- Synthesis of redox-active fluorinated 5-hydroxytryptophans as molecular reporters for biological electron transfer
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Fluorinated 5-hydroxytryptophans (Fn-5HOWs) were synthesized in gram scale quantities and incorporated into a β-hairpin peptide and the protein azurin. The redox-active Fn-5HOWs exhibit unique radical spectroscopic signatures that expand the function of 5HOW as probes for biological electron transfer.
- Ohler, Amanda,Long, Hanna,Ohgo, Kei,Tyson, Kristin,Murray, David,Davis, Amanda,Whittington, Chris,Hvastkovs, Eli G.,Duffy, Liam,Haddy, Alice,Sargent, Andrew L.,Allen, William E.,Offenbacher, Adam R.
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supporting information
p. 3107 - 3110
(2021/04/02)
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- Biocatalysts from cyanobacterial hapalindole pathway afford antivirulent isonitriles against MRSA
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Abstract: The emergence of resistance to frontline antibiotics has called for novel strategies to combat serious pathogenic infections. Methicillin-resistant Staphylococcus aureus [MRSA] is one such pathogen. As opposed to traditional antibiotics, bacteriostatic anti-virulent agents disarm MRSA, without exerting pressure, that cause resistance. Herein, we employed a thermophilic Thermotoga maritima tryptophan synthase (TmTrpB1) enzyme followed by an isonitrile synthase and Fe(II)-α-ketoglutarate-dependent oxygenase, in sequence as biocatalysts to produce antivirulent indole vinyl isonitriles. We report on conversion of simple derivatives of indoles to their C3-vinyl isonitriles, as the enzymes employed here demonstrated broader substrate tolerance. In toto, eight distinct L-Tryptophan derived α-amino acids (7) were converted to their bioactive vinyl isonitriles (3) by action of an isonitrile synthase (WelI1) and an Fe(II)-α-ketoglutarate-dependent oxygenase (WelI3) yielding structural variants possessing antivirulence against MRSA. These indole vinyl isonitriles at 10 μg/mL are effective as antivirulent compounds against MRSA, as evidenced through analysis of rabbit blood hemolysis assay. Based on a homology modelling exercise, of enzyme-substrate complexes, we deduced potential three dimensional alignments of active sites and glean mechanistic insights into the substrate tolerance of the Fe(II)-α-ketoglutarate-dependent oxygenase. Graphic abstract: [Figure not available: see fulltext.]
- Bunn, Brittney M,Xu, Mizhi,Webb, Chase M,Viswanathan, Rajesh
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- Mutagenesis of an Active-Site Loop in Tryptophan Hydroxylase Dramatically Slows the Formation of an Early Intermediate in Catalysis
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Solution studies of the aromatic amino acid hydroxylases are consistent with the FeIVO intermediate not forming until both the amino acid and tetrahydropterin substrates have bound. Structural studies have shown that the positions of active-site loops differs significantly between the free enzyme and the enzyme-amino acid-tetrahydropterin complex. In tryptophan hydroxylase (TrpH) these mobile loops contain residues 124-134 and 365-371, with a key interaction involving Ile366. The I366N mutation in TrpH results in decreases of 1-2 orders of magnitude in the kcat and kcat/Km values. Single turnover analyses establish that the limiting rate constant for turnover is product release for the wild-type enzyme but is formation of the first detectable intermediate I in catalysis in the mutant enzyme. The mutation does not alter the kinetics of NO binding to the ternary complex nor does it uncouple FeIVO formation from amino acid hydroxylation. The effects on the kcat value of wild-type TrpH of changing viscosity are consistent with rate-limiting product release. While the effect of viscosity on the kcat/KO2 value is small, consistent with reversible oxygen binding, the effects on the kcat/Km values for tryptophan and the tetrahydropterin are large, with the latter value exceeding the expected limit and varying with the identity of the viscogen. In contrast, the kinetic parameters of I366N TrpH show small changes with viscosity. The results are consistent with binding of the amino acid and pterin substrate to form the ternary complex being directly coupled to closure of loops over the active site and formation of the reactive complex. The mutation destabilizes this initial event.
- Subedi, Bishnu P.,Fitzpatrick, Paul F.
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supporting information
p. 5185 - 5192
(2018/04/23)
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- Biocatalytic Production of Psilocybin and Derivatives in Tryptophan Synthase-Enhanced Reactions
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Psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine) is the main alkaloid of the fungal genus Psilocybe, the so-called “magic mushrooms.” The pharmaceutical interest in this psychotropic natural product as a future medication to treat depression and anxiety is strongly re-emerging. Here, we present an enhanced enzymatic route of psilocybin production by adding TrpB, the tryptophan synthase of the mushroom Psilocybe cubensis, to the reaction. We capitalized on its substrate flexibility and show psilocybin formation from 4-hydroxyindole and l-serine, which are less cost-intensive substrates, compared to the previous method. Furthermore, we show enzymatic production of 7-phosphoryloxytryptamine (isonorbaeocystin), a non-natural congener of the Psilocybe alkaloid norbaeocystin (4-phosphoryloxytryptamine), and of serotonin (5-hydroxytryptamine) by means of the same in vitro approach.
- Blei, Felix,Baldeweg, Florian,Fricke, Janis,Hoffmeister, Dirk
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p. 10028 - 10031
(2018/07/29)
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- A Panel of TrpB Biocatalysts Derived from Tryptophan Synthase through the Transfer of Mutations that Mimic Allosteric Activation
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Naturally occurring enzyme homologues often display highly divergent activity with non-natural substrates. Exploiting this diversity with enzymes engineered for new or altered function, however, is laborious because the engineering must be replicated for each homologue. A small set of mutations of the tryptophan synthase β-subunit (TrpB) from Pyrococcus furiosus, which mimics the activation afforded by binding of the α-subunit, was demonstrated to have a similar activating effect in different TrpB homologues with as little as 57 % sequence identity. Kinetic and spectroscopic analyses indicate that the mutations function through the same mechanism: mimicry of α-subunit binding. From these enzymes, we identified a new TrpB catalyst that displays a remarkably broad activity profile in the synthesis of 5-substituted tryptophans. This demonstrates that allosteric activation can be recapitulated throughout a protein family to explore natural sequence diversity for desirable biocatalytic transformations.
- Murciano-Calles, Javier,Romney, David K.,Brinkmann-Chen, Sabine,Buller, Andrew R.,Arnold, Frances H.
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supporting information
p. 11577 - 11581
(2016/10/24)
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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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- Similarities and differences of serotonin and its precursors in their interactions with model membranes studied by molecular dynamics simulation
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In this work, we report a molecular dynamics (MD) simulations study of relevant biological molecules as serotonin (neutral and protonated) and its precursors, tryptophan and 5-hydroxy-tryptophan, in a fully hydrated bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-choline (POPC). The simulations were carried out at the fluid lamellar phase of POPC at constant pressure and temperature conditions. Two guest molecules of each type were initially placed at the water phase. We have analyzed, the main localization, preferential orientation and specific interactions of the guest molecules within the bilayer. During the simulation run, the four molecules were preferentially found at the water-lipid interphase. We found that the interactions that stabilized the systems are essentially hydrogen bonds, salt bridges and cation-π. None of the guest molecules have access to the hydrophobic region of the bilayer. Besides, zwitterionic molecules have access to the water phase, while protonated serotonin is anchored in the interphase. Even taking into account that these simulations were done using a model membrane, our results suggest that the studied molecules could not cross the blood brain barrier by diffusion. These results are in good agreement with works that show that serotonin and Trp do not cross the BBB by simple diffusion.
- Wood, Irene,Martini, M. Florencia,Pickholz, Mónica
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p. 124 - 130
(2013/07/27)
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- TRP/HIS EXCHANGE AND KYNURENIN INDUCED TRP TRANSPORT
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The present invention provides methods for detecting changes in tryptophan concentrations in a cell and methods for identifying agents that modulate cellular tryptophan concentrations. In particular, the present invention provides methods for detecting cellular exchange between tryptophan and kynurenine, and methods for identifying agents that modulate this exchange. The present invention also provides methods for treating a disease associated with immunosuppression in a subject in need thereof. In particular, the present invention is directed toward a method of treating a disease associated with immunosuppression comprising contacting the disease with an agent that modulates cellular Trp/kynurenine exchange. Furthermore, the present invention provides methods for identifying an agent that modulates an immunosuppression.
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- Plant phenolics affect oxidation of tryptophan
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The effect of berry phenolics such as anthocyanins, ellagitannins, and proanthocyanidins from raspberry (Rubus idaeus), black currant (Ribes nigrum), and cranberry (Vaccinium oxycoccus) and byproducts of deoiling processes rich in phenolics such as rapese
- Salminen, Hanna,Heinonen, Marina
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experimental part
p. 7472 - 7481
(2010/04/29)
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- Chemo-enzymatic synthesis and characterization of L-tryptophans selectively 13C-enriched or hydroxylated in the six-membered ring using transformed Escherichia coli cells
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L-(3a-13C)- and L-(6-13C)tryptophan have been synthesized from simple labelled compounds via a single reaction scheme based on the conversion of 1,3-cyclohexanedione into indole.The labelled indoles have been converted in one step into the corresponding L-tryptophans using transformed Escherichia coli cells with large amounts of the enzyme, tryptophan synthease.The same reaction scheme has been used for the synthesis of 4- and 7-indolol.These hydroxyindoles together with 5-indolol have been converted into 4-, 7- and 5-hydroxy-L-tryptophan, respectively, using theEscherichia coli cells.The latter compound is the immediate precursor of the neurotransmitter, serotonin.It appears that 7-indolol is the only indole derivative which is converted faster than unsubstituted indole by the enzyme, tryptophan synthease.With the preparation of L-(3a-13C)- and L-(6-13C)tryptophan, we have completed the series of indoles and L-tryptophans with a stable isotope (13C, 15N or 2H) in the aromatic ring.In this paper, we also discuss the NMR parameters of these mono-isotopically labelled systems.
- Berg, E. M. M. van den,Jansen, F. J. H. M.,Goede, A. T. J. W. de,Baldew, A. U.,Lugtenburg, J.
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p. 287 - 297
(2007/10/02)
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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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- Pharmaceutical compositions capable of increasing cerebral serotonin concentration
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A pharmaceutical composition suitable for increasing cerebral serotonin concentration, comprising a serotonin precursor selected from the group consisting of 5-hydroxytryptophan and derivatives of 5-hydroxytryptophan having the formula: STR1 wherein R represent hydrogen; a C1 -C12 alkyl group; or a C5 -C16 alicyclic, monocyclic aromatic hydrocarbyl, or polycyclic aromatic hydrocarbyl group; and a nitrogenous heterocyclic compound selected from the group consisting of inosine, theophylline, theobromine, allopurinol, pyridoxine, hypoxanthine, folic acid, adenine, nicotinamide, caffeine, and orotic acid.
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- Syntheses of Substituted L- and D-Tryptophans
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Several 5-substituted nb-methoxycarbonyl-L- and -D-tryptophan derivatives were synthesized from proline by a new route involving electrochemical oxidation, as well as by the known procedures.Removal of the Nb-methoxycarbonyl group was accomplished by treatment with Me3SiI in refluxing chloroform, then alkaline hydrolysis of the methyl esters afforded 5-substituted L- and D-tryptophans with high optical purities.Keywords - L-tryptophan 5-substituted; D-tryptophan 5-substituted; anodic oxidation; N-methoxycarbonyl group deprotection; Fischer indole synthesis; iodotrimethylsilane
- Irie, Kunihiko,Ishida, Akihiko,Nakamura, Tohru,Oh-ishi, Tokuro
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p. 2126 - 2139
(2007/10/02)
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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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