Mechanistic Studies on Tryptophan Lyase (NosL): Identification of Cyanide as a Reaction Product.
Ontology highlight
ABSTRACT:
SUBMITTER: Bhandari DM
PROVIDER: S-EPMC6078386 | biostudies-literature | 2018 Jan
REPOSITORIES: biostudies-literature
Publications
Mechanistic Studies on Tryptophan Lyase (NosL): Identification of Cyanide as a Reaction Product.
Bhandari Dhananjay M DM Fedoseyenko Dmytro D Begley Tadhg P TP
Journal of the American Chemical Society 20180102 2
Tryptophan lyase (NosL) catalyzes the formation of 3-methylindole-2-carboxylic acid and 3-methylindole from l-tryptophan. In this paper, we provide evidence supporting a formate radical intermediate and demonstrate that cyanide is a byproduct of the NosL-catalyzed reaction with l-tryptophan. These experiments require a major revision of the NosL mechanism and uncover an unanticipated connection between NosL and HydG, the radical SAM enzyme that forms cyanide and carbon monoxide from tyrosine dur ...[more]
Similar Datasets
Project description:Tryptophan 7-halogenase catalyzes chlorination of free tryptophan to 7-chlorotryptophan, which is the first step in the antibiotic pyrrolnitrin biosynthesis. Many biologically and pharmaceutically active natural products contain chlorine and thus, an understanding of the mechanism of its introduction into organic molecules is important. Whilst enzyme-catalyzed chlorination is accomplished with ease, it remains a difficult task for the chemists. Therefore, utilizing enzymes in the synthesis of chlorinated organic compounds is important, and providing atomistic mechanistic insights about the reaction mechanism of tryptophan 7-halogenase is vital and timely. In this work, we examined a mechanism for the reaction of tryptophan chlorination, performed by tryptophan 7-halogenase, by calculating potential energy and free energy surfaces using two different Combined Quantum Mechanical/Molecular Mechanical (QM/MM) methods both employing Density Functional Theory (DFT) for the QM region. Both computational strategies agree on the nature of the rate-limiting step and provided close results for the reaction barriers of the two reaction steps. The calculations for both the potential energy and the free energy profiles showed very similar geometric features and hydrogen bonding interactions for the characterized stationary points.
| S-EPMC5727139 | biostudies-literature
Project description:The prenyltransferase CymD catalyzes the reverse N-prenylation of tryptophan using dimethylallyl diphosphate (DMAPP) in the biosynthesis of the cyclic peptides cyclomarin and cyclomarazine. The mechanism is of interest because a non-nucleophilic indole nitrogen must be alkylated in this process. Three mechanisms were initially considered, including (A) a direct addition of a carbocation to the nitrogen, (B) an addition of a carbocation to C-3 followed by an aza-Cope rearrangement, and (C) deprotonation of the indole followed by an S(N)2' addition to DMAPP. The use of 4-fluorotryptophan and 6-fluorotryptophan revealed that the reaction kinetics are only modestly affected by these substitutions, consistent with the notion that positive charge does not accumulate on the indole ring during catalysis. When (E)-3-(fluoromethyl)-2-buten-1-yl diphosphate was used in place of DMAPP, the maximal rate was reduced by a factor of 100, consistent with the development of positive charge on the dimethylallyl moiety. Positional isotope exchange (PIX) experiments show that the reaction with Trp proceeds without isotopic scrambling of the label in the starting material [1-(18)O]DMAPP. However, in the case of 4-fluorotryptophan, significant isotopic scrambling is observed (v(PIX)/v(rxn) = 1.1). This is consistent with a mechanism involving a discrete carbocation intermediate. Finally, a significant solvent kinetic isotope effect of 2.3 was observed in D(2)O, indicating that a proton transfer step is rate-limiting. Taken together, these observations support a mechanism that is a hybrid of mechanisms A and C. Ionization of DMAPP generates a dimethylallyl carbocation, and deprotonation of the indole nitrogen accompanies or precedes the nucleophilic attack.
| S-EPMC3475536 | biostudies-literature
Project description:A kinetic analysis of a "declick" reaction is described. Compound 1, previously reported to couple an amine and a thiol (i.e. "click") under mild aqueous conditions to create 2, undergoes release of the unaltered coupling partners upon triggering with dithiothreitol (DTT). In the study reported herein various aniline derivatives possessing para-electron donating and withdrawing groups were used as the amines. UV/vis spectroscopy of the declick reaction shows time-dependent spectra lacking isosbestic points, implying a multi-step mechanism. Global data fitting using numerical integration of rate equations and singular value decomposition afforded the spectra and time-dependence of each species, as well as rate constants for each step. The kinetic analysis reveals a multi-step process with an intermediate where both thiols of DTT have added prior to expulsion of the aniline leaving group, followed by rearrangement to the final product. Hammett plots show a negative rho value on two of the steps, indicating positive charge building (i.e. reduction of a negative charge) in the step leading to the intermediate and its rate-determining breakdown. Overall, the kinetic study reported herein gives a complete mechanistic picture of the declick reaction.
| S-EPMC6849640 | biostudies-literature
Project description:Actinobacterial 2-hydroxyacyl-CoA lyase reversibly catalyzes the thiamine diphosphate-dependent cleavage of 2-hydroxyisobutyryl-CoA to formyl-CoA and acetone. This enzyme has great potential for use in synthetic one-carbon assimilation pathways for sustainable production of chemicals, but lacks details of substrate binding and reaction mechanism for biochemical reengineering. We determined crystal structures of the tetrameric enzyme in the closed conformation with bound substrate, covalent postcleavage intermediate, and products, shedding light on active site architecture and substrate interactions. Together with molecular dynamics simulations of the covalent precleavage complex, the complete catalytic cycle is structurally portrayed, revealing a proton transfer from the substrate acyl Cβ hydroxyl to residue E493 that returns it subsequently to the postcleavage Cα-carbanion intermediate. Kinetic parameters obtained for mutants E493A, E493Q, and E493K confirm the catalytic role of E493 in the WT enzyme. However, the 10- and 50-fold reduction in lyase activity in the E493A and E493Q mutants, respectively, compared with WT suggests that water molecules may contribute to proton transfer. The putative catalytic glutamate is located on a short α-helix close to the active site. This structural feature appears to be conserved in related lyases, such as human 2-hydroxyacyl-CoA lyase 2. Interestingly, a unique feature of the actinobacterial 2-hydroxyacyl-CoA lyase is a large C-terminal lid domain that, together with active site residues L127 and I492, restricts substrate size to ≤C5 2-hydroxyacyl residues. These details about the catalytic mechanism and determinants of substrate specificity pave the ground for designing tailored catalysts for acyloin condensations for one-carbon and short-chain substrates in biotechnological applications.
| S-EPMC8760513 | biostudies-literature
Project description:5-Thyminyl-5,6-dihydrothymine (also called spore photoproduct or SP) is the exclusive DNA photodamage product in bacterial endospores. It is repaired by a radical SAM (S-adenosylmethionine) enzyme, the spore photoproduct lyase (SPL), at the bacterial early germination phase. Our previous studies proved that SPL utilizes the 5'-dA• generated by the SAM cleavage reaction to abstract the H(6proR) atom to initiate the SP repair process. The resulting thymine allylic radical was suggested to take an H atom from an unknown protein source, most likely cysteine 141. Here we show that C141 can be readily alkylated in the native SPL by an iodoacetamide treatment, suggesting that it is accessible to the TpT radical. SP repair by the SPL C141A mutant yields TpTSO(2)(-) and TpT simultaneously from the very beginning of the reaction; no lag phase is observed for TpTSO(2)(-) formation. Should any other protein residue serve as the H donor, its presence would result in TpT being the major product at least for the first enzyme turnover. These observations provide strong evidence to support C141 as the direct H atom donor. Moreover, because of the lack of this intrinsic H donor, the C141A mutant produces TpT via an unprecedented thymine cation radical reduction (proton-coupled electron transfer) process, contrasting to the H atom transfer mechanism in the wild-type (WT) SPL reaction. The C141A mutant repairs SP at a rate that is ~3-fold slower than that of the WT enzyme. Formation of TpTSO(2)(-) and TpT exhibits a V(max) deuterium kinetic isotope effect (KIE) of 1.7 ± 0.2, which is smaller than the (D)V(max) KIE of 2.8 ± 0.3 determined for the WT SPL reaction. These findings suggest that removing the intrinsic H atom donor disturbs the rate-limiting process during enzyme catalysis. As expected, the prereduced C141A mutant supports only ~0.4 turnover, which is in sharp contrast to the >5 turnovers exhibited by the WT SPL reaction, suggesting that the enzyme catalytic cycle (SAM regeneration) is disrupted by this single mutation.
| S-EPMC3448869 | biostudies-literature
Project description:Enantiomerically enriched, deuterated branched carbonates (Z)-(S)-PhCH(OCO(2)Me)-CH = CHD (1-D), (Z)-(R)-PhCH(OCO(2)Me)CH = CHD (2-D), and linear carbonate (E)-(S)-PhCH = CHCHD(OCO(2)Me) (3-D) were used as probes in the Mo-catalyzed asymmetric allylic alkylation with sodium dimethyl malonate, catalyzed by ligand-complex 11 derived from the mixed benzamide/picolinamide of (S,S)-transdiaminocyclohexane and (norbornadiene)Mo(CO)(4). The results of these studies, along with x-ray crystallography and solution NMR structural analysis of the pi-allyl intermediate, conclusively established the reaction proceeded by a retention-retention pathway. This mechanism contrasts with that defined for Pd-catalyzed allylic alkylations, which proceed by an inversion-inversion pathway. A proposed rationale for the retention pathway for nucleophilic substitution involves CO-coordination to form a tri-CO intermediate, followed by complexation with the anion of dimethyl malonate to produce a seven-coordinate intermediate, which reductively eliminates to afford product with retention of configuration.
| S-EPMC397389 | biostudies-literature
Project description:Toxoflavin (an azapteridine) is degraded to a single product by toxoflavin lyase (TflA) in a reaction dependent on reductant, Mn(II), and oxygen. The isolated product was fully characterized by NMR and MS and was identified as a triazine in which the pyrimidine ring was oxidatively degraded. A mechanism for toxoflavin degradation based on the identification of the enzymatic product and the recently determined crystal structure of toxoflavin lyase is proposed.
| S-EPMC3332044 | biostudies-literature
Project description:(S)-2-hydroxypropylphosphonate ((S)-2-HPP) epoxidase (HppE) is a mononuclear non-haem-iron-dependent enzyme responsible for the final step in the biosynthesis of the clinically useful antibiotic fosfomycin. Enzymes of this class typically catalyse oxygenation reactions that proceed via the formation of substrate radical intermediates. By contrast, HppE catalyses an unusual dehydrogenation reaction while converting the secondary alcohol of (S)-2-HPP to the epoxide ring of fosfomycin. Here we show that HppE also catalyses a biologically unprecedented 1,2-phosphono migration with the alternative substrate (R)-1-HPP. This transformation probably involves an intermediary carbocation, based on observations with additional substrate analogues, such as (1R)-1-hydroxyl-2-aminopropylphosphonate, and model reactions for both radical- and carbocation-mediated migration. The ability of HppE to catalyse distinct reactions depending on the regio- and stereochemical properties of the substrate is given a structural basis using X-ray crystallography. These results provide compelling evidence for the formation of a substrate-derived cation intermediate in the catalytic cycle of a mononuclear non-haem-iron-dependent enzyme. The underlying chemistry of this unusual phosphono migration may represent a new paradigm for the in vivo construction of phosphonate-containing natural products that can be exploited for the preparation of new phosphonate derivatives.
| S-EPMC3725809 | biostudies-literature
Project description:Free radical chain oxidation of highly oxidizable 7-dehydrocholesterol (7-DHC), initiated by 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile), was carried out at 37 degrees C in benzene for 24 h. Fifteen oxysterols derived from 7-DHC were isolated and characterized with 1D and 2D NMR spectroscopy and mass spectrometry. A mechanism that involves abstraction of hydrogen atoms at C-9 and/or C-14 is proposed to account for the formation of all of the oxysterols and the reaction progress profile. In either the H-9 or H-14 mechanism, a pentadienyl radical intermediate is formed after abstraction of H-9 or H-14 by a peroxyl radical. This step is followed by the well-precedented transformations observed in peroxidation reactions of polyunsaturated fatty acids such as oxygen addition, peroxyl radical 5-exo cyclization, and S(H)i carbon radical attack on the peroxide bond. The mechanism for peroxidation of 7-DHC also accounts for the formation of numerous oxysterol natural products isolated from fungal species, marine sponges, and cactaceous species. In a cell viability test, the oxysterol mixture from 7-DHC peroxidation was found to be cytotoxic to Neuro2a neuroblastoma cells in the micromolar concentration range. We propose that the high reactivity of 7-DHC and the oxysterols generated from its peroxidation may play important roles in the pathogenesis of Smith-Lemli-Opitz syndrome, X-linked dominant chondrodysplasia punctata, and cerebrotendinous xanthomatosis, all of these being metabolic disorders characterized by an elevated level of 7-DHC.
| S-EPMC2839323 | biostudies-literature
Project description:The homogeneous cycloaddition reaction of CO2 and epichlorohydrin catalyzed by amine-functionalized ionic liquid (AFIL) to yield cyclic carbonate is reported in this study. The AFIL has the dual function of ionic liquid and organic base. The experimental study indicates that AFIL can efficiently catalyze the conversion of CO2 and epichlorohydrin to the product 3-chloro-1,2-propylene. The mechanistic study based on DFT calculations reveals that the imidazolium ring in AFIL primarily catalyzes the ring-opening reaction of epichlorohydrin, while the protonated amine group is responsible for stabilizing the Br- anion in the nucleophilic attack. This study provides a deep insight into the catalytic roles of AFIL and also inspires us to design efficient dual function catalysts for CO2 utilization.
| S-EPMC6747045 | biostudies-literature