RESUMO
Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in vitro and in vivo serotonin detection, respectively.
Assuntos
Evolução Molecular Direcionada , Aprendizado de Máquina , Serotonina/metabolismo , Algoritmos , Sequência de Aminoácidos , Tonsila do Cerebelo/fisiologia , Animais , Comportamento Animal , Sítios de Ligação , Encéfalo/metabolismo , Células HEK293 , Humanos , Cinética , Modelos Lineares , Camundongos , Camundongos Endogâmicos C57BL , Fótons , Ligação Proteica , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Sono/fisiologia , Vigília/fisiologiaRESUMO
Bacterial phytases have attracted industrial interest as animal feed supplement due to their high activity and sufficient thermostability (required for feed pelleting). We devised an approach named KeySIDE, an iterative Key-residues interrogation of the wild type with Substitutions Identified in Directed Evolution for improving Yersinia mollaretii phytase (Ymphytase) thermostability by combining key beneficial substitutions and elucidating their individual roles. Directed evolution yielded in a discovery of nine positions in Ymphytase and combined iteratively to identify key positions. The "best" combination (M6: T77K, Q154H, G187S, and K289Q) resulted in significantly improved thermal resistance; the residual activity improved from 35 % (wild type) to 89 % (M6) at 58 °C and 20-min incubation. Melting temperature increased by 3 °C in M6 without a loss of specific activity. Molecular dynamics simulation studies revealed reduced flexibility in the loops located next to helices (B, F, and K) which possess substitutions (Helix-B: T77K, Helix-F: G187S, and Helix-K: K289E/Q). Reduced flexibility in the loops might be caused by strengthened hydrogen bonding network (e.g., G187S and K289E/K289Q) and a salt bridge (T77K). Our results demonstrate a promising approach to design phytases in food research, and we hope that the KeySIDE might become an attractive approach for understanding of structure-function relationships of enzymes.
Assuntos
6-Fitase/genética , 6-Fitase/metabolismo , Evolução Molecular Direcionada/métodos , Engenharia de Proteínas/métodos , Yersinia/enzimologia , Yersinia/genética , 6-Fitase/química , Substituição de Aminoácidos , Estabilidade Enzimática , Simulação de Dinâmica Molecular , TemperaturaRESUMO
Baeyer-Villiger monooxygenase-catalysed reactions are attractive for industrial processes. Here we report on expanding the substrate scope of phenylacetone monooxygenase (PAMO). In order to introduce activity on alicyclic ketones in PAMO, we generated and screened a library of 1,500 mutants. Based on recently published structures of PAMO and its mutants, we selected previously uncharacterised positions as well as known hot-spots to be targeted by focused mutagenesis. We were able to mutate 11 positions in a single step by using the OmniChange method for the mutant library generation. Screening of the library using a phosphate-based activity detection method allowed identification of a quadruple mutant (P253F/G254A/R258M/L443F) active on cyclopentanone. The substrate scope of this mutant is extended to several aliphatic ketones while activity on aromatic compounds typical for PAMO was preserved. Moreover, the mutant is as thermostable as PAMO. Our results demonstrate the power of screening structure-inspired, focused mutant libraries for creating Baeyer-Villiger monooxygenases with new specificities.
Assuntos
Cetonas/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Mutagênese , Acetona/análogos & derivados , Acetona/metabolismo , Testes Genéticos , Especificidade por SubstratoRESUMO
Phytase improves as a feed supplement the nutritional quality of phytate-rich diets (e.g., cereal grains, legumes, and oilseeds) by hydrolyzing indigestible phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) and increasing abdominal absorption of inorganic phosphates, minerals, and trace elements. Directed phytase evolution was reported for improving industrial relevant properties such as thermostability (pelleting process) or activity. In this study, we report the cloning, characterization, and directed evolution of the Yersinia mollaretii phytase (Ymphytase). Ymphytase has a tetrameric structure with positive cooperativity (Hill coefficient was 2.3) and a specific activity of 1,073 U/mg which is â¼10 times higher than widely used fungal phytases. High-throughput prescreening methods using filter papers or 384-well microtiter plates were developed. Precise subsequent screening for thermostable and active phytase variants was performed by combining absorbance and fluorescence-based detection system in 96-well microtiter plates. Directed evolution yielded after mutant library generation (SeSaM method) and two-step screening (in total â¼8,400 clones) a phytase variant with â¼20% improved thermostability (58°C for 20 min; residual activity wild type â¼34%; variant â¼53%) and increased melting temperature (1.5°C) with a slight loss of specific activity (993 U/mg).
Assuntos
6-Fitase/genética , 6-Fitase/metabolismo , Evolução Molecular Direcionada , Yersinia/enzimologia , 6-Fitase/química , Clonagem Molecular , Estabilidade Enzimática , Ensaios de Triagem em Larga Escala , Multimerização Proteica , TemperaturaRESUMO
Subcellular pharmacokinetic measurements have informed the study of central nervous system (CNS)-acting drug mechanisms. Recent investigations have been enhanced by the use of genetically encoded fluorescent biosensors for drugs of interest at the plasma membrane and in organelles. We describe screening and validation protocols for identifying hit pairs comprising a drug and biosensor, with each screen including 13-18 candidate biosensors and 44-84 candidate drugs. After a favorable hit pair is identified and validated via these protocols, the biosensor is then optimized, as described in other papers, for sensitivity and selectivity to the drug. We also show sample hit pair data that may lead to future intensity-based drug-sensing fluorescent reporters (iDrugSnFRs). These protocols will assist scientists to use fluorescence responses as criteria in identifying favorable fluorescent biosensor variants for CNS-acting drugs that presently have no corresponding biosensor partner. This protocol was validated in: eLife (2022), DOI: 10.7554/eLife.74648 Graphical abstract.
RESUMO
Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug-sensing fluorescent reporters (iDrugSnFRs) for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives - 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by >30-fold. The new nicotinic iDrugSnFRs provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.
Assuntos
Alcaloides/química , Azepinas/química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Agonistas Nicotínicos/química , Abandono do Hábito de Fumar , Alcaloides/metabolismo , Animais , Azocinas/química , Azocinas/metabolismo , Fluorescência , Humanos , Ligantes , Camundongos , Quinolizinas/química , Quinolizinas/metabolismoRESUMO
The infectious disease burden imposed by trypanosomatidae family continues to create burden in countries that are least equipped to bring new medicines to the clinic. For sickness caused by this family of parasites (African trypanosomiasis, Chagas disease, and leishmaniasis) no vaccines are available, and currently available drugs suffer from insufficient efficacy, excessive toxicity, and steady loss of effectiveness due to resistance. Availability of the genome sequence of pathogens of this family offers a unique avenue for the identification of novel common drug targets for all three pathogens. Sirtuin family of nicotinamide adenine dinucleotide (NAD)-dependent deacetylases are remarkably conserved throughout evolution from archaebacteria to eukaryotes and plays an important role in trypanosomatidae biology and virulence. In order to gain insight for selective drug design, three-dimensional (3D) models of L. major, L. infantum, T. brucie, and T. cruzi sirtuin were constructed by homology modeling and compared with human sirtuin. The molecular electrostatic potentials and cavity depth analysis of these models suggest that the inhibitor binding catalytic domain has various minor structural differences in the active site of trypanosomal and human sirtuin, regardless of sequence similarity. These studies have implications for designing effective strategies to identify inhibitors that can be developed as novel broad-spectrum antitrypanosomal drugs.
Assuntos
Desenho de Fármacos , Sirtuína 2/química , Trypanosomatina/efeitos dos fármacos , Trypanosomatina/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Simulação por Computador , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Sirtuína 1/química , Sirtuína 1/genética , Sirtuína 2/antagonistas & inibidores , Sirtuína 2/genética , Especificidade da Espécie , Eletricidade Estática , Trypanosomatina/genéticaRESUMO
The tandem BRCT domains (tBRCT) of BRCA1 engage phosphoserine-containing motifs in target proteins to propagate intracellular signals initiated by DNA damage, thereby controlling cell cycle arrest and DNA repair. Recently, we identified Bractoppin, the first small-molecule inhibitor of the BRCA1 tBRCT domain, which selectively interrupts BRCA1-mediated cellular responses evoked by DNA damage. Here, we combine structure-guided chemical elaboration, protein mutagenesis and cellular assays to define the structural features responsible for Bractoppin's activity. Bractoppin fails to bind mutant forms of BRCA1 tBRCT bearing K1702A, a key residue mediating phosphopeptide recognition, or F1662R or L1701K that adjoin the pSer-recognition site. However, the M1775R mutation, which engages the Phe residue in the consensus phosphopeptide motif pSer-X-X-Phe, does not affect Bractoppin binding, confirming a binding mode distinct from the substrate phosphopeptide binding. We explored these structural features through structure-guided chemical elaboration and characterized structure-activity relationships (SARs) in biochemical assays. Two analogues, CCBT2088 and CCBT2103 were effective in abrogating BRCA1 foci formation and inhibiting G2 arrest induced by irradiation of cells. Collectively, our findings reveal structural features underlying the activity of a novel inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domain, providing fresh insights to guide the development of inhibitors that target protein-protein interactions.
Assuntos
Proteína BRCA1/antagonistas & inibidores , Imidazóis/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína BRCA1/isolamento & purificação , Proteína BRCA1/metabolismo , Relação Dose-Resposta a Droga , Imidazóis/síntese química , Imidazóis/química , Modelos Moleculares , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
The target for the "rapid" (<24 h) antidepressant effects of S-ketamine is unknown, vitiating programs to rationally develop more effective rapid antidepressants. To describe a drug's target, one must first understand the compartments entered by the drug, at all levels-the organ, the cell, and the organelle. We have, therefore, developed molecular tools to measure the subcellular, organellar pharmacokinetics of S-ketamine. The tools are genetically encoded intensity-based S-ketamine-sensing fluorescent reporters, iSKetSnFR1 and iSKetSnFR2. In solution, these biosensors respond to S-ketamine with a sensitivity, S-slope = delta(F/F0)/(delta[S-ketamine]) of 0.23 and 1.9/µM, respectively. The iSKetSnFR2 construct allows measurements at <0.3 µM S-ketamine. The iSKetSnFR1 and iSKetSnFR2 biosensors display >100-fold selectivity over other ligands tested, including R-ketamine. We targeted each of the sensors to either the plasma membrane (PM) or the endoplasmic reticulum (ER). Measurements on these biosensors expressed in Neuro2a cells and in human dopaminergic neurons differentiated from induced pluripotent stem cells (iPSCs) show that S-ketamine enters the ER within a few seconds after appearing in the external solution near the PM, then leaves as rapidly after S-ketamine is removed from the extracellular solution. In cells, S-slopes for the ER and PM-targeted sensors differ by <2-fold, indicating that the ER [S-ketamine] is less than 2-fold different from the extracellular [S-ketamine]. Organelles represent potential compartments for the engagement of S-ketamine with its antidepressant target, and potential S-ketamine targets include organellar ion channels, receptors, and transporters.
RESUMO
Nicotine dependence is thought to arise in part because nicotine permeates into the endoplasmic reticulum (ER), where it binds to nicotinic receptors (nAChRs) and begins an "inside-out" pathway that leads to up-regulation of nAChRs on the plasma membrane. However, the dynamics of nicotine entry into the ER are unquantified. Here, we develop a family of genetically encoded fluorescent biosensors for nicotine, termed iNicSnFRs. The iNicSnFRs are fusions between two proteins: a circularly permutated GFP and a periplasmic choline-/betaine-binding protein engineered to bind nicotine. The biosensors iNicSnFR3a and iNicSnFR3b respond to nicotine by increasing fluorescence at [nicotine] <1 µM, the concentration in the plasma and cerebrospinal fluid of a smoker. We target iNicSnFR3 biosensors either to the plasma membrane or to the ER and measure nicotine kinetics in HeLa, SH-SY5Y, N2a, and HEK293 cell lines, as well as mouse hippocampal neurons and human stem cell-derived dopaminergic neurons. In all cell types, we find that nicotine equilibrates in the ER within 10 s (possibly within 1 s) of extracellular application and leaves as rapidly after removal from the extracellular solution. The [nicotine] in the ER is within twofold of the extracellular value. We use these data to run combined pharmacokinetic and pharmacodynamic simulations of human smoking. In the ER, the inside-out pathway begins when nicotine becomes a stabilizing pharmacological chaperone for some nAChR subtypes, even at concentrations as low as â¼10 nM. Such concentrations would persist during the 12 h of a typical smoker's day, continually activating the inside-out pathway by >75%. Reducing nicotine intake by 10-fold decreases activation to â¼20%. iNicSnFR3a and iNicSnFR3b also sense the smoking cessation drug varenicline, revealing that varenicline also permeates into the ER within seconds. Our iNicSnFRs enable optical subcellular pharmacokinetics for nicotine and varenicline during an early event in the inside-out pathway.
Assuntos
Retículo Endoplasmático/metabolismo , Nicotina/farmacocinética , Animais , Técnicas Biossensoriais/métodos , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Feminino , Células HEK293 , Células HeLa , Hipocampo/metabolismo , Humanos , Mamíferos , Camundongos , Neurônios/metabolismo , Gravidez , Transporte Proteico/fisiologia , Receptores Nicotínicos/metabolismo , Fumar/metabolismo , Vareniclina/farmacocinéticaRESUMO
Plasmodium falciparum sirtuin, PfSir2, contains histone deacetylase (HDAC) activity that may be central to the regulation of virulence gene expression in the parasites. Although a few reports have been published recently regarding in vitro and in vivo function of PfSir2, expression of the endogenous protein (c. 30 kDa) has not been shown yet. Here we report the presence of PfSir2 in the parasite at the protein level by specific antibodies. HDAC activity of PfSir2 can be inhibited by nicotinamide, a product of sirtuin reaction. Surprisingly, we find that nicotinamide also delays parasite growth significantly in culture. These findings further our knowledge on PfSir2 and raise the possibility of using an inexpensive agent like nicotinamide as an antimalarial in combination with other antiparasitic drugs.
Assuntos
Antimaláricos , Inibidores de Histona Desacetilases , Niacinamida/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Sirtuínas/metabolismo , Animais , Anticorpos Antiprotozoários , DNA de Protozoário , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimentoRESUMO
Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: (i) identification of conserved and highly variable regions; (ii) protein sequence design by substituting residues in the highly variable regions, and gene synthesis; (iii) in vitro DNA recombination of synthetic genes; and (iv) screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the technique's utility for directed evolution, the surface of a phytase enzyme from Yersinia mollaretii (Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering-guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzyme's specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.
Assuntos
Evolução Molecular Direcionada/métodos , Engenharia de Proteínas/métodos , 6-Fitase/química , 6-Fitase/genética , 6-Fitase/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Alinhamento de Sequência , Yersinia/enzimologia , Yersinia/genéticaRESUMO
Cyclic peptides are increasingly desired for their enhanced stability and pharmacologic properties. Due to their limited conformational flexibility, cyclic peptides with C-to-N-terminal peptide bond and a disulfide bridge can confer high target binding affinity and resistance to proteolytic enzymes. Challenging drug targets including protein interaction surfaces can be successfully targeted using peptides rather than small molecules or proteins. Peptides, capable of antibody-like affinities with increased potency, can be designed to fill in the gap between small molecules and larger proteins. However, cysteine-rich peptides with several disulfide bonds have limitations in production and purification. Therefore, we devised a strategy to identify cyclic peptides with single disulfide connectivity that offers desired properties along with ease in synthesis and production. Here, de novo design of cyclic peptides is demonstrated through screening of peptide libraries using bacterial display and cell sorting. Herein, a step-by-step protocol is presented to design and screen diverse peptide libraries to identify cyclic peptides with desired specificity and affinity towards arbitrary target proteins.
Assuntos
Bactérias , Dissulfetos , Citometria de Fluxo , Biblioteca de Peptídeos , Peptídeos Cíclicos , Animais , Bactérias/genética , Bactérias/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Humanos , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/química , Peptídeos Cíclicos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Directed evolution of Yersinia mollaretii phytase (Ymphytase) yielded an improved variant SM2P3E4 (also named M1; D52N, T77K, K139E, G187S, V298M) in our previous study. Variant M1 retained high specific activity (993U/mg; equivalent to 93% of wild-type activity) and improved thermal resistance (T50 improved by 1.5°C compared to wild-type at 58°C; 20min incubation time), making variant M1 an attractive enzyme for industrial applications. Recently, the OmniChange method was developed for multi-site saturation mutagenesis. The five sites identified in variant M1 were subjected to OmniChange saturation in order to explore whether a variant with higher activity, higher thermal resistance, and higher resistance at low pH (2-3h incubation was performed to mimic the gastric residence time of phytase) could be identified. Screening of a small library of 1100 clones, covering <0.004% of the theoretical sequence space of 3.35×10(7) variants, yielded a Ymphytase variant with 32% improved residual activity (58°C for 20min), 2°C increased apparent melting temperature (Tm), and 2-fold higher pH stability (pH 2.8; 3h incubation time) when compared to the wild-type Ymphytase. Compared to the M1 variant, the pH stability (pH 2.8; 3h incubation time) was improved by 3-fold, and thermal resistance as well as activity was improved slightly (residual activity: 32% compared to 20%; apparent Tm: 2°C compared to 1.5°C; activity difference <4%).
Assuntos
6-Fitase/metabolismo , Mutagênese Sítio-Dirigida , Yersinia/enzimologia , 6-Fitase/genética , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Variação Genética , Mutagênese , Especificidade por Substrato , TemperaturaRESUMO
Fusion protein construction is a widely employed biochemical technique, especially when it comes to multi-component enzymes such as cytochrome P450s. Here we describe a novel method for generating fusion proteins with variable linker lengths, protein fusion with variable linker insertion (P-LinK), which was validated by fusing P450cin monooxygenase (CinA) to the flavodoxin shuttle protein (CinC). CinC was fused to the C terminus of CinA through a series of 16 amino acid linkers of different lengths in a single experiment employing 3 PCR amplifications. Screening for 2-ß-hydroxy-1,8-cineole production by CinA-CinC fusion proteins revealed that enzymatically active variants possessed linker lengths of more than 5 amino acids, reaching optimum enzyme activity at a linker length of 10 amino acids. Our P-LinK method not only minimizes experimental effort and significantly reduces time demands but also requires only a single cloning and transformation step in order to generate multiple linker variants (1 to 16 amino acids long), making the approach technically simple and robust.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos , Sistema Enzimático do Citocromo P-450/metabolismo , Escherichia coli/genética , Biblioteca Gênica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The carbonyl reductase from Candida parapsilosis (CPCR2) is a versatile biocatalyst for the production of optically pure alcohols from ketones. Prochiral ketones like 2-methyl cyclohexanone are, however, only poorly accepted, despite CPCR2's large substrate spectrum. The substrate spectrum of CPCR2 was investigated by selecting five amino positions (55, 92, 118, 119 and 262) and exploring them by single site-saturation mutagenesis. Screening of CPCR2 libraries with poor (14 compounds) and well-accepted (2 compounds) substrates showed that only position 55 and position 119 showed an influence on activity. Saturation of positions 92, 118 and 262 yielded only wild-type sequences for the two well-accepted substrates and no variant converted one of the 14 other compounds. Only the variant (L119M) showed a significantly improved activity (7-fold on 2-methyl cyclohexanone; vmax = 33.6 U/mg, Km = 9.7 mmol/l). The L119M substitution exhibited also significantly increased activity toward reduction of 3-methyl (>2-fold), 4-methyl (>5-fold) and non-substituted cyclohexanone (>4-fold). After docking 2-methyl cyclohexanone into the substrate-binding pocket of a CPCR2 homology model, we hypothesized that the flexible side chain of M119 provides more space for 2-methyl cyclohexanone than branched L119. This report represents the first study on CPCR2 engineering and provides first insights how to redesign CPCR2 toward a broadened substrate spectrum.
Assuntos
Oxirredutases do Álcool/química , Candida/enzimologia , Conformação Proteica , Engenharia de Proteínas , Oxirredutases do Álcool/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Cicloexanonas/química , Humanos , Mutagênese Sítio-Dirigida , Estereoisomerismo , Especificidade por SubstratoRESUMO
The carbonyl reductase from Candida parapsilosis (CPCR2) is an industrially attractive biocatalyst for producing chiral alcohols from ketones. The homodimeric enzyme has a broad substrate spectrum and an excellent stereoselectivity, but is rapidly inactivated at aqueous-organic interfaces. The latter limits CPCR2's application in biphasic reaction media. Reengineering the protein surface of CPCR2 yielded a variant CPCR2-(A275N, L276Q) with 1.5-fold increased activity, 1.5-fold higher interfacial stability (cyclohexane/buffer system), and increased thermal resistance (ΔT50=+2.7 °C). Site-directed and site-saturation mutagenesis studies discovered that position 275 mainly influences stability and position 276 governs activity. After single site-saturation of position 275, amino acid exchanges to asparagine and threonine were discovered to be stabilizing. Interestingly, both positions are located at the dimer interface and close to the active site and computational analysis identified an inter-subunit hydrogen bond formation at position 275 to be responsible for stabilization. Finally, the variant CPCR2-(A275S, L276Q) was found by simultaneous site-saturation of positions 275 and 276. CPCR2-(A275S, L276Q) has compared to wtCPCR2 a 1.4-fold increased activity, a 1.5-fold higher interfacial stability, and improved thermal resistance (ΔT50=+5.2 °C).
Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Álcoois/metabolismo , Candida/enzimologia , Oxirredutases do Álcool/isolamento & purificação , Álcoois/química , Candida/química , Estabilidade Enzimática/genética , Fermentação , Humanos , Cetonas/química , Cetonas/metabolismo , Engenharia Metabólica , Mutagênese Sítio-DirigidaRESUMO
Focused mutant library generation methods have been developed to improve mainly "localizable" enzyme properties such as activity and selectivity. Current multi-site saturation methods are restricted by the gene sequence, require subsequent PCR steps and/or additional enzymatic modifications. Here we report, a multiple site saturation mutagenesis method, OmniChange, which simultaneously and efficiently saturates five independent codons. As proof of principle, five chemically cleaved DNA fragments, each carrying one NNK-degenerated codon, were generated and assembled to full gene length in a one-pot-reaction without additional PCR-amplification or use of restriction enzymes or ligases. Sequencing revealed the presence of up to 27 different codons at individual positions, corresponding to 84.4% of the theoretical diversity offered by NNK-degeneration. OmniChange is absolutely sequence independent, does not require a minimal distance between mutated codons and can be accomplished within a day.
Assuntos
Códon , Sequência de Bases , Mutagênese , Reação em Cadeia da PolimeraseRESUMO
Phytases catalyze the release of phosphate by stepwise hydrolysis of phytate, a major source of phosphate in cereal grains, legumes, and oilseeds. Phytase improves, as a feed supplement, the nutritional quality of phytate rich diets and eventually reduce environmental pollution. Recently, phytases from enterobacteriaceae family have attracted industrial interest due to their high specific activity (2500-4000 U/mg). However, only limited information is available concerning structural dynamics of this class of enzymes. In this study, 50 nanosecond molecular dynamics simulation was performed on two Escherichia coli phytase structures (closed and open active site loop) to investigate conformational dynamics of the active site loop. Cluster analysis and principal component analysis (PCA) reveal significant difference in the conformational dynamics of active site compared to reported crystal structure. Molecular dynamic studies indicated that the movement in the active site of E. coli phytase is mainly confined by the active site loop resulted in wider opening of the loop in absence of phytate. The molecular dynamics studies highlight the possible role of loop residues as prerequisite for highly active phytases.