RESUMO
Elucidating the underlying photochemical mechanisms of action (MoA) of photodynamic therapy (PDT) may allow its efficacy to be improved and could set the stage for the development of new classes of PDT photosensitizers. Here, we provide evidence that "photoredox catalysis in cells," wherein key electron transport pathways are disrupted, could constitute a general MoA associated with PDT. Taking the cellular electron donor nicotinamide adenine dinucleotide as an example, we have found that well-known photosensitizers, such as Rose Bengal, BODIPY, phenoselenazinium, phthalocyanine, and porphyrin derivatives, are able to catalyze its conversion to NAD+. This MoA stands in contrast to conventional type I and type II photoactivation mechanisms involving electron and energy transfer, respectively. A newly designed molecular targeting photocatalyst (termed CatER) was designed to test the utility of this mechanism-based approach to photosensitizer development. Photoexcitation of CatER induces cell pyroptosis via the caspase 3/GSDME pathway. Specific epidermal growth factor receptor positive cancer cell recognition, high signal-to-background ratio tumor imaging (SBRTI = 12.2), and good tumor growth inhibition (TGI = 77.1%) are all hallmarks of CatER. CatER thus constitutes an effective near-infrared pyroptotic cell death photo-inducer. We believe the present results will provide the foundation for the synthesis of yet-improved phototherapeutic agents that incorporate photocatalytic chemistry into their molecular design.
Assuntos
Antineoplásicos , Neoplasias , Fotoquimioterapia , Fármacos Fotossensibilizantes , Antineoplásicos/farmacologia , Catálise , Linhagem Celular Tumoral , Humanos , Neoplasias/diagnóstico por imagem , Neoplasias/terapia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacologiaRESUMO
Cancer stem cells (CSCs) are associated with the invasion and metastatic relapse of various cancers. However, current cancer therapies are limited to targeting the bulk of primary tumor cells while remaining the CSCs untouched. Here, we report a new proton (H+) modulation approach to selectively eradicate CSCs via cutting off the H+ leaks on the inner mitochondrial membrane (IMM). Based on the fruit extract of Gardenia jasminoides, a multimodal molecule channel blocker with high biosafety, namely, Bo-Mt-Ge, is developed. Importantly, in this study, we successfully identify that mitochondrial uncoupling protein UCP2 is closely correlated with the stemness of CSCs, which may offer a new perspective for selective CSC drug discovery. Mechanistic studies show that Bo-Mt-Ge can specifically inhibit the UCP2 activities, decrease the H+ influx in the matrix, regulate the electrochemical gradient, and deplete the endogenous GSH, which synergistically constitute a unique MoA to active apoptotic CSC death. Intriguingly, Bo-Mt-Ge also counteracts the therapeutic resistance via a two-pronged tactic: drug efflux pump P-glycoprotein downregulation and antiapoptotic factor (e.g., Bcl-2) inhibition. With these merits, Bo-Mt-Ge proved to be one of the safest and most efficacious anti-CSC agents, with ca. 100-fold more potent than genipin alone in vitro and in vivo. This study offers new insights and promising solutions for future CSC therapies in the clinic.
Assuntos
Membranas Mitocondriais , Neoplasias , Humanos , Membranas Mitocondriais/metabolismo , Prótons , Neoplasias/patologia , Células-Tronco Neoplásicas/metabolismoRESUMO
O-Methylation catalyzed by O-methyltransferases (OMTs) is an important modification of flavonoids for improving the transport efficiency across membranes and metabolic stability in mammalian cells. Chrysoeriol, also known as 3'-O-methylated luteolin, is a methylated flavonoid compound with health-promoting activities. The generation of chrysoeriol from luteolin can be catalyzed by a rice-derived 3'-OMT named ROMT-9, which has a high regiospecificity and activity toward flavonoids in vitro. Herein, we explored the potential of ROMT-9 for in vivo biosynthesis of chrysoeriol in Escherichia coli and adopted semi-rational enzyme engineering guided by homology modeling and molecular docking to improve the bio-production. Two positive variants including L34Q and W284A were obtained which promoted chrysoeriol formation to more than 85 mg/L from 200 mg/L of luteolin in 24 h compared with a titer of 55 mg/L for the strain expressing the native enzyme. Further biochemical analysis confirmed that such improvement in production stemmed from a higher enzyme expression level for the L34Q variant and higher efficiency in substrate binding and catalysis for the W284A variant. This study provides some insights into the engineering of other flavonoid OMTs and will facilitate high-level biosynthesis of methylated flavonoids in engineered microorganisms. KEY POINTS: ⢠Biosynthesis of chrysoeriol from luteolin in E. coli using ROMT-9 ⢠Engineering of ROMT-9 for better bio-production ⢠ROMT-9 variants promote production via better expression or better catalysis.
Assuntos
Flavonoides , Metiltransferases , Animais , Flavonoides/metabolismo , Metiltransferases/metabolismo , Escherichia coli/metabolismo , Luteolina/metabolismo , Simulação de Acoplamento Molecular , Mamíferos/metabolismoRESUMO
Engineering Taq DNA polymerase (TaqPol) for improved activity, stability and sensitivity was critical for its wide applications. Multiple sequence alignment (MSA) has been widely used in engineering enzymes for improved properties. Here, we first designed TaqPol mutations based on MSA of 2756 sequences from both thermophilic and non-thermophilic organisms. Two double mutations were generated including a variant H676F/R677G showing a decrease in both activity and stability, and a variant Y686R/E687K showing an improved activity, but a decreased stability. Mutations targeted on coevolutionary residues of Arg677 and Tyr686 were then applied to rescue stability or activity loss of the double mutants, which achieved a partial success. Sequence analysis revealed that the two mutations are abundant in non-thermophilic sequences but not in thermophilic homologues. Then, a small-scale MSA containing sequences from only thermophilic organisms was applied to predict 13 single variants and two of them, E507Q and E734N showed a simultaneous increase in both stability and activity, even in sensitivity. A customized MSA was hence more effective in engineering a thermophilic enzyme and could be used in engineering other enzymes. Molecular dynamics simulations revealed the impact of mutations on the protein dynamics and interactions between TaqPol and substrates. KEY POINTS: ⢠The pool of sequence for alignment is critical to engineering Taq DNA polymerase. ⢠The variants with low properties can be rescued by mutations in coevolving network. ⢠Improving binding with DNA can improve DNA polymerase stability and activity.
RESUMO
l-threonine aldolase (LTA) catalyzes C-C bond synthesis with moderate diastereoselectivity. In this study, with LTA from Cellulosilyticum sp (CpLTA) as an object, a mutability landscape was first constructed by performing saturation mutagenesis at substrate access tunnel amino acids. The combinatorial active-site saturation test/iterative saturation mutation (CAST/ISM) strategy was then used to tune diastereoselectivity. As a result, the diastereoselectivity of mutant H305L/Y8H/V143R was improved from 37.2 %syn to 99.4 %syn . Furthermore, the diastereoselectivity of mutant H305Y/Y8I/W307E was inverted to 97.2 %anti . Based on insight provided by molecular dynamics simulations and coevolution analysis, the Prelog rule was employed to illustrate the diastereoselectivity regulation mechanism of LTA, holding that the asymmetric formation of the C-C bond was caused by electrons attacking the carbonyl carbon atom of the substrate aldehyde from the re or si face. The study would be useful to expand LTA applications and guide engineering of other C-C bond-forming enzymes.
Assuntos
Aminoácidos , Glicina Hidroximetiltransferase , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Mutação , Mutagênese , Aminoácidos/química , Domínio Catalítico , Especificidade por SubstratoRESUMO
Pyroptosis, a newly characterized form of immunogenic cell death, is attracting increasing attention as a promising approach to cancer immunotherapy. However, biocompatible strategies to activate pyroptosis remain rare. Here, we show that a photocatalytic superoxide radical (O2-â¢) generator, NI-TA, triggers pyroptosis in cancer cells. NI-TA was designed to take advantage of an intramolecular triplet-ground state splitting energy modulation approach. Detailed studies revealed that the pyroptosis triggered by NI-TA under conditions of photoexcitation proceeds through a caspase-3/gasdermin E (GSDME) pathway rather than via canonical processes involving caspase-1/gasdermin-D (GSDMD). NI-TA was found to function via a partial-O2-recycling mode of action and to trigger cell pyroptosis and provide for effective cancer cell ablation even under conditions of hypoxia (≤2% O2). In the case of T47D 3D multicellular spheroids, good antitumor efficiency and stemness inhibition are achieved. This work highlights how photocatalytic chemistry may be leveraged to develop effective pyroptosis-inducing agents.
Assuntos
Neoplasias , Piroptose , Caspase 1/metabolismo , Caspase 1/farmacologia , SuperóxidosRESUMO
The transformational effect of photoredox catalytic chemistries has inspired new opportunities, enabling us to interrogate nature in ways that are not possible otherwise and to unveil new biotechnologies in therapy and diagnosis. However, the deployment of artificial photoredox catalysis in living systems remains challenging, mired by the off-target risk and safety concerns of photocatalyst toxicity. Here, we present an appealing approach, namely conditionally activatable photoredox catalysis (ConAPC), and as a proof of concept design the first ConAPC architecture (Se-NO2) based upon classic self-immolative chemistry, in which the inherent photocatalytic properties can be temporarily caged while the species becomes active only at the tumor sites via sensing to specific biomarkers. Such a masking strategy allows a spatial-temporal control of photoresponsivity in vitro and in vivo. In particular, for ConAPC design, a new biologically benign metal-free photocatalyst (Se-NH2), which is able to initiate NIR photoredox catalysis to manipulate the cellular electron pool in an O2-independent mechanism of action, is identified. With this unique strategy, potent tumor-specific targeting photocatalytic eradication (TGI: 95%) is obtained in a mouse model. Impressively, favorable features such as high-resolution tumor recognition (SBR: 33.6) and excellent biocompatibility and safety are also achieved. This work therefore offers a new possibility for chemists to leverage artificial photocatalytic reactions toward the development of facile and intelligent photocatalytic theranostics.
Assuntos
Processos FotoquímicosRESUMO
Bacillus coagulans, a thermophilic facultative anaerobe, is a favorable chassis strain for the biosynthesis of desired products. In this study, B. coagulans was converted into an efficient malic acid producer by metabolic engineering and promoter engineering. Promoter mapping revealed that the endogenous promoter Pldh was a tandem promoter. Accordingly, a promoter library was developed, covering a wide range of relative transcription efficiencies with small increments. A reductive tricarboxylic acid pathway was established in B. coagulans by introducing the genes encoding pyruvate carboxylase (pyc), malate dehydrogenase (mdh), and phosphoenolpyruvate carboxykinase (pckA). Five promoters of various strengths within the library were screened to fine-tune the expression of pyc to improve the biosynthesis of malic acid. In addition, genes involved in the competitive metabolic pathways were deleted to focus the substrate and energy flux toward malic acid. Dual-phase fed-batch fermentation was performed to increase the biomass of the strain, further improving the titer of malic acid to 25.5 g/L, with a conversion rate of 0.3 g/g glucose. Our study is a pioneer research using promoter engineering and genetically modified B. coagulans for the biosynthesis of malic acid, providing an effective approach for the industrialized production of desired products using B. coagulans.
Assuntos
Bacillus coagulans , Proteínas de Bactérias , Ciclo do Ácido Cítrico , Malatos/metabolismo , Engenharia Metabólica , Regiões Promotoras Genéticas , Bacillus coagulans/genética , Bacillus coagulans/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genéticaRESUMO
κ-Carrageenase (EC3.2.1.83) is a class of glycoside hydrolase, which can be used for hydrolysis of κ-carrageenan to κ-carrageenan oligosaccharides. In this study, a bacterium, identified as Pseudoalteromonas sp. ZDY3 isolated from rotten algae, was capable to degrade κ-carrageenan. The κ-carrageenase produced by Pseudoalteromonas sp. ZDY3 was purified to homogeneity and named as CgkZDY3. The accurate molecular mass of CgkZDY3 was determined through LC-HRMS, and a posttranslational removal of C-terminal end of the protein was discovered. CgkZDY3 had strict hydrolysis specificity to κ-carrageenan, the values of Km and kcat/Km of CgkZDY3 were 3.67 mg mL-1 and 53.0 mL mg-1 s-1, respectively. CgkZDY3 was a cold-adapted κ-carrageenase with excellent storage stability of both the temperature below 35 °C and a wide pH range, and was an endo-type κ-carrageenase with high hydrolysis rate, oligosaccharides with different degrees of polymerization can be obtained by controlling the hydrolysis time, and the final products were κ-neocarrabiose and κ-neocarratetraose. These properties are of great significance for production of κ-carrageenan oligosaccharides with different polymerization degrees under process control.
Assuntos
Aclimatação , Proteínas de Bactérias , Pseudoalteromonas/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Temperatura Baixa , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificaçãoRESUMO
Tumor hypoxia has proven to be the major bottleneck of photodynamic therapy (PDT) to clinical transformation. Different from traditional O2 delivery approaches, here we describe an innovative binary photodynamic O2-economizer (PDOE) tactic to reverse hypoxia-driven resistance by designing a superoxide radical (O2â¢-) generator targeting mitochondria respiration, termed SORgenTAM. This PDOE system is able to block intracellular O2 consumption and down-regulate HIF-1α expression, which successfully rescues cancer cells from becoming hypoxic and relieves the intrinsic hypoxia burden of tumors in vivo, thereby sparing sufficient endogenous O2 for the PDT process. Photosensitization mechanism studies demonstrate that SORgenTAM has an ideal intersystem crossing rate and triplet excited state lifetime for generating O2â¢- through type-I photochemistry, and the generated O2â¢- can further trigger a biocascade to reduce the PDT's demand for O2 in an O2-recycble manner. Furthermore, SORgenTAM also serves to activate the AMPK metabolism signaling pathway to inhibit cell repair and promote cell death. Consequently, using this two-step O2-economical strategy, under relatively low light dose irradiation, excellent therapeutic responses toward hypoxic tumors are achieved. This study offers a conceptual while practical paradigm for overcoming the pitfalls of phototherapeutics.
Assuntos
Neoplasias/tratamento farmacológico , Fenotiazinas/uso terapêutico , Fármacos Fotossensibilizantes/uso terapêutico , Hipóxia Tumoral/efeitos dos fármacos , Animais , Respiração Celular/efeitos dos fármacos , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Luz , Células MCF-7 , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Fenotiazinas/síntese química , Fenotiazinas/efeitos da radiação , Fotoquimioterapia , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/efeitos da radiação , Superóxidos/metabolismoRESUMO
meso-Diaminopimelate dehydrogenase ( meso-DAPDH) is a good candidate for one-step synthesis of d-amino acid from 2-keto acids. Our previous research revealed the classification of meso-DAPDH family and showed that type II meso-DAPDH, such as the meso-DAPDH from Symbiobacterium thermophilum (StDAPDH), could catalyze reductive amination. In this article, seven residues of StDAPDH, which are highly conserved in each subfamily but are different between two subfamilies, were targeted to explore the relationships between structure and function. Determination of kinetic parameters showed that the amino acid residues, including P69, K159, V68, S90, V14, and V156, played very important roles in the catalytic function of StDAPDH. Molecular dynamics simulation revealed that these point mutations reduced the productive conformations by the newly formed or eliminated interactions between the residues and ligands. These results strengthen our understanding of the catalytic mechanism and evolution of meso-DAPDH and can aid future endeavors in enzyme engineering.
Assuntos
Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/metabolismo , Coenzimas/metabolismo , Sequência Conservada , Modelos Moleculares , Aminoácido Oxirredutases/genética , Sequência de Aminoácidos , Domínio Catalítico , Clostridiales/enzimologia , Cinética , Mutação , Especificidade por SubstratoRESUMO
In an effort to identify agents from natural products that inhibit protein tyrosine phosphatase 1B (PTP1B), 5 new prenylated stilbenes, (±)-styrastilbene A (1: ), styrastilbene B (2: ), and (±)-styrastilbenes Câ-âE (3, 4: , and 7: ), along with 4 known structurally related compounds (5, 6, 8: , and 9: ), were isolated from the roots of Artocarpus styracifolius. Their structures were elucidated by spectroscopic methods, including 1D and 2D nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass spectrometry (HRESIMS), ultraviolet (UV), and infrared (IR). Based on these isolates, a new plausible biosynthetic pathway for the unusual stilbene derivatives 3: -8: with a tetracyclic ring system is proposed. Among these compounds, 1: -3, 8: , and 9: displayed significant PTP1B inhibitory effects with IC50 values ranging from 2.40 (95% confidence interval [CI]: 2.21â-â2.59) to 8.80 (95% CI: 8.28â-â9.32) µM. Moreover, kinetic analysis and molecular docking simulations were performed to provide insight into the inhibition type as well as the interaction and binding mode of these active isolates with PTP1B. Our results revealed mixed-type PTP1B inhibition for all compounds tested. Docking simulations of these stilbene derivatives showed negative binding energies and close proximity to residues at the allosteric and catalytic sites of PTP1B. These findings suggest that these compounds may have a potential to be further developed as agents for the management of type 2 diabetes mellitus.
Assuntos
Artocarpus/química , Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular , Estrutura Molecular , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Estilbenos/farmacologia , Raízes de Plantas/química , Prenilação , Estilbenos/químicaRESUMO
N-acetyl-d-neuraminic acid (Neu5Ac) is a valuable resource that has seen increasing demand in both medicine and biotechnology. Although enzymatic systems and whole-cell biocatalysts have been developed for the synthesis of Neu5Ac, low yield and productivity still hamper the use of these methods on larger scales. We report the creation of an Escherichia coli biocatalyst for the efficient synthesis of Neu5Ac using a metabolic and protein engineering strategy. Expression of the two enzymes, N-acetyl-D-glucosamine 2-epimerase (AGE) and Neu5Ac lyase (NAL), was balanced using promoter engineering. Genes encoding competing pathways and GlcNAc catabolism were deleted, and then a structure-guided process was used to identify a more efficient NAL and an AGE mutant with a higher rate of Neu5Ac synthesis. The resulting biocatalyst produced 351.8â¯mM Neu5Ac with a yield of 58.6% from GlcNAc. This work exemplifies the use of rational design and protein engineering to construct a complex bacterial biocatalyst that can serve as a platform for the large-scale synthesis of a useful biological material.
Assuntos
Biocatálise , Escherichia coli , Microrganismos Geneticamente Modificados , Ácido N-Acetilneuramínico , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Ácido N-Acetilneuramínico/biossíntese , Ácido N-Acetilneuramínico/genética , Engenharia de Proteínas/métodosRESUMO
By screening 25 different psychrophilic strains isolated from the Arctic habitat, we isolated a strain capable of producing lipase. We identified this strain as Psychrobacter sp. ZY124 based on the amplified 16S rDNA sequence. The lipase, named as Lipase ZC12, produced from the supernatant of Psychrobacter sp. ZY124 cultured at 15 °C was purified to homogeneity by ammonium sulfate precipitation followed by Phenyl Sepharose FF gel hydrophobic chromatography. Based on the obtained amino acid sequence, Lipase ZC12 is classified as a member of the Proteus/psychrophilic subfamily of lipase family I.1; it has a molecular weight of 37.9 kDa. We also determined that the apparent optimum temperature for Lipase ZC12 activity is 40 °C. Lipase ZC12 shows remarkable organic solvent tolerance by remaining more 50% after incubated with 10-90% different organic solvents. In addition, acyl chain esters with C12 or longer were confirmed to be preferable substrates for Lipase ZC12. Lipase ZC12 also shows better stereoselectivity for (R, S)-1-phenylethanol chiral resolution in n-hexane solvent with (S)-1-phenylethanol (eep 92%) and conversion rate (39%) by transesterification reactions. These properties may provide potential applications in biocatalysis and biotransformation in non-aqueous media, such as in detergent, transesterification or esterification and chiral resolution.
Assuntos
Proteínas de Bactérias/metabolismo , Lipase/metabolismo , Psychrobacter/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Estabilidade Enzimática , Hexanos/química , Lipase/química , Lipase/genética , Psychrobacter/genética , Solventes/química , Especificidade por SubstratoRESUMO
Acetoin is an important physiological metabolite excreted by microbes. Its functions include avoiding acidification, participating in regulation of the NAD+/NADH ratio, and storing carbon. Acetolactate decarboxylase is a well-characterized anabolic enzyme involved with 3-hydroxy butanone (acetoin). It catalyzes conversion of the (R)- and (S)-enantiomers of acetolactate to generate the single product, (R)-acetoin. In addition to the X-ray crystal structure of acetolactate decarboxylase from Bacillus brevis, although the enzyme is widely present in microorganisms, very few atomic structures of acetolactate decarboxylase are reported. In this paper, we solved and reported a 1.5 Å resolution crystal structure of acetolactate decarboxylase from Bacillus subtilis. Dimeric assembly is observed in the solved structure, which is consistent with the elution profile conducted by molecular filtration. A zinc ion is coordinated by highly conserved histidines (191, 193, and 204) and conserved glutamates (62 and 251). We performed kinetic studies on acetolactate decarboxylase from Bacillus subtilis using circular dichroism, allowing the conversion of acetolactate to chiral acetoin for real-time tracking, yielding a Km value of 21 mM and a kcat value of 2.2 s-1. Using the two enantiomers of acetolactate as substrates, we further investigated the substrate preference of acetolactate decarboxylase from Bacillus subtilis by means of molecular docking and dynamic simulation in silico. The binding free energy of (S)-acetolactate was found to be ~ 30 kcal/mol greater than that of (R)-acetolactate, indicating a more stable binding for (S)-acetolactate.
Assuntos
Bacillus subtilis/enzimologia , Carboxiliases/química , Carboxiliases/metabolismo , Modelos Moleculares , Acetoína/metabolismo , Bacillus subtilis/genética , Cinética , Simulação de Acoplamento Molecular , Estrutura Terciária de ProteínaRESUMO
Leaf spot disease caused by the fungus Fusarium proliferatum (Matsushima) Nirenberg is a destructive disease of tomato plants in China. Typical symptoms of infected tomato plants are softened and wilted stems and leaves, leading to the eventual death of the entire plant. In this study, we resorted to transcriptional profile analysis to gain insight into the repertoire of effectors involved in F. proliferatum-tomato interactions. A total of 61,544,598 clean reads were de novo assembled to provide a F. proliferatum reference transcriptome. From these, 75,044 unigenes were obtained, with 19.46% of the unigenes being assigned to 276 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, with 22.3% having a homology with genes from F. fujikuroi. A total of 18,075 differentially expressed genes (DEGs) were identified, 720 of which were found to code for secreted proteins. Of these, 184 were identified as candidate effectors, while 79.89% had an upregulated expression. Moreover, 17 genes that were differentially expressed in RNA-seq studies were randomly selected for validation by quantitative real-time polymerase chain reaction (qRT-PCR). The study demonstrates that transcriptome analysis could be an effective method for identifying the repertoire of candidate effectors and may provide an invaluable resource for future functional analyses of F. proliferatum pathogenicity in F. proliferatum and tomato plant-host interactions.
Assuntos
Fusarium/genética , Doenças das Plantas/microbiologia , Solanum lycopersicum/microbiologia , Transcriptoma , Fusarium/patogenicidade , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Folhas de Planta/microbiologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The complete enzyme catalytic cycle includes substrate binding, chemical reaction and product release, in which different dynamic conformations are adopted. Due to the complex relationship among enzyme activity, stability and dynamics, the directed evolution of enzymes for improved activity or stability commonly leads to a trade-off in stability or activity. It hence remains a challenge to engineer an enzyme to have both enhanced activity and stability. Here, we have attempted to reconstruct the dynamics correlation network involved with active center to improve both activity and stability of a 2,3-butanediol dehydrogenase (2,3-BDH) by introducing inter-chain disulfide bonds. A computational strategy was first applied to evaluate the effect of introducing inter-chain disulfide bond on activity and stability of three 2,3-BDHs, and the N258C mutation of 2,3-BDH from Corynebacterium glutamicum (CgBDH) was proved to be effective in improving both activity and stability. In the results, CgBDH-N258C showed a different unfolding curve from the wild type, with two melting temperatures (Tm) of 68.3 °C and 50.8 °C, 19.7 °C and 2 °C higher than 48.6 °C of the wild type. Its half-life was also improved by 14.8-fold compared to the wild type. Catalytic efficiency (kcat/Km) of the mutant was increased by 7.9-fold toward native substrate diacetyl and 8.8-fold toward non-native substrate 2,5-hexanedione compared to the wild type. Molecular dynamics simulations revealed that an interaction network formed by Cys258, Arg162, Ala144 and the catalytic residues was reconstructed in the mutant and the dynamics change caused by the disulfide bond could be propagated through the interactions network. This improved the enzyme stability and activity by decreasing the flexibility and locking more "reactive" pose, respectively. Further construction of mutations including A144G showing a 44-fold improvement in catalytic efficiency toward meso-2,3-BD confirmed the role of modifying dynamics correlation network in tunning enzyme activity and selectivity. This study provided important insights into the relationship among dynamics, enzyme catalysis and stability, and will be useful in the designing new enzymes with co-evolution of stability, activity and selectivity.
Assuntos
Oxirredutases do Álcool , Corynebacterium glutamicum , Dissulfetos , Estabilidade Enzimática , Simulação de Dinâmica Molecular , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Dissulfetos/química , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , Mutação , Domínio Catalítico , Cinética , Conformação Proteica , Engenharia de Proteínas/métodosRESUMO
As one of rare high-value ocotillol (OCT)-type ginsenosides, pseudoginsenoside Rt5 has been identified with significant pharmacological activities. UDP-glycosyltransferases (UGTs) play pivotal roles in catalyzing the transfer of a glycosyl moiety from a donor to an acceptor. In this study, the novel UGT, PjUGT10, was screened from the transcriptome database of Panax japonicus and identified with the enzymatic activity of transferring a glucosyl group on OCT to produce Rt5. The catalytic efficiency of PjUGT10 was further enhanced by employing site-directed mutation. Notably, the variant M7 exhibited a remarkable 6.16 × 103-fold increase in kcat/Km towards 20S,24R-ocotillol and a significant 2.02 × 103-fold increase to UDP-glucose, respectively. Moreover, molecular dynamics simulations illustrated a reduced distance between 20S,24R-ocotillol and the catalytic residue His15 or UDP-glucose, favoring conformation interactions between the enzyme and substrates. Subsequently, Rt5 was synthesized in an engineered Escherichia coli strain M7 coupled with a UDP-glucose synthetic system. This study not only shed light on the protein engineering that can enhance the catalytic activity of PjUGT10, but also established a whole-cell approach for the production of Rt5.
RESUMO
The advent of generative artificial intelligence (AI) technologies marks a transformative moment for the scientific sphere, unlocking novel avenues to elevate scientific writing's efficiency and quality, expedite insight discovery, and enhance code development processes. Essential to leveraging these advancements is prompt engineering, a method that enhances AI interaction efficiency and quality. Despite its benefits, effective application requires blending researchers' expertise with AI, avoiding overreliance. A balanced strategy of integrating AI with independent critical thinking ensures the advancement and quality of scientific research, leveraging innovation while maintaining research integrity.
RESUMO
Optically pure 1,2,3,4-tetrahydroquinolines (THQs) represent a class of important motifs in many natural products and pharmaceutical agents. While recent advances on redox biocatalysis have demonstrated the great potential of amine oxidases, all the transformations focused on 2-substituted THQs. The corresponding biocatalytic method for the preparation of chiral 4-substituted THQs is still challenging due to the poor activity and stereoselectivity of the available enzyme. Herein, we developed a biocatalytic kinetic resolution approach for enantiodivergent synthesis of 4-phenyl- or alkyl-substituted THQs. Through structure-guided protein engineering of cyclohexylamine oxidase derived from Brevibacterium oxidans IH-35 A (CHAO), the variant of CHAO (Y215H/Y214S) displayed improved specific activity toward model substrate 4-phenyl substituted THQ (0.14 U/mg, 13-fold higher than wild-type CHAO) with superior (R)-stereoselectivity (E > 200). Molecular dynamics simulations show that CHAO Y215H/Y214S allows a suitable substrate positioning in the expanded binding pocket to be facilely accessed, enabling enhanced activity and stereoselectivity. Furthermore, a series of 4-alkyl-substituted THQs can be transformed by CHAO Y215H/Y214S, affording R-isomers with good yields (up to 50 %) and excellent enantioselectivity (up to ee > 99 %). Interestingly, the monoamine oxidase from Pseudomonas fluorescens Pf0-1 (PfMAO1) with opposite enantioselectivity was also mined. Together, this system enriches the kinetic resolution methods for the synthesis of chiral THQs.