- Straightforward Regeneration of Reduced Flavin Adenine Dinucleotide Required for Enzymatic Tryptophan Halogenation
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Flavin-dependent halogenases are known to regioselectively introduce halide substituents into aromatic moieties, for example, the indole ring of tryptophan. The process requires halide salts and oxygen instead of molecular halogen in the chemical halogena
- Ismail, Mohamed,Schroeder, Lea,Frese, Marcel,Kottke, Tilman,Hollmann, Frank,Paul, Caroline E.,Sewald, Norbert
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p. 1389 - 1395
(2019/02/10)
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- Dynamic Kinetic Resolution for Asymmetric Synthesis of L-Noncanonical Amino Acids from D-Ser Using Tryptophan Synthase and Alanine Racemase
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L-Ser is often used to synthesize some significant l-noncanonical α-amino acids(l-ncAAs), which are the prevalent intermediates and precursors for functional synthetic compounds. In this study, threonine aldolase from Escherichia coli k-12 MG1655 has been used to synthesize l-Ser. In contrast to the maximum catalytic capacity (20 g/L) for l-threonine aldolase(LTA), d-Ser was synthesized with high yield (240 g/L) from cheap Gly and paraformaldehyde using d-threonine aldolase (DTA) from Arthrobacter sp ATCC. In order to fully utilize d-Ser and expand the resource of l-Ser, a dynamic kinetic resolution system was constructed to convert d/dl-Ser to l-Ser through combining alanine racemase (Alr) from Bacillus subtilis with l-tryptophan synthase (TrpS) from Escherichia coli k-12 MG1655, and l-ncAAs including l-Trp and l-Cys derivatives were synthesized with excellent enantioselectivity and in high yields. The results indicated l-ncAAs could be efficiently synthesized from d-Ser using this original and green dynamic kinetic resolution system, and the reliable l-Ser resource has been established from simple and achiral substrates.
- Yu, Jinhai,Li, Jing,Gao, Xia,Zeng, Shuiyun,Zhang, Hongjuan,Liu, Junzhong,Jiao, Qingcai
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p. 6618 - 6625
(2019/11/03)
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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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- Aromatic Halogenation by Using Bifunctional Flavin Reductase–Halogenase Fusion Enzymes
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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.
- Andorfer, Mary C.,Belsare, Ketaki D.,Girlich, Anna M.,Lewis, Jared C.
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p. 2099 - 2103
(2017/10/07)
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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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- Extending the biocatalytic scope of regiocomplementary flavin-dependent halogenase enzymes
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Flavin-dependent halogenases are potentially valuable biocatalysts for the regioselective halogenation of aromatic compounds. These enzymes, utilising benign inorganic halides, offer potential advantages over traditional non-enzymatic halogenation chemistry that often lacks regiocontrol and requires deleterious reagents. Here we extend the biocatalytic repertoire of the tryptophan halogenases, demonstrating how these enzymes can halogenate a range of alternative aryl substrates. Using structure guided mutagenesis we also show that it is possible to alter the regioselectivity as well as increase the activity of the halogenases with non-native substrates including anthranilic acid; an important intermediate in the synthesis and biosynthesis of pharmaceuticals and other valuable products. This journal is
- Shepherd, Sarah A.,Karthikeyan, Chinnan,Latham, Jonathan,Struck, Anna-Winona,Thompson, Mark L.,Menon, Binuraj R. K.,Styles, Matthew Q.,Levy, Colin,Leys, David,Micklefield, Jason
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p. 3454 - 3460
(2015/05/27)
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- Improving the stability and catalyst lifetime of the halogenase RebH by directed evolution
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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. Evolving halos: We have used directed evolution to engineer an RebH halogenase variant with a Topt more than 5-°C higher than that of wild-type RebH, and a second variant with a Tm 18-°C higher. These enzymes provided significantly improved conversion for halogenation of tryptophan and several non-natural substrates.
- Poor, Catherine B.,Andorfer, Mary C.,Lewis, Jared C.
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p. 1286 - 1289
(2014/06/24)
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- Regioselective enzymatic halogenation of substituted tryptophan derivatives using the FAD-dependent halogenase RebH
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Regioselective methods to establish carbon-halide bonds are still rare, although halogenation is considered as a commonly used methodology for the functionalization of organic compounds. The incorporation of halogen substituents by organic synthesis usually requires hazardous conditions, shows poor regioselectivity and results in the formation of unwanted byproducts. In addition, halogenation by electrophilic aromatic substitution (SEAr) obeys distinct rules depending on electron-withdrawing or -donating groups already present in the aromatic ring. We employed the tryptophan-7-halogenase RebH for regioselective enzymatic halogenation to overcome these limitations. In combination with a tryptophan synthase, an array of C5- and C6-substituted tryptophan derivatives was synthesized and halogenated by RebH. The halogenase is able override these directing effects and halogenates at the electronically unfavored C7-meta-position, even in presence of ortho/para-directing groups. No business as usual: The tryptophan halogenase RebH from Lechevalieria aerocolonigenes is able to halogenate at the electronically unfavored C7-meta-position of C5-substituted tryptophan derivatives, even in presence of deactivating ortho/para-directing groups.
- Frese, Marcel,Guzowska, Paulina H.,Voss, Hauke,Sewald, Norbert
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p. 1270 - 1276
(2014/05/20)
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- Regioselective arene halogenation using the FAD-dependent halogenase RebH
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Together we're strong: Co-expression of the halogenase RebH with GroEL/ES and fusion of the flavin reductase RebF to MBP enabled production of both enzymes on scales sufficient for preparative regioselective oxidative halogenation of arenes. The activity and selectivity of RebH contrasts with those reported for the structurally homologous halogenase PrnA, which only enabled halogenation of nonnatural substrates at their most electronically activated positions. Copyright
- Payne, James T.,Andorfer, Mary C.,Lewis, Jared C.
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supporting information
p. 5271 - 5274
(2013/06/26)
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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; experimental part
p. 14024 - 14025
(2009/03/11)
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- New insights into the mechanism of enzymatic chlorination of tryptophan
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(Chemical Equation Presented) It takes two: Both a lysine and a glutamate residue in the active site of tryptophan halogenase are essential for its chlorination activity. A mechanism for the regioselective enzymatic chlorination of tryptophan involving both amino acids is suggested (see scheme).
- Flecks, Silvana,Patallo, Eugenio P.,Zhu, Xiaofeng,Ernyei, Aliz J.,Seifert, Gotthard,Schneider, Alexander,Dong, Changjiang,Naismith, James H.,Van Pee, Karl-Heinz
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experimental part
p. 9533 - 9536
(2009/05/06)
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- FADH2-dependence of tryptophan 7-halogenase
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Tryptophan 7-halogenase (Trp 7-hal) catalyses the regioselective chlorination and bromination of tryptophan. For halogenating activity, Trp 7-hal requires FADH2 produced from FAD and NADH by a flavin reductase, halide ions (chloride or bromide), molecular oxygen and tryptophan as the organic substrate. Investigations of the flavin dependence showed that purified Trp 7-hal itself does not contain flavin. Keeping the Trp 7-hal separated from the flavin reductase during the reaction revealed that Trp 7-hal can use diffusible FADH2 produced by a flavin reductase showing that direct contact between the halogen ase and the flavin reductase is not absolutely necessary. Thus, the reaction also proceeds when chemically reduced flavin is used. For the catalytic regeneration of FADH2, the organometallic complex (pentamethylcyclopentadienyl)rhodium-bipyridine {[Cp*Rh(bpy) (H2O)]2+} can be employed as the redox catalyst with formate as the electron donor. With this chemoenzymatic system about 85% yields of the product formed by the two-component enzyme system consisting of Trp 7-hal and a flavin reductase were obtained.
- Unversucht, Susanne,Hollmann, Frank,Schmid, Andreas,Van Pee, Karl-Heinz
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p. 1163 - 1167
(2007/10/03)
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- Structure-activity relationship study of novel necroptosis inhibitors
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Necroptosis is a regulated caspase-independent cell death mechanism that results in morphological features resembling necrosis. It can be induced in a FADD-deficient variant of human Jurkat T cells treated with TNF-α. 5-(1H-Indol-3-ylmethyl)-2-thiohydantoins and 5-(1H-indol-3-ylmethyl)hydantoins were found to be potent necroptosis inhibitors (called necrostatins). A SAR study revealed that several positions of the indole were intolerant of substitution, while small substituents at the 7-position resulted in increased inhibitory activity. The hydantoin ring was also quite sensitive to structural modifications. A representative member of this compound class demonstrated moderate pharmacokinetic characteristics and readily entered the central nervous system upon intravenous administration.
- Teng, Xin,Degterev, Alexei,Jagtap, Prakash,Xing, Xuechao,Choi, Sungwoon,Denu, Regine,Yuan, Junying,Cuny, Gregory D.
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p. 5039 - 5044
(2007/10/03)
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- Inhibitors of cellular necrosis
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The present invention relates to compounds and pharmaceutical preparations and their use in therapy for preventing or treating trauma, ischemia, stroke and degenerative diseases associated with cell death. Methods and compositions of the invention are particularly useful for treating neurological disorders associated with cellular necrosis.
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Page/Page column 40
(2008/06/13)
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- Synthesis of 7-Chloro-L- and 7-Chloro-D-tryptophan; Biosynthesis of Pyrrolnitrin
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The article describes the synthesis of 7-chloro-DL-tryptophan (1 with R=Cl) via the intermediates 5, 6, 7 and 8.The separation of the optical antipodes was achieved by treatment of 7-chloro-N-chloroacetyl-DL-tryptophan (9) with carboxypeptidase.DL-Tryptophan (1 with R=H), 1 with R=Cl and its anomers, 1a with R=Cl and 1b with R=Cl, are substrates of L-tryptophan-2,3-dioxygenase from Pseudomonas aureofaciens.
- Pee, Karl-Heinz van,Salcher, Olga,Lingens, Franz
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p. 233 - 239
(2007/10/02)
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