utilizing F
- Kim, Young-Ah,Han, So-Yeop
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p. 2931 - 2943
(2007/10/03)
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- Tryptophane and 3-indolepyruvic acid, methods of production therefor
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3-indolepyruvic acid derivatives substituted on the benzene moiety show good antagonizing activity on excitatory aminoacids and on free radicals, for which reason they are therapeutic agents in cerebral and peripheral degenerative pathologies. Their metho
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- 3-indolepyruvic acid derivatives their method of production and therapeutic use
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3-indolepyruvic acid derivatives substituted on the benzene moiety show good antagonizing activity on excitatory aminoacids and on free radicals, for which reason they are therapeutic agents in cerebral and peripheral degenerative pathologies. Their metho
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- Process for preparing optically active tryptophans
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Biochemical optical resolution of DL-tryptophans in which DL-tryptophan amides are interacted with the culture products, or their treated products, of a microorganism capable of producing amidase is described. L-Tryptophan amides in racemic DL-tryptophan amides are asymmetrically hydrolyzed to form optically active L-tryptophans at a high yield and almost all D-tryptophan amides remain without being subjected to hydrolysis. The resultant D-tryptophan amides are readily hydrolyzed, after separating L-tryptophans, to form optically active D-tryptophans at a high yield.
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- Process for preparing optically active tryptophanes
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Biochemical optical resolution of DL-tryptophanes in which DL-tryptophane amides are interacted with the culture products, or their treated products, of a microorganism capable of producing amidase is described. L-tryptophane amides in racemic DL-tryptophane amides are asymmetrically hydrolyzed to form optically active L-tryptophanes at a high yield and almost all D-tryptophane amides remain without being subjected to hydrolysis. The resultant D-tryptophane amides are readily hydrolyzed, after separating L-tryptophanes, to form optically active D-tryptophanes at a high yield.
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- Process for the resolution of DL-6-chlorotryptophan
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Seed crystals of one optically active enantiomer of 6-chlorotryptophan methanesulfonate or 6-chlorotryptophan benzenesulfonate are added to a supersaturated solution of DL-6-chlorotryptophan methanesulfonate or DL-6-chlorotryptophan benzenesulfonate. Crys
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- Salts of the γ-lactone of (-)-threo-hydroxycitric acid with R(+)-α-methyl-p-nitrobenzylamine
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Racemic organic carboxylic acids are efficiently resolved into their enantiomers with antipodes of α-methyl-p-nitrobenzylamine.
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