RESUMO
We characterise a reversible bacterial zinc-containing benzyl alcohol dehydrogenase (BaDH) accepting either NAD+ or NADP+ as a redox cofactor. Remarkably, its redox cofactor specificity is pH-dependent with the phosphorylated cofactors favored at lower and the dephospho-forms at higher pH. BaDH also shows different steady-state kinetic behavior with the two cofactor forms. From a structural model, the pH-dependent shift may affect the charge of a histidine in the 2'-phosphate-binding pocket of the redox cofactor binding site. The enzyme is phylogenetically affiliated to a new subbranch of the Zn-containing alcohol dehydrogenases, which share this conserved residue. BaDH appears to have some specificity for its substrate, but also turns over many substituted benzyl alcohol and benzaldehyde variants, as well as compounds containing a conjugated C=C double bond with the aldehyde carbonyl group. However, compounds with an sp3-hybridised C next to the alcohol/aldehyde group are not or only weakly turned over. The enzyme appears to contain a Zn in its catalytic site and a mixture of Zn and Fe in its structural metal-binding site. Moreover, we demonstrate the use of BaDH in an enzyme cascade reaction with an acid-reducing tungsten enzyme to reduce benzoate to benzyl alcohol. KEY POINTS: â¢Zn-containing BaDH has activity with either NAD + or NADP+ at different pH optima. â¢BaDH converts a broad range of substrates. â¢BaDH is used in a cascade reaction for the reduction of benzoate to benzyl alcohol.
Assuntos
Oxirredutases do Álcool , Álcool Benzílico , Coenzimas , NADP , Oxirredução , Zinco , Concentração de Íons de Hidrogênio , NADP/metabolismo , Especificidade por Substrato , Álcool Benzílico/metabolismo , Álcool Benzílico/química , Cinética , Zinco/metabolismo , Coenzimas/metabolismo , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , NAD/metabolismo , Benzaldeídos/metabolismo , Benzaldeídos/química , Domínio Catalítico , Sítios de Ligação , Filogenia , Modelos MolecularesRESUMO
3-Ketosteroid Δ1-dehydrogenases (KstD) are important microbial flavin enzymes that initiate the metabolism of steroid ring A and find application in the synthesis of steroid drugs. We present a structure of the KstD from Sterolibacterium denitrificans (AcmB), which contains a previously uncharacterized putative membrane-associated domain and extended proton-relay system. The experimental and theoretical studies show that the steroid Δ1-dehydrogenation proceeds according to the Ping-Pong bi-bi kinetics and a two-step base-assisted elimination (E2cB) mechanism. The mechanism is validated by evaluating the experimental and theoretical kinetic isotope effect for deuterium-substituted substrates. The role of the active-site residues is quantitatively assessed by point mutations, experimental activity assays, and QM/MM MD modeling of the reductive half-reaction (RHR). The pre-steady-state kinetics also reveals that the low pH (6.5) optimum of AcmB is dictated by the oxidative half-reaction (OHR), while the RHR exhibits a slight optimum at the pH usual for the KstD family of 8.5. The modeling confirms the origin of the enantioselectivity of C2-H activation and substrate specificity for Δ4-3-ketosteroids. Finally, the cholest-4-en-3-one turns out to be the best substrate of AcmB in terms of ΔG of binding and predicted rate of dehydrogenation.
Assuntos
Oxirredutases , Prótons , Oxirredutases/metabolismo , Catálise , Esteroides/metabolismo , Mutagênese , Cetosteroides , Cinética , Especificidade por SubstratoRESUMO
In contrast to their molybdenum dependent relatives, tungsten enzymes operate at significantly lower redox potentials, and in some cases they can carry out reversible redox transformations of their substrates and products. Still, the electrochemical properties of W enzymes have received much less attention than their Mo relatives. Herein we analyse the tungsten enzyme aldehyde oxidoreductase (AOR) from the mesophilic bacterium Aromatoleum aromaticum which has been immobilised on a glassy carbon working electrode. This generates a functional system that electrochemically oxidises a wide variety of aromatic and aliphatic aldehydes in the presence of the electron transfer mediators benzyl viologen, methylene blue or dichlorophenol indophenol. Simulation of the cyclic voltammetry has enabled a thorough kinetic analysis of the system, which reveals that methylene blue acts as a two-electron acceptor. In contrast, the other two mediators act as single electron oxidants. The different electrochemical driving forces imparted by these mediators also lead to significantly different outer sphere electron transfer rates with AOR. This work shows that electrocatalytic aldehyde oxidation can be achieved at a low applied electrochemical potential leading to an extremely energy efficient catalytic process.
Assuntos
Aldeído Oxirredutases , Aldeídos , Aldeído Oxirredutases/química , Aldeído Oxirredutases/metabolismo , Tungstênio , Azul de Metileno , Cinética , Oxirredução , Aldeído DesidrogenaseRESUMO
This research aimed at obtaining new derivatives of pregn-1,4-diene-3,20-dione (Δ1-progesterone) (2) through microbiological transformation. For the role of catalysts, we used six strains of entomopathogenic filamentous fungi (Beauveria bassiana KCh J1.5, Beauveria caledonica KCh J3.3, Isaria fumosorosea KCh J2, Isaria farinosa KCh KW1.1, Isaria tenuipes MU35, and Metarhizium robertsii MU4). The substrate (2) was obtained by carrying out an enzymatic 1,2-dehydrogenation on an increased scale (3.5 g/L) using a recombinant cholest-4-en-3-one Δ1-dehydrogenase (AcmB) from Sterolibacterium denitrificans. All selected strains were characterized by the high biotransformation capacity for the used substrate. As a result of the biotransformation, six steroid derivatives were obtained: 11α-hydroxypregn-1,4-diene-3,20-dione (3), 6ß,11α-dihydroxypregn-1,4-diene-3,20-dione (4), 6ß-hydroxypregn-1,4-diene-3,11,20-trione (5), 6ß,17α-dihydroxypregn-1,4-diene-3,20-dione (6), 6ß,17ß-dihydroxyandrost-1,4-diene-3-one (7), and 12ß,17α-dihydroxypregn-1,4-diene-3,20-dione (8). The results show evident variability of the biotransformation process between strains of the tested biocatalysts from different species described as entomopathogenic filamentous fungi. The obtained products were tested in silico using cheminformatics tools for their pharmacokinetic and pharmacodynamic properties, proving their potentially high biological activities. This study showed that the obtained compounds may have applications as effective inhibitors of testosterone 17ß-dehydrogenase. Most of the obtained products should, also with a high probability, find potential uses as androgen antagonists, a prostate as well as menopausal disorders treatment. They should also demonstrate immunosuppressive, erythropoiesis-stimulating, and anti-inflammatory properties.
Assuntos
Antagonistas de Androgênios , Progesterona , Masculino , Humanos , Biotransformação , Imunossupressores , QuimioinformáticaRESUMO
Bacteria and fungi that are able to metabolize steroids express 3-ketosteroid-Δ1-dehydrogenases (KstDs). KstDs such as AcmB form Sterolibacterium denitrificans Chol-1 catalyze the enantioselective 1α,2ß-dehydrogenation of steroids to their desaturated analogues, e.g., the formation of 1,4-androstadiene-3,17-dione (ADD) from 4-androsten-3,17-dione (AD). The reaction catalyzed by KstD can be reversed if the appropriate electron donor, such as benzyl viologen radical cation, is present. Furthermore, KstDs can also catalyze transhydrogenation, which is the transfer of H atoms between 3-ketosteroids and 1-dehydrosteroids. In this paper, we showed that AcmB exhibits lower pH optima for hydrogenation and dehydrogenation by 3.5-4 pH units than those observed for KstD from Nocardia corallina. We confirmed the enantiospecificity of 1α,2ß-hydrogenation and 1α,2ß-transhydrogenation catalyzed by AcmB and showed that, under acidic pH conditions, deuterons are introduced not only at 2ß but also at the 1α position. We observed a higher degree of H/D exchange at Y363, which activates the C2-H bond, compared to that at FAD, which is responsible for redox at the C1 position. Furthermore, for the first time, we observed the introduction of the third deuteron into the steroid core. This effect was explained through a combination of LC-MS experiments and QM:MM modelling, and we attribute it to a decrease in the enantioselectivity of C2-H activation upon the deuteration of the 2ß position. The increase in the activation barrier resulting from isotopic substitution increases the chance of the formation of d3-substituted 3-ketosteroids. Finally, we demonstrate a method for the synthesis of 3-ketosteroids chirally deuterated at 1α,2ß positions, obtaining 1α,2ß-d2-4-androsten-3,17-dione with a 51% yield (8.61 mg).
Assuntos
Betaproteobacteria , Oxirredutases , Marcação por Isótopo , Oxirredutases/metabolismo , Cinética , Betaproteobacteria/metabolismo , Esteroides/metabolismoRESUMO
The microbiome's significance in chronic rhinosinusitis (CRS) is unclear. Antimicrobials are recommended in acute exacerbations of the disease (AECRS). Increasing rates of antibiotic resistance have stimulated research on alternative therapeutic options, including silver nanoparticles (AgNPs). However, there are concerns regarding the safety of silver administration. The aim of this study was to assess the biological activity of tannic acid-prepared AgNPs (TA-AgNPs) towards sinonasal pathogens and nasal epithelial cells (HNEpC). The minimal inhibitory concentration (MIC) for pathogens isolated from patients with AECRS was approximated using the well diffusion method. The cytotoxicity of TA-AgNPswas evaluated using an MTT assay and trypan blue exclusion. A total of 48 clinical isolates and 4 reference strains were included in the study (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Klebsiellaoxytoca, Acinetobacter baumannii, Serratia marcescens, Enterobacter cloacae). The results of the studies revealed that the MIC values differed between isolates, even within the same species. All the isolates were sensitive to TA-AgNPs in concentrations non-toxic to human cells during 24 h exposition. However, 48 h exposure to TA-AgNPs increased toxicity to HNEpC, narrowing their therapeutic window and enabling 19% of pathogens to resist the TA-AgNPs' biocidal action. It was concluded that TA-AgNPs are non-toxic for the investigated eukaryotic cells after short-term exposure and effective against most pathogens isolated from patients with AECRS, but sensitivity testing may be necessary before application.
Assuntos
Nanopartículas Metálicas , Prata , Humanos , Prata/farmacologia , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Taninos/farmacologia , Escherichia coliRESUMO
BACKGROUND: 3-Ketosteroid Δ1-dehydrogenases (KSTDs) are the enzymes involved in microbial cholesterol degradation and modification of steroids. They catalyze dehydrogenation between C1 and C2 atoms in ring A of the polycyclic structure of 3-ketosteroids. KSTDs substrate spectrum is broad, even though most of them prefer steroids with small substituents at the C17 atom. The investigation of the KSTD's substrate specificity is hindered by the poor solubility of the hydrophobic steroids in aqueous solutions. In this paper, we used 2-hydroxpropyl-ß-cyclodextrin (HBC) as a solubilizing agent in a study of the KSTDs steady-state kinetics and demonstrated that substrate bioavailability has a pivotal impact on enzyme specificity. RESULTS: Molecular dynamics simulations on KSTD1 from Rhodococcus erythropolis indicated no difference in ΔGbind between the native substrate, androst-4-en-3,17-dione (AD; - 8.02 kcal/mol), and more complex steroids such as cholest-4-en-3-one (- 8.40 kcal/mol) or diosgenone (- 6.17 kcal/mol). No structural obstacle for binding of the extended substrates was also observed. Following this observation, our kinetic studies conducted in the presence of HBC confirmed KSTD1 activity towards both types of steroids. We have compared the substrate specificity of KSTD1 to the other enzyme known for its activity with cholest-4-en-3-one, KSTD from Sterolibacterium denitrificans (AcmB). The addition of solubilizing agent caused AcmB to exhibit a higher affinity to cholest-4-en-3-one (Ping-Pong bi bi KmA = 23.7 µM) than to AD (KmA = 529.2 µM), a supposedly native substrate of the enzyme. Moreover, we have isolated AcmB isoenzyme (AcmB2) and showed that conversion of AD and cholest-4-en-3-one proceeds at a similar rate. We demonstrated also that the apparent specificity constant of AcmB for cholest-4-en-3-one (kcat/KmA = 9.25â106 M-1 s-1) is almost 20 times higher than measured for KSTD1 (kcat/KmA = 4.71â105 M-1 s-1). CONCLUSIONS: We confirmed the existence of AcmB preference for a substrate with an undegraded isooctyl chain. However, we showed that KSTD1 which was reported to be inactive with such substrates can catalyze the reaction if the solubility problem is addressed.
Assuntos
2-Hidroxipropil-beta-Ciclodextrina/metabolismo , Betaproteobacteria/enzimologia , Betaproteobacteria/metabolismo , Colestenonas/metabolismo , Oxirredutases/metabolismo , Rhodococcus/enzimologia , Rhodococcus/metabolismo , Proteínas de Bactérias/metabolismo , Betaproteobacteria/genética , Catálise , Clonagem Molecular , DNA Bacteriano , Isoenzimas/metabolismo , Cetosteroides/metabolismo , Cinética , Simulação de Dinâmica Molecular , Proteínas Recombinantes/metabolismo , Rhodococcus/genética , Compostos de Espiro/metabolismo , Esteroides/metabolismo , Especificidade por Substrato , Triterpenos/metabolismoRESUMO
PURPOSE: Chronic rhinosinusitis (CRS) is a highly prevalent multifactorial disorder. Culture-directed antibiotics are frequently prescribed to patients with CRS and the middle nasal meatus (MM) is traditionally believed to be a representative sampling site of the sinuses as a whole. The purpose of our study was to reevaluate the reliability of the MM as a sampling site in patients with CRS who suffer from impaired drainage from the sinuses to the MM. METHODS: Swabs and tissue biopsies were collected from the MM, maxillary sinus and frontal sinus from 50 patients with CRS. The results of bacterial culture were compared between sampling methods and sites in relation to the patency of the sinus ostia. RESULTS: 782 bacterial isolates were cultured from the samples. Concordant results between the MM and the sinus cavity were noted in 80% of patients for the maxillary sinus, but only 66% for the frontal sinus and 76% for the sinuses a whole. The differences were similarly prevalent in patients with open and occluded sinus ostia. Notably, swabs from all three sites provided representative information in 92% of patients and tissue biopsies did not provide additional information compared to multiple swabs. CONCLUSION: The traditional method of sampling from the middle meatus provides inadequate information in 24% of patients with CRS, which may result in inadequate antibiotic therapy and contribute to increasing antibiotic resistance. Additional sampling from the sinuses should be recommended whenever possible, while invasive sampling is not necessary.
Assuntos
Seios Paranasais , Rinite , Sinusite , Doença Crônica , Humanos , Seio Maxilar , Cavidade Nasal , Reprodutibilidade dos Testes , Rinite/diagnóstico , Sinusite/diagnósticoRESUMO
The electrochemically driven catalysis of the complex molybdoenzyme steroid C25 dehydrogenase (S25DH) from the ß-Proteobacterium Sterolibacterium denitrificans is reported. S25DH catalyses the oxygen-independent regioselective hydroxylation of the tertiary C25 atom of sterols and also their derivatives. Cholest-4-en-3-one is a native substrate for S25DH, which produces 25-hydroxycholest-4-en-3-one as a product of catalytic turnover. Cholecalciferol (vitD3 ) is also a substrate. S25DH was immobilised on a modified gold working electrode with the co-adsorbent chitosan. The complexes ferricyanide ([Fe(CN)6 ]3- ) and ferrocenium methanol (FM+ ) are effective artificial electron acceptors from S25DH and act as mediators of electron transfer between the electrode and the enzyme. 2-Hydroxypropyl-ß-cyclodextrin (HPCD) was employed as a sterol solubiliser, in addition to 2-methoxyethanol. The catalytic activity varied, depending upon the concentration of solubiliser in the reaction mixture. Parallel studies with [Fe(CN)6 ]3- as a chemical (as opposed to electrochemical) oxidant coupled to HPLC analysis show that S25DH is capable of oxidising both vitD3 and its less stable isomer, pre-vitD3 , and that the former substrate is stabilised by HPCD.
Assuntos
Colestenonas/química , Bactérias Gram-Negativas/química , Oxirredutases/química , Oxirredutases/metabolismo , Rhodocyclaceae/química , Esteroides/química , Esteróis/química , Catálise , Hidroxilação , Oxirredução , Esteroides/metabolismo , Esteróis/metabolismoRESUMO
The steroid superfamily includes a wide range of compounds that are essential for living organisms of the animal and plant kingdoms. Structural modifications of steroids highly affect their biological activity. In this review, we focus on hydroxylation of steroids by bacterial hydroxylases, which take part in steroid catabolic pathways and play an important role in steroid degradation. We compare three distinct classes of metalloenzymes responsible for aerobic or anaerobic hydroxylation of steroids, namely: cytochrome P450, Rieske-type monooxygenase 3-ketosteroid 9α-hydroxylase, and molybdenum-containing steroid C25 dehydrogenases. We analyze the available literature data on reactivity, regioselectivity, and potential application of these enzymes in organic synthesis of hydroxysteroids. Moreover, we describe mechanistic hypotheses proposed for all three classes of enzymes along with experimental and theoretical evidences, which have provided grounds for their formulation. In case of the 3-ketosteroid 9α-hydroxylase, such a mechanistic hypothesis is formulated for the first time in the literature based on studies conducted for other Rieske monooxygenases. Finally, we provide comparative analysis of similarities and differences in the reaction mechanisms utilized by bacterial steroid hydroxylases.
Assuntos
Proteínas de Bactérias/metabolismo , Esteroide Hidroxilases/metabolismo , Animais , Catálise , Humanos , Hidroxilação/fisiologia , Esteroides/metabolismoRESUMO
The published online version contains mistake in the author list. The correct presentation should have been "Rita Bernhardt" instead of "Rita Bernhard". There was a missing "t" on the original publication.
RESUMO
Molybdenum is an essential nutrient for metabolism in plant, bacteria, and animals. Molybdoenzymes are involved in nitrogen assimilation and oxidoreductive detoxification, and bioconversion reactions of environmental, industrial, and pharmaceutical interest. Molybdoenzymes contain a molybdenum cofactor (Moco), which is a pyranopterin heterocyclic compound that binds a molybdenum atom via a dithiolene group. Because Moco is a large and complex compound deeply buried within the protein, molybdoenzymes are accompanied by private chaperone proteins responsible for the cofactor's insertion into the enzyme and the enzyme's maturation. An efficient recombinant expression and purification of both Moco-free and Moco-containing molybdoenzymes and their chaperones is of paramount importance for fundamental and applied research related to molybdoenzymes. In this work, we focused on a D1 protein annotated as a chaperone of steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S. The D1 protein is presumably involved in the maturation of S25DH engaged in oxygen-independent oxidation of sterols. As this chaperone is thought to be a crucial element that ensures the insertion of Moco into the enzyme and consequently, proper folding of S25DH optimization of the chaperon's expression is the first step toward the development of recombinant expression and purification methods for S25DH. We have identified common E. coli strains and conditions for both expression and purification that allow us to selectively produce Moco-containing and Moco-free chaperones. We have also characterized the Moco-containing chaperone by EXAFS and HPLC analysis and identified conditions that stabilize both forms of the protein. The protocols presented here are efficient and result in protein quantities sufficient for biochemical studies.
Assuntos
Proteínas de Bactérias , Coenzimas , Escherichia coli/metabolismo , Expressão Gênica , Metaloproteínas , Chaperonas Moleculares , Nitrosomonadaceae/genética , Pteridinas , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Coenzimas/biossíntese , Coenzimas/química , Coenzimas/genética , Coenzimas/isolamento & purificação , Escherichia coli/química , Escherichia coli/genética , Metaloproteínas/biossíntese , Metaloproteínas/química , Metaloproteínas/genética , Metaloproteínas/isolamento & purificação , Chaperonas Moleculares/biossíntese , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Cofatores de Molibdênio , Nitrosomonadaceae/metabolismo , Pteridinas/química , Pteridinas/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Molecular modeling techniques and density functional theory calculations were performed to study the mechanism of enzymatic radical C-C coupling catalyzed by benzylsuccinate synthase (BSS). BSS has been identified as a glycyl radical enzyme that catalyzes the enantiospecific fumarate addition to toluene initiating its anaerobic metabolism in the denitrifying bacterium Thauera aromatica, and this reaction represents the general mechanism of toluene degradation in all known anaerobic degraders. In this work docking calculations, classical molecular dynamics (MD) simulations, and DFT+D2 cluster modeling was employed to address the following questions: (i) What mechanistic details of the BSS reaction yield the most probable molecular model? (ii) What is the molecular basis of enantiospecificity of BSS? (iii) Is the proposed mechanism consistent with experimental observations, such as an inversion of the stereochemistry of the benzylic protons, syn addition of toluene to fumarate, exclusive production of (R)-benzylsuccinate as a product and a kinetic isotope effect (KIE) ranging between 2 and 4? The quantum mechanics (QM) modeling confirms that the previously proposed hypothetical mechanism is the most probable among several variants considered, although C-H activation and not C-C coupling turns out to be the rate limiting step. The enantiospecificity of the enzyme seems to be enforced by a thermodynamic preference for binding of fumarate in the pro(R) orientation and reverse preference of benzyl radical attack on fumarate in pro(S) pathway which results with prohibitively high energy barrier of the radical quenching. Finally, the proposed mechanism agrees with most of the experimental observations, although the calculated intrinsic KIE from the model (6.5) is still higher than the experimentally observed values (4.0) which suggests that both C-H activation and radical quenching may jointly be involved in the kinetic control of the reaction.
Assuntos
Carbono-Carbono Liases/metabolismo , Thauera/enzimologia , Carbono-Carbono Liases/química , Domínio Catalítico , Fumaratos/metabolismo , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação Proteica , Especificidade por Substrato , Succinatos/metabolismo , Thauera/química , Thauera/metabolismo , Tolueno/metabolismoRESUMO
Benzylsuccinate synthase is a glycyl radical enzyme that initiates anaerobic toluene metabolism by adding fumarate to the methyl group of toluene to yield (R)-benzylsuccinate. To investigate whether the reaction occurs with retention or inversion of configuration at the methyl group of toluene, we synthesized both enantiomers of chiral toluene with all three Hâ isotopes in their methyl groups. The chiral toluenes were converted into benzylsuccinates preferentially containing (2) H and (3) H at their benzylic Câ atoms, owing to a kinetic isotope effect favoring hydrogen abstraction from the methyl groups. The configuration of the products was analyzed by enzymatic CoA-thioester synthesis and stereospecific oxidation using enzymes involved in benzylsuccinate degradation. Assessment of the configurations of the benzylsuccinate isomers based on loss or retention of tritium showed that inversion of configuration at the methyl group occurs when the chiral toluenes react with fumarate.
Assuntos
Carbono-Carbono Liases/metabolismo , Succinatos/metabolismo , Tolueno/química , Fumaratos/química , Oxirredução , Estereoisomerismo , Succinatos/química , Tolueno/metabolismo , Trítio/químicaRESUMO
The reaction of benzylsuccinate synthase, the radical-based addition of toluene to a fumarate cosubstrate, is initiated by hydrogen transfer from a conserved cysteine to the nearby glycyl radical in the active center of the enzyme. In this study, we analyze this step by comprehensive computer modeling, predicting (i) the influence of bound substrates or products, (ii) the energy profiles of forward- and backward hydrogen-transfer reactions, (iii) their kinetic constants and potential mechanisms, (iv) enantiospecificity differences, and (v) kinetic isotope effects. Moreover, we support several of the computational predictions experimentally, providing evidence for the predicted H/D-exchange reactions into the product and at the glycyl radical site. Our data indicate that the hydrogen transfer reactions between the active site glycyl and cysteine are principally reversible, but their rates differ strongly depending on their stereochemical orientation, transfer of protium or deuterium, and the presence or absence of substrates or products in the active site. This is particularly evident for the isotope exchange of the remaining protium atom of the glycyl radical to deuterium, which appears dependent on substrate or product binding, explaining why the exchange is observed in some, but not all, glycyl-radical enzymes.
Assuntos
Biocatálise , Cinética , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/metabolismo , Domínio Catalítico , Modelos Moleculares , Cisteína/química , Cisteína/metabolismo , Hidrogênio/química , Radicais Livres/química , Radicais Livres/metabolismo , Carbono-Carbono LiasesRESUMO
Good substrate gone bad! BN/CC isosterism of ethylbenzene leads to N-ethyl-1,2-azaborine and B-ethyl-1,2-azaborine. In contrast to ethylbenzene, which is the substrate for ethylbenzene dehydrogenase (EbDH), N-ethyl-1,2-azaborine (see scheme; Fc=Ferricenium tetrafluoroborate) and B-ethyl-1,2-azaborine are strong inhibitors of EbDH. Thus, the changes provided by BN/CC isosterism can lead to new biochemical reactivity.
Assuntos
Derivados de Benzeno/química , Compostos de Boro/química , Compostos de Boro/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Derivados de Benzeno/antagonistas & inibidores , Derivados de Benzeno/metabolismo , Hidroxilação/efeitos dos fármacos , Oxirredutases/antagonistas & inibidores , Oxirredutases/química , Oxirredutases/metabolismoRESUMO
Aldehyde oxidoreductases (AORs) are tungsten enzymes catalyzing the oxidation of many different aldehydes to the corresponding carboxylic acids. In contrast to other known AORs, the enzyme from the denitrifying betaproteobacterium Aromatoleum aromaticum (AORAa) consists of three different subunits (AorABC) and uses nicotinamide adenine dinucleotide (NAD) as an electron acceptor. Here, we reveal that the enzyme forms filaments of repeating AorAB protomers that are capped by a single NAD-binding AorC subunit, based on solving its structure via cryo-electron microscopy. The polyferredoxin-like subunit AorA oligomerizes to an electron-conducting nanowire that is decorated with enzymatically active and W-cofactor (W-co) containing AorB subunits. Our structure further reveals the binding mode of the native substrate benzoate in the AorB active site. This, together with quantum mechanics:molecular mechanics (QM:MM)-based modeling for the coordination of the W-co, enables formulation of a hypothetical catalytic mechanism that paves the way to further engineering for applications in synthetic biology and biotechnology.
Assuntos
Aldeído Oxirredutases , Nanofios , Aldeído Oxirredutases/química , Aldeído Oxirredutases/metabolismo , Tungstênio/metabolismo , NAD , Microscopia Crioeletrônica , Aldeído DesidrogenaseRESUMO
Ethylbenzene dehydrogenase (EbDH) catalyzes the initial step in anaerobic degradation of ethylbenzene in denitrifying bacteria, namely, the oxygen-independent hydroxylation of ethylbenzene to (S)-1-phenylethanol. In our study we investigate the kinetic properties of 46 substrate analogs acting as substrates or inhibitors of the enzyme. The apparent kinetic parameters of these compounds give important insights into the function of the enzyme and are consistent with the predicted catalytic mechanism based on a quantum chemical calculation model. In particular, the existence of the proposed substrate-derived radical and carbocation intermediates is substantiated by the formation of alternative dehydrogenated and hydroxylated products from some substrates, which can be regarded as mechanistic models. In addition, these results also show the surprisingly high diversity of EbDH in hydroxylating different kinds of alkylaromatic and heterocyclic compounds to the respective alcohols. This may lead to attractive industrial applications of ethylbenzene dehydrogenase for a new process of producing alcohols via hydroxylation of the corresponding aromatic hydrocarbons rather than the customary procedure of reducing the corresponding ketones.
Assuntos
Bactérias/enzimologia , Inibidores Enzimáticos/metabolismo , Oxirredutases/metabolismo , Derivados de Benzeno/metabolismo , Cinética , Oxirredutases/química , Álcool Feniletílico/metabolismo , Especificidade por SubstratoRESUMO
The enzyme-catalyzed reactions are traditionally studied with experimental kinetic assays. The modern theoretical modeling techniques provide a complementary way to investigate these catalytic reactions. Experimental assay frequently does not allow an unequivocal answer to the factors controlling the reaction mechanism. On the other hand, the theoretical experiments provide a precise understanding of the molecular-level steps involved in catalytic reactions. However, modeling requires at least structural data on the enzyme and reactant, and the complexity of the enzyme systems can still be a challenge.In this chapter, we are going to describe how to apply theoretical modeling methods, such as MD simulation, QM-only cluster models of enzyme active site, or QM:MM multiscale modeling to study enzyme kinetics and even to predict kinetic isotope effect (KIE). We present a full protocol that starts from the PDB structure of the enzyme, through MD simulation of enzyme: substrate complex and statistical analysis of MD trajectory, selection of a model of the active site, and study of reaction pathways. We show how theoretical predictions basing on QM-only cluster models, QM:MM model, or multiple QM:MM models derived from QM:MM:MD simulations can be correlated with experimental kinetic results. Finally, we show how one can calculate intrinsic KIE associated with an individual molecular step.
Assuntos
Simulação de Dinâmica Molecular , Teoria Quântica , Catálise , Domínio Catalítico , Simulação por Computador , CinéticaRESUMO
Tungsten-dependent aldehyde oxidoreductases (AORs) catalyze the oxidation of aldehydes to acids and are the only known enzymes reducing non-activated acids using electron donors with low redox potentials. We report here that AOR from Aromatoleum aromaticum (AOR Aa ) catalyzes the reduction of organic acids not only with low-potential Eu(II) or Ti(III) complexes but also with H2 as an electron donor. Additionally, AOR Aa catalyzes the H2-dependent reduction of NAD+ or benzyl viologen. The rate of H2-dependent NAD+ reduction equals to 10% of that of aldehyde oxidation, representing the highest H2 turnover rate observed among the Mo/W enzymes. As AOR Aa simultaneously catalyzes the reduction of acids and NAD+, we designed a cascade reaction utilizing a NAD(P)H-dependent alcohol dehydrogenase to reduce organic acids to the corresponding alcohols with H2 as the only reductant. The newly discovered W-hydrogenase side activity of AOR Aa may find applications in either NADH recycling or conversion of carboxylic acids to more useful biochemicals.