RESUMO
Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and progression of hepatocellular carcinoma (HCC). HCC accounts for approximately 75% of primary liver cancers and is within the top three causes of cancer death worldwide. As a result of treatment limitations, the overall 5-year survival rate for all patients with HCC is under 20%. The prevalence of HCC necessitates continued development of novel and effective treatment methods. In recent years, the therapeutic potential of selective inactivation of hOAT has been demonstrated for the treatment of HCC. Inspired by previous increased selectivity for hOAT by the expansion of the cyclopentene ring scaffold to a cyclohexene, we designed, synthesized, and evaluated a series of novel fluorinated cyclohexene analogues and identified (R)-3-amino-5,5-difluorocyclohex-1-ene-1-carboxylic acid as a time-dependent inhibitor of hOAT. Structural and mechanistic studies have elucidated the mechanism of inactivation of hOAT by 5, resulting in a PLP-inactivator adduct tightly bound to the active site of the enzyme. Intact protein mass spectrometry, 19F NMR spectroscopy, transient state kinetic studies, and X-ray crystallography were used to determine the structure of the final adduct and elucidate the mechanisms of inactivation. Interestingly, despite the highly electrophilic intermediate species conferred by fluorine and structural evidence of solvent accessibility in the hOAT active site, Lys292 and water did not participate in nucleophilic addition during the inactivation mechanism of hOAT by 5. Instead, rapid aromatization to yield the final adduct was favored.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos , Ornitina-Oxo-Ácido Transaminase , Humanos , Ornitina-Oxo-Ácido Transaminase/metabolismo , Ornitina-Oxo-Ácido Transaminase/química , Ornitina-Oxo-Ácido Transaminase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Ácidos Carboxílicos/química , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/farmacologia , Cicloexenos/química , Cicloexenos/síntese química , Cicloexenos/farmacologia , Cicloexenos/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Cristalografia por Raios X , Modelos MolecularesRESUMO
Probiotics hold promise as a potential therapy for colorectal cancer (CRC), but encounter obstacles related to tumor specificity, drug penetration, and dosage adjustability. In this study, genetic circuits based on the E. coli Nissle 1917 (EcN) chassis were developed to sense indicators of tumor microenvironment and control the expression of therapeutic payloads. Integration of XOR gate amplify gene switch into EcN biosensors resulted in a 1.8-2.3-fold increase in signal output, as confirmed by mathematical model fitting. Co-culturing programmable EcNs with CRC cells demonstrated a significant reduction in cellular viability ranging from 30% to 50%. This approach was further validated in a mouse subcutaneous tumor model, revealing 47%-52% inhibition of tumor growth upon administration of therapeutic strains. Additionally, in a mouse tumorigenesis model induced by AOM and DSS, the use of synthetic bacterial consortium (SynCon) equipped with multiple sensing modules led to approximately 1.2-fold increased colon length and 2.4-fold decreased polyp count. Gut microbiota analysis suggested that SynCon maintained the abundance of butyrate-producing bacteria Lactobacillaceae NK4A136, whereas reducing the level of gut inflammation-related bacteria Bacteroides. Taken together, engineered EcNs confer the advantage of specific recognition of CRC, while SynCon serves to augment the synergistic effect of this approach.
Assuntos
Colite , Neoplasias Colorretais , Microbioma Gastrointestinal , Animais , Camundongos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/microbiologia , Colite/induzido quimicamente , Escherichia coli/genética , Inflamação , Microambiente TumoralRESUMO
ADP-glucose pyrophosphorylase is a key regulatory enzyme involved in starch and glycogen synthesis in plants and bacteria, respectively. It has been hypothesized that inter-subunit communications are important for the allosteric effect in this enzyme. However, no specific interactions have been identified as part of the regulatory signal. The enzyme from Agrobacterium tumefaciens is a homotetramer allosterically regulated by fructose 6-phosphate and pyruvate. Three pairs of distinct subunit-subunit interfaces are present. Here we focus on an interface that features two symmetrical interactions between Arg11 and Asp141 from one subunit with residues Asp141 and Arg11 of the neighbor subunit, respectively. Previously, scanning mutagenesis showed that a mutation at the Arg11 position disrupted the activation of the enzyme. Considering the distance of these residues from the allosteric and catalytic sites, we hypothesized that the interaction between Arg11 and Asp141 is critical for allosteric signaling rather than effector binding. To prove our hypothesis, we mutated those two sites (D141A, D141E, D141N, D141R, R11D, and R11K) and performed kinetic and binding analysis. Mutations that altered the charge affected the regulation the most. To prove that the interaction per se (rather than the presence of specific residues) is critical, we partially rescued the R11D protein by introducing a second mutation (R11D/D141R). This could not restore the activator effect on kcat , but it did rescue the effect on substrate affinity. Our results indicate the critical functional role of Arg11 and Asp141 to relay the allosteric signal in this subunit interface.
Assuntos
Agrobacterium tumefaciens , Amido , Glucose-1-Fosfato Adenililtransferase/genética , Glucose-1-Fosfato Adenililtransferase/química , Glucose-1-Fosfato Adenililtransferase/metabolismo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Mutação , Ácido Pirúvico , Cinética , Regulação Alostérica/genéticaRESUMO
Ornithine aminotransferase (OAT) is overexpressed in hepatocellular carcinoma (HCC), and we previously showed that inactivation of OAT inhibits the growth of HCC. Recently, we found that (3S,4S)-3-amino-4-fluorocyclopentenecarboxylic acid (5) was a potent inactivator of γ-aminobutyric acid aminotransferase (GABA-AT), proceeding by an enamine mechanism. Here we describe our investigations into the activity and mechanism of 5 as an inactivator of human OAT. We have found that 5 exhibits 10-fold less inactivation efficiency (kinact/KI) against hOAT than GABA-AT. A comprehensive mechanistic study was carried out to understand its inactivation mechanism with hOAT. pKa and electrostatic potential calculations were performed to further support the notion that the α,ß-unsaturated alkene of 5 is critical for enhancing acidity and nucleophilicity of the corresponding intermediates and ultimately responsible for the improved inactivation efficiency of 5 over the corresponding saturated analogue (4). Intact protein mass spectrometry and the crystal structure complex with hOAT provide evidence to conclude that 5 mainly inactivates hOAT through noncovalent interactions, and that, unlike with GABA-AT, covalent binding with hOAT is a minor component of the total inhibition which is unique relative to other monofluoro-substituted derivatives. Furthermore, based on the results of transient-state measurements and free energy calculations, it is suggested that the α,ß-unsaturated carboxylate group of PLP-bound 5 may be directly involved in the inactivation cascade by forming an enolate intermediate. Overall, compound 5 exhibits unusual structural conversions which are catalyzed by specific residues within hOAT, ultimately leading to an enamine mechanism-based inactivation of hOAT through noncovalent interactions and covalent modification.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Aminoácidos/farmacologia , Inibidores Enzimáticos/farmacologia , Ornitina-Oxo-Ácido Transaminase/química , Ornitina-Oxo-Ácido Transaminase/metabolismo , Ácido gama-Aminobutírico , Ácidos Carboxílicos/farmacologia , Ácidos Carboxílicos/química , OrnitinaRESUMO
To clarify the mechanism of the response of sugar beet (Beta vulgaris L.) to cadmium (Cd) stress, this study investigated changes in the phenotype, physiological indexes, and subcellular structure of B. vulgaris under Cd treatment and the transcriptional pattern of the BvHIPP24 gene (a heavy metal-associated isoprenylated plant protein involved in heavy metal detoxification). The plant height and shoot and root growth of B. vulgaris seedlings were inhibited to some extent under 0.5 and 1 mM Cd, with gradually wilting and yellowing of leaves and dark brown roots. When the Cd concentration was increased, malondialdehyde content and the activities of peroxidase, superoxide dismutase, and glutathione S-transferase increased differentially. qPCR indicated that the expression of BvHIPP24 was induced by different concentrations of Cd. Although transmission electron microscopy revealed damage to nuclei, mitochondria, and chloroplasts, B. vulgaris exhibited strong adaptability to 0.5 mM Cd according to a comprehensive analysis using the membership function. The results showed that B. vulgaris may reduce cell damage and improve its Cd tolerance by regulating functional gene expression and antioxidant enzymes. This study increases our understanding of the Cd-tolerance mechanism of B. vulgaris and provides insights into the use of B. vulgaris in Cd bioremediation.
Sugar beet is a novel energy crop with superior characteristics for both heavy metal phytoremediation and biomass energy development. This work is the first to investigate both the morphological, physiological, and ultrastructural response of sugar beet to cadmium stress and the induction of a functional metallochaperone gene by cadmium. This study explains the cadmium tolerance mechanism of sugar beet based on a comprehensive evaluation and provides an important theoretical basis for further application of beet in heavy metal bioremediation.
Assuntos
Beta vulgaris , Metais Pesados , Cádmio/toxicidade , Cádmio/metabolismo , Beta vulgaris/genética , Beta vulgaris/metabolismo , Biodegradação Ambiental , Expressão Gênica , Açúcares/metabolismo , Açúcares/farmacologia , Raízes de PlantasRESUMO
The allosteric regulation of ADP-glucose pyrophosphorylase is critical for the biosynthesis of glycogen in bacteria and starch in plants. The enzyme from Agrobacterium tumefaciens is activated by fructose 6-phosphate (Fru6P) and pyruvate (Pyr). The Pyr site has been recently found, but the site where Fru6P binds has remained unknown. We hypothesize that a sulfate ion previously found in the crystal structure reveals a part of the regulatory site mimicking the presence of the phosphoryl moiety of the activator Fru6P. Ser72 interacts with this sulfate ion and, if the hypothesis is correct, Ser72 would affect the interaction with Fru6P and activation of the enzyme. Here, we report structural, binding, and kinetic analysis of Ser72 mutants of the A. tumefaciens ADP-glucose pyrophosphorylase. By X-ray crystallography, we found that when Ser72 was replaced by Asp or Glu side chain carboxylates protruded into the sulfate-binding pocket. They would present a strong steric and electrostatic hindrance to the phosphoryl moiety of Fru6P, while being remote from the Pyr site. In agreement, we found that Fru6P could not activate or bind to S72E or S72D mutants, whereas Pyr was still an effective activator. These mutants also blocked the binding of the inhibitor AMP. This could potentially have biotechnological importance in obtaining enzyme forms insensitive to inhibition. Other mutations in this position (Ala, Cys, and Trp) confirmed the importance of Ser72 in regulation. We propose that the ADP-glucose pyrophosphorylase from A. tumefaciens have two distinct sites for Fru6P and Pyr working in tandem to regulate glycogen biosynthesis.
Assuntos
Agrobacterium tumefaciens , Serina , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Frutose , Glucose-1-Fosfato Adenililtransferase/química , Glucose-1-Fosfato Adenililtransferase/genética , Glucose-1-Fosfato Adenililtransferase/metabolismo , Glicogênio/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Fosfatos , Serina/genética , SulfatosRESUMO
Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver and occurs predominantly in patients with underlying chronic liver diseases. Over the past decade, human ornithine aminotransferase (hOAT), which is an enzyme that catalyzes the metabolic conversion of ornithine into an intermediate for proline or glutamate synthesis, has been found to be overexpressed in HCC cells. hOAT has since emerged as a promising target for novel anticancer therapies, especially for the ongoing rational design effort to discover mechanism-based inactivators (MBIs). Despite the significance of hOAT in human metabolism and its clinical potential as a drug target against HCC, there are significant knowledge deficits with regard to its catalytic mechanism and structural characteristics. Ongoing MBI design efforts require in-depth knowledge of the enzyme active site, in particular, pKa values of potential nucleophiles and residues necessary for the molecular recognition of ligands. Here, we conducted a study detailing the fundamental active-site properties of hOAT using stopped-flow spectrophotometry and X-ray crystallography. Our results quantitatively revealed the pH dependence of the multistep reaction mechanism and illuminated the roles of ornithine α-amino and δ-amino groups in substrate recognition and in facilitating catalytic turnover. These findings provided insights of the catalytic mechanism that could benefit the rational design of MBIs against hOAT. In addition, substrate recognition and turnover of several fragment-sized alternative substrates of hOATs, which could serve as structural templates for MBI design, were also elucidated.
Assuntos
Ornitina-Oxo-Ácido Transaminase/metabolismo , Carcinoma Hepatocelular , Cristalografia por Raios X , Humanos , Concentração de Íons de Hidrogênio , Cinética , Neoplasias Hepáticas , Modelos Moleculares , Ornitina/química , Ornitina-Oxo-Ácido Transaminase/química , Especificidade por SubstratoRESUMO
Human ornithine aminotransferase (hOAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that contains a similar active site to that of γ-aminobutyric acid aminotransferase (GABA-AT). Recently, pharmacological inhibition of hOAT was recognized as a potential therapeutic approach for hepatocellular carcinoma. In this work, we first studied the inactivation mechanisms of hOAT by two well-known GABA-AT inactivators (CPP-115 and OV329). Inspired by the inactivation mechanistic difference between these two aminotransferases, a series of analogues were designed and synthesized, leading to the discovery of analogue 10b as a highly selective and potent hOAT inhibitor. Intact protein mass spectrometry, protein crystallography, and dialysis experiments indicated that 10b was converted to an irreversible tight-binding adduct (34) in the active site of hOAT, as was the unsaturated analogue (11). The comparison of kinetic studies between 10b and 11 suggested that the active intermediate (17b) was only generated in hOAT and not in GABA-AT. Molecular docking studies and pKa computational calculations highlighted the importance of chirality and the endocyclic double bond for inhibitory activity. The turnover mechanism of 10b was supported by mass spectrometric analysis of dissociable products and fluoride ion release experiments. Notably, the stopped-flow experiments were highly consistent with the proposed mechanism, suggesting a relatively slow hydrolysis rate for hOAT. The novel second-deprotonation mechanism of 10b contributes to its high potency and significantly enhanced selectivity for hOAT inhibition.
Assuntos
4-Aminobutirato Transaminase , Neoplasias Hepáticas , Ácidos Carboxílicos , Inibidores Enzimáticos/química , Humanos , Cinética , Simulação de Acoplamento Molecular , Ornitina-Oxo-Ácido Transaminase , Fenilacetatos , Fosfato de Piridoxal/química , Ácido gama-AminobutíricoRESUMO
The inhibition of human ornithine δ-aminotransferase (hOAT) is a potential therapeutic approach to treat hepatocellular carcinoma. In this work, (S)-3-amino-4,4-difluorocyclopent-1-enecarboxylic acid (SS-1-148, 6) was identified as a potent mechanism-based inactivator of hOAT while showing excellent selectivity over other related aminotransferases (e.g., GABA-AT). An integrated mechanistic study was performed to investigate the turnover and inactivation mechanisms of 6. A monofluorinated ketone (M10) was identified as the primary metabolite of 6 in hOAT. By soaking hOAT holoenzyme crystals with 6, a precursor to M10 was successfully captured. This gem-diamine intermediate, covalently bound to Lys292, observed for the first time in hOAT/ligand crystals, validates the turnover mechanism proposed for 6. Co-crystallization yielded hOAT in complex with 6 and revealed a novel noncovalent inactivation mechanism in hOAT. Native protein mass spectrometry was utilized for the first time in a study of an aminotransferase inactivator to validate the noncovalent interactions between the ligand and the enzyme; a covalently bonded complex was also identified as a minor form observed in the denaturing intact protein mass spectrum. Spectral and stopped-flow kinetic experiments supported a lysine-assisted E2 fluoride ion elimination, which has never been observed experimentally in other studies of related aminotransferase inactivators. This elimination generated the second external aldimine directly from the initial external aldimine, rather than the typical E1cB elimination mechanism, forming a quinonoid transient state between the two external aldimines. The use of native protein mass spectrometry, X-ray crystallography employing both soaking and co-crystallization methods, and stopped-flow kinetics allowed for the detailed elucidation of unusual turnover and inactivation pathways.
Assuntos
Ornitina-Oxo-Ácido Transaminase/metabolismo , Humanos , Estrutura Molecular , Ornitina-Oxo-Ácido Transaminase/químicaRESUMO
Human ornithine aminotransferase (hOAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that was recently found to play an important role in the metabolic reprogramming of hepatocellular carcinoma (HCC) via the proline and glutamine metabolic pathways. The selective inhibition of hOAT by compound 10 exhibited potent in vivo antitumor activity. Inspired by the discovery of the aminotransferase inactivator (1S,3S)-3-amino-4-(difluoromethylene)cyclopentane-1-carboxylic acid (5), we rationally designed, synthesized, and evaluated a series of six-membered-ring analogs. Among them, 14 was identified as a new selective hOAT inactivator, which demonstrated a potency 22× greater than that of 10. Three different types of protein mass spectrometry approaches and two crystallographic approaches were employed to identify the structure of hOAT-14 and the formation of a remarkable final adduct (32') in the active site. These spectral studies reveal an enzyme complex heretofore not observed in a PLP-dependent enzyme, which has covalent bonds to two nearby residues. Crystal soaking experiments and molecular dynamics simulations were carried out to identify the structure of the active-site intermediate 27' and elucidate the order of the two covalent bonds that formed, leading to 32'. The initial covalent reaction of the activated warhead occurs with *Thr322 from the second subunit, followed by a subsequent nucleophilic attack by the catalytic residue Lys292. The turnover mechanism of 14 by hOAT was supported by a mass spectrometric analysis of metabolites and fluoride ion release experiments. This novel mechanism for hOAT with 14 will contribute to the further rational design of selective inactivators and an understanding of potential inactivation mechanisms by aminotransferases.
Assuntos
Inibidores Enzimáticos/farmacologia , Ornitina-Oxo-Ácido Transaminase/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Cinética , Espectrometria de Massas , Modelos Moleculares , Estrutura Molecular , Ornitina-Oxo-Ácido Transaminase/metabolismoRESUMO
The native function of dihydropyrimidine dehydrogenase (DPD) is to reduce the 5,6-vinylic bond of pyrimidines uracil and thymine with electrons obtained from NADPH. NADPH and pyrimidines bind at separate active sites separated by â¼60 Å that are bridged by four Fe4S4 centers. We have shown that DPD undergoes reductive activation, taking up two electrons from NADPH [Beaupre, B. A., et al. (2020) Biochemistry 59, 2419-2431]. pH studies indicate that the rate of turnover is not controlled by the protonation state of the general acid, cysteine 671. The activation of the C671 variants is delineated into two phases particularly at low pH values. Spectral deconvolution of the delineated reductive activation reaction reveals that the initial phase results in the accumulation of charge transfer absorption added to the binding difference spectrum for NADPH. The second phase results in reduction of one of the two flavins. X-ray crystal structure analysis of the C671S variant soaked with NADPH and the slow substrate, thymine, in a low-oxygen atmosphere resolved the presumed activated form of the enzyme that has the FMN cofactor reduced. These data reveal that charge transfer arises from the proximity of the NADPH and FAD bases and that the ensuing flavin is a result of rapid transfer of electrons to the FMN without accumulation of reduced forms of the FAD or Fe4S4 centers. These data suggest that the slow rate of turnover of DPD is governed by the movement of a mobile structural feature that carries the C671 residue.
Assuntos
Di-Hidrouracila Desidrogenase (NADP)/química , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Hidrogênio/metabolismo , Animais , Domínio Catalítico , Di-Hidrouracila Desidrogenase (NADP)/fisiologia , Flavina-Adenina Dinucleotídeo/química , Flavinas/química , Hidrogênio/química , Cinética , NADP/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Oxirredução , Estrutura Terciária de Proteína , Pirimidinas/química , Pirimidinas/metabolismo , Espectrofotometria/métodos , SuínosRESUMO
Dihydropyrimidine dehydrogenase (DPD) is a complex enzyme that reduces the 5,6-vinylic bond of pyrimidines, uracil, and thymine. 5-Fluorouracil (5FU) is also a substrate for DPD and a common chemotherapeutic agent used to treat numerous cancers. The reduction of 5FU to 5-fluoro-5,6-dihydrouracil negates its toxicity and efficacy. Patients with high DPD activity levels typically have poor outcomes when treated with 5FU. DPD is thus a central mitigating factor in the treatment of a variety of cancers. 5-Ethynyluracil (5EU) covalently inactivates DPD by cross-linking with the active-site general acid cysteine in the pyrimidine binding site. This reaction is dependent on the simultaneous binding of 5EU and nicotinamide adenine dinucleotide phosphate (NADPH). This ternary complex induces DPD to become activated by taking up two electrons from the NADPH. The covalent inactivation of DPD by 5EU occurs concomitantly with this reductive activation with a rate constant of â¼0.2 s-1. This kinact value is correlated with the rate of reduction of one of the two flavin cofactors and the localization of a mobile loop in the pyrimidine active site that places the cysteine that serves as the general acid in catalysis proximal to the 5EU ethynyl group. Efficient cross-linking is reliant on enzyme activation, but this process appears to also have a conformational aspect in that nonreductive NADPH analogues can also induce a partial inactivation. Cross-linking then renders DPD inactive by severing the proton-coupled electron transfer mechanism that transmits electrons 56 Å across the protein.
Assuntos
Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Uracila/análogos & derivados , Animais , Domínio Catalítico , Di-Hidrouracila Desidrogenase (NADP)/química , Sinergismo Farmacológico , Ligação Proteica , Suínos , Uracila/metabolismo , Uracila/farmacologiaRESUMO
In this work, a novel label-free photoelectrochemical (PEC) biosensor based on Au/CsxWO3 heterogeneous films with low background noise and excellent sensitive detection of alpha-fetoprotein (AFP) was proposed. We prepared Au/CsxWO3 heterogeneous films by different methods including template removal and spin coating. The surface plasmon resonances (SPR) of Au film and CsxWO3 semiconductor nanocrystals largely improved the photoelectrochemical properties of the electrodes and enhanced the photocurrent. Under the optimal conditions, the prepared PEC biosensor showed a linear response between photocurrent variation and the logarithm of AFP concentration in the range from 0.01 ng/mL to 500 ng/mL with a low detection limit of 7 pg/mL. In addition, the proposed PEC biosensor had excellent specificity, repeatability, long-term stability and showed satisfactory results in the analysis of human serum samples, which would have great prospect in clinical application and biological analysis in the future.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Césio , Técnicas Eletroquímicas , Ouro , Humanos , Limite de Detecção , Óxidos , Ressonância de Plasmônio de Superfície , Tungstênio , alfa-FetoproteínasRESUMO
Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the leading cause of death among people with cirrhosis. HCC is typically diagnosed in advanced stages when tumors are resistant to both radio- and chemotherapy. Human ornithine aminotransferase (hOAT) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme involved in glutamine and proline metabolism. Because hOAT is overexpressed in HCC cells and a contributing factor for the uncontrolled cellular division that propagates malignant tumors (Ueno et al. J. Hepatol. 2014, 61, 1080-1087), it is a potential drug target for the treatment of HCC. (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidenyl)-cyclopentane-1-carboxylic acid (BCF3) has been shown in animal models to slow the progression of HCC by acting as a selective and potent mechanism-based inactivator of OAT (Zigmond et al. ACS Med. Chem. Lett. 2015, 6, 840-844). Previous studies have shown that the BCF3-hOAT reaction has a bifurcation in which only 8% of the inhibitor inactivates the enzyme while the remaining 92% ultimately acts as a substrate and undergoes hydrolysis to regenerate the active PLP form of the enzyme. In this manuscript, the rate-limiting step of the inactivation mechanism was determined by stopped-flow spectrophotometry and time-dependent 19F NMR experiments to be the decay of a long-lived external aldimine species. A crystal structure of this transient complex revealed both the structural basis for fractional irreversible inhibition and the principal mode of inhibition of hOAT by BCF3, which is to trap the enzyme in this transient but quasi-stable external aldimine form.
Assuntos
Carcinoma Hepatocelular/patologia , Inibidores Enzimáticos/química , Neoplasias Hepáticas/patologia , Ornitina-Oxo-Ácido Transaminase/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Cinética , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Camundongos , Estrutura Molecular , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate-dependent enzyme, plays a critical role in the progression of hepatocellular carcinoma (HCC). Pharmacological selective inhibition of hOAT has been shown to be a potential therapeutic approach for HCC. Inspired by the discovery of the nonselective aminotransferase inactivator (1R,3S,4S)-3-amino-4-fluoro cyclopentane-1-carboxylic acid (1), in this work, we rationally designed, synthesized, and evaluated a novel series of fluorine-substituted cyclohexene analogues, thereby identifying 8 and 9 as novel selective hOAT time-dependent inhibitors. Intact protein mass spectrometry and protein crystallography demonstrated 8 and 9 as covalent inhibitors of hOAT, which exhibit two distinct inactivation mechanisms resulting from the difference of a single fluorine atom. Interestingly, they share a similar turnover mechanism, according to the mass spectrometry-based analysis of metabolites and fluoride ion release experiments. Molecular dynamics (MD) simulations and electrostatic potential (ESP) charge calculations were conducted, which elucidated the significant influence of the one-fluorine difference on the corresponding intermediates, leading to two totally different inactivation pathways. The novel addition-aromatization inactivation mechanism for 9 contributes to its significantly enhanced potency, along with excellent selectivity over other aminotransferases.
Assuntos
Ácidos Cicloexanocarboxílicos/química , Cicloexilaminas/química , Inibidores Enzimáticos/química , Hidrocarbonetos Fluorados/química , Ornitina-Oxo-Ácido Transaminase/antagonistas & inibidores , Ácidos Cicloexanocarboxílicos/síntese química , Ácidos Cicloexanocarboxílicos/metabolismo , Cicloexilaminas/síntese química , Cicloexilaminas/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Humanos , Hidrocarbonetos Fluorados/síntese química , Hidrocarbonetos Fluorados/metabolismo , Modelos Químicos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ornitina-Oxo-Ácido Transaminase/química , Ornitina-Oxo-Ácido Transaminase/metabolismo , Ligação Proteica , Fosfato de Piridoxal/química , Ácido gama-Aminobutírico/análogos & derivadosRESUMO
Bacterial ADP-glucose pyrophosphorylases are allosterically regulated by metabolites that are key intermediates of central pathways in the respective microorganism. Pyruvate (Pyr) and fructose 6-phosphate (Fru6P) activate the enzyme from Agrobacterium tumefaciens by increasing Vmax about 10- and 20-fold, respectively. Here, we studied the combined effect of both metabolites on the enzyme activation. Our results support a model in which there is a synergistic binding of these two activators to two distinct sites and that each activator leads the enzyme to distinct active forms with different properties. In presence of both activators, Pyr had a catalytically dominant effect over Fru6P determining the active conformational state. By mutagenesis we obtained enzyme variants still sensitive to Pyr activation, but in which the allosteric signal by Fru6P was disrupted. This indicated that the activation mechanism for each effector was not the same. The ability for this enzyme to have more than one allosteric activator site, active forms, and allosteric signaling mechanisms is critical to expand the evolvability of its regulation. These synergistic interactions between allosteric activators may represent a feature in other allosteric enzymes.
Assuntos
Agrobacterium tumefaciens/enzimologia , Proteínas de Bactérias/metabolismo , Frutosefosfatos/metabolismo , Glucose-1-Fosfato Adenililtransferase/metabolismo , Ácido Pirúvico/metabolismo , Regulação Alostérica , Sítio Alostérico , Ativação Enzimática , Cinética , Modelos MolecularesRESUMO
The inhibition of ornithine aminotransferase (OAT), a pyridoxal 5'-phosphate-dependent enzyme, has been implicated as a treatment for hepatocellular carcinoma (HCC), the most common form of liver cancer, for which there is no effective treatment. From a previous evaluation of our aminotransferase inhibitors, (1S,3S)-3-amino-4-(perfluoropropan-2-ylidene)cyclopentane-1-carboxylic acid hydrochloride (1) was found to be a selective and potent inactivator of human OAT (hOAT), which inhibited the growth of HCC in athymic mice implanted with human-derived HCC, even at a dose of 0.1 mg/kg. Currently, investigational new drug (IND)-enabling studies with 1 are underway. The inactivation mechanism of 1, however, has proved to be elusive. Here we propose three possible mechanisms, based on mechanisms of known aminotransferase inactivators: Michael addition, enamine addition, and fluoride ion elimination followed by conjugate addition. On the basis of crystallography and intact protein mass spectrometry, it was determined that 1 inactivates hOAT through fluoride ion elimination to an activated 1,1'-difluoroolefin, followed by conjugate addition and hydrolysis. This result was confirmed with additional studies, including the detection of the cofactor structure by mass spectrometry and through the identification of turnover metabolites. On the basis of this inactivation mechanism and to provide further evidence for the mechanism, analogues of 1 (19, 20) were designed, synthesized, and demonstrated to have the predicted selective inactivation mechanism. These analogues highlight the importance of the trifluoromethyl group and provide a basis for future inactivator design.
Assuntos
Ciclopentanos/química , Ciclopentanos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ornitina-Oxo-Ácido Transaminase/antagonistas & inibidores , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , Carcinoma Hepatocelular/enzimologia , Halogenação , Humanos , Neoplasias Hepáticas/enzimologia , Modelos Moleculares , Ornitina-Oxo-Ácido Transaminase/química , Ornitina-Oxo-Ácido Transaminase/metabolismoRESUMO
The pathways for biosynthesis of glycogen in bacteria and starch in plants are evolutionarily and biochemically related. They are regulated primarily by ADP-glucose pyrophosphorylase, which evolved to satisfy metabolic requirements of a particular organism. Despite the importance of these two pathways, little is known about the mechanism that controls pyrophosphorylase activity or the location of its allosteric sites. Here, we report pyruvate-bound crystal structures of ADP-glucose pyrophosphorylase from the bacterium Agrobacterium tumefaciens, identifying a previously elusive activator site for the enzyme. We found that the tetrameric enzyme binds two molecules of pyruvate in a planar conformation. Each binding site is located in a crevice between the C-terminal domains of two subunits where they stack via a distinct ß-helix region. Pyruvate interacts with the side chain of Lys-43 and with the peptide backbone of Ser-328 and Gly-329 from both subunits. These structural insights led to the design of two variants with altered regulatory properties. In one variant (K43A), the allosteric effect was absent, whereas in the other (G329D), the introduced Asp mimicked the presence of pyruvate. The latter generated an enzyme that was preactivated and insensitive to further activation by pyruvate. Our study furnishes a deeper understanding of how glycogen biosynthesis is regulated in bacteria and the mechanism by which transgenic plants increased their starch production. These insights will facilitate rational approaches to enzyme engineering for starch production in crops of agricultural interest and will promote further study of allosteric signal transmission and molecular evolution in this important enzyme family.
Assuntos
Agrobacterium tumefaciens/enzimologia , Glucose-1-Fosfato Adenililtransferase/química , Glucose-1-Fosfato Adenililtransferase/metabolismo , Piruvatos/metabolismo , Sítios de Ligação , Glucose-1-Fosfato Adenililtransferase/genética , Glicogênio/biossíntese , Glicogênio/química , Modelos Moleculares , Estrutura MolecularRESUMO
DJ-1 is a Parkinson's disease associated protein endowed with enzymatic, redox sensing, regulatory, chaperoning, and neuroprotective activities. Although DJ-1 has been vigorously studied for the past decade and a half, its exact role in the progression of the disease remains uncertain. In addition, little is known about the spatiotemporal regulation of DJ-1, or the biochemical basis explaining its numerous biological functions. Progress has been hampered by the lack of inhibitors with precisely known mechanisms of action. Herein, we have employed biophysical methodologies and X-ray crystallography to identify and to optimize a family of compounds inactivating the critical Cys106 residue of human DJ-1. We demonstrate these compounds are potent inhibitors of various activities of DJ-1 in vitro and in cell-based assays. This study reports a new family of DJ-1 inhibitors with a defined mechanism of action, and contributes toward the understanding of the biological function of DJ-1.
Assuntos
Doença de Parkinson/tratamento farmacológico , Proteína Desglicase DJ-1/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Cristalografia por Raios X , Cisteína/química , Cisteína/metabolismo , Descoberta de Drogas , Células HEK293 , Células HeLa , Humanos , Simulação de Acoplamento Molecular , Conformação Proteica/efeitos dos fármacos , Proteína Desglicase DJ-1/química , Proteína Desglicase DJ-1/metabolismoRESUMO
Cystathionine ß-synthase (CBS) is a key regulator of sulfur amino acid metabolism, taking homocysteine from the methionine cycle to the biosynthesis of cysteine via the trans-sulfuration pathway. CBS is also a predominant source of H2S biogenesis. Roles for CBS have been reported for neuronal death pursuant to cerebral ischemia, promoting ovarian tumor growth, and maintaining drug-resistant phenotype by controlling redox behavior and regulating mitochondrial bioenergetics. The trans-sulfuration pathway is well-conserved in eukaryotes, but the analogous enzymes have different enzymatic behavior in different organisms. CBSs from the higher organisms contain a heme in an N-terminal domain. Though the presence of the heme, whose functions in CBSs have yet to be elucidated, is biochemically interesting, it hampers UV-vis absorption spectroscopy investigations of pyridoxal 5'-phosphate (PLP) species. CBS from Saccharomyces cerevisiae (yCBS) naturally lacks the heme-containing N-terminal domain, which makes it an ideal model for spectroscopic studies of the enzymological reaction catalyzed and allows structural studies of the basic yCBS catalytic core (yCBS-cc). Here we present the crystal structure of yCBS-cc, solved to 1.5 Å. Crystal structures of yCBS-cc in complex with enzymatic reaction intermediates have been captured, providing a structural basis for residues involved in catalysis. Finally, the structure of the yCBS-cc cofactor complex generated by incubation with an inhibitor shows apparent off-pathway chemistry not normally seen with CBS.