Kinetic characterization of Cas9 enzymes.
Ontology highlight
ABSTRACT:
SUBMITTER: Liu MS
PROVIDER: S-EPMC8364395 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes. We used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach also enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The "Big Papi" (paired aureus and pyogenes for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.
| S-EPMC5800952 | biostudies-literature
Project description:Measuring the catalytic activity of immobilized enzymes underpins development of biosensing, bioprocessing, and analytical chemistry tools. To expand the range of approaches available for measuring enzymatic activity, we report on a technique to probe activity of enzymes immobilized in porous materials in the absence of confounding mass transport artifacts. We measured reaction kinetics of calf intestinal alkaline phosphatase (CIAP) immobilized in benzophenone-modified polyacrylamide (BPMA-PAAm) gel films housed in an array of fluidically isolated chambers. To ensure kinetics measurements are not confounded by mass transport limitations, we employed Weisz's modulus (?), which compares observed enzyme-catalyzed reaction rates to characteristic substrate diffusion times. We characterized activity of CIAP immobilized in BPMA-PAAm gels in a reaction-limited regime (? ? 0.15 for all measurements), allowing us to isolate the effect of immobilization on enzymatic activity. Immobilization of CIAP in BPMA-PAAm gels produced a ?2× loss in apparent enzyme-substrate affinity (Km) and ?200× decrease in intrinsic catalytic activity (kcat) relative to in-solution measurements. As estimating Km and kcat requires multiple steps of data manipulation, we developed a computational approach (bootstrapping) to propagate uncertainty in calibration data through all data manipulation steps. Numerical simulation revealed that calibration error is only negligible when the normalized root-mean-squared error (NRMSE) in the calibration falls below 0.05%. Importantly, bootstrapping is independent of the mathematical model, and thus generalizable beyond enzyme kinetics studies. Furthermore, the measurement tool presented can be readily adapted to study other porous immobilization supports, facilitating rational design (immobilization method, geometry, enzyme loading) of immobilized-enzyme devices.
| S-EPMC6275122 | biostudies-literature
Project description:Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9-guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family.
| S-EPMC4636735 | biostudies-literature
Project description:Patients with hereditary glutathione synthetase deficiency suffer from haemolytic anaemia, 5-oxoprolinuria, metabolic acidosis, recurrent bacterial infections and various degrees of central nervous system dysfunction. To investigate the molecular basis of the mutations associated with this disease, seven naturally occurring missense mutations [L188P (Leu188-->Pro), D219A, D219G, Y270C, Y270H, R283C and P314L] were expressed using a His-tagged, Escherichia coli-based expression system. Effects of the mutations on kinetic properties, including negative co-operative binding of gamma-glutamyl substrate, were evaluated. The mutation P314L did not have any major effect on these parameters and was classified as a neutral mutation. The remaining mutations decreased V(max) to 2-27% of wild-type activity. Negative co-operativity for gamma-gluABA (L-gamma-glutamyl-L-alpha-aminobutyric acid) was abolished in five mutant recombinant enzymes, whereas for one mutant enzyme, this co-operativity changed from negative to positive. The structural consequences of the mutations were interpreted on the basis of the known structure of the wild-type enzyme.
| S-EPMC1133856 | biostudies-literature
Project description:The spectroscopic and kinetic characterization of two intermediates from the H₂O₂ oxidation of three dimethyl ester [(proto), (meso), (deuteroporphyrinato) (picdien)]Fe(III) complexes ([FePPPic], [FeMPPic] and [FeDPPic], respectively) pinch-porphyrin peroxidase enzyme models, with s = 5/2 and 3/2 Fe(III) quantum mixed spin (qms) ground states is described herein. The kinetic study by UV/Vis at λmax = 465 nm showed two different types of kinetics during the oxidation process in the guaiacol test for peroxidases (1-3 + guaiacol + H₂O₂ → oxidation guaiacol products). The first intermediate was observed during the first 24 s of the reaction. When the reaction conditions were changed to higher concentration of pinch-porphyrins and hydrogen peroxide only one type of kinetics was observed. Next, the reaction was performed only between pinch-porphyrins-Fe(III) and H₂O₂, resulting in only two types of kinetics that were developed during the first 0-4 s. After this time a self-oxidation process was observed. Our hypotheses state that the formation of the π-cation radicals, reaction intermediates of the pinch-porphyrin-Fe(III) family with the ligand picdien [N,N'-bis-pyridin-2-ylmethyl-propane-1,3-diamine], occurred with unique kinetics that are different from the overall process and was involved in the oxidation pathway. UV-Vis, ¹H-NMR and ESR spectra confirmed the formation of such intermediates. The results in this paper highlight the link between different spectroscopic techniques that positively depict the kinetic traits of artificial compounds with enzyme-like activity.
| S-EPMC6273987 | biostudies-literature
Project description:Human mortalin is an Hsp70 chaperone that has been implicated in cancer, Alzheimer's and Parkinson's disease, and involvement has been suggested in cellular iron-sulfur cluster biosynthesis. However, study of this important human chaperone has been hampered by a lack of active material sufficient for biochemical characterization. Herein, we report the successful purification and characterization of recombinant human mortalin in Escherichia coli. The recombinant protein was expressed in the form of inclusion bodies and purified by Ni-NTA affinity chromatography. The subsequently refolded protein was confirmed to be active by its ATPase activity, a characteristic blue-shift in the fluorescence emission maximum following the addition of ATP, and its ability to bind to a likely physiological substrate. Single turnover kinetic experiments of mortalin were performed and compared with another Hsp70 chaperone, Thermotogamaritima DnaK; with each exhibiting slow ATP turnover rates. Secondary structures for both chaperones were similar by circular dichroism criteria. This work describes an approach to functional expression of human mortalin that provides sufficient material for detailed structure-function studies of this important Hsp70 chaperone.
| S-EPMC2887293 | biostudies-literature
Project description:Xylanase is an industrially important enzyme having wide range of applications especially in paper industry. It is crucial to gain an understanding about the structure and functional aspects of various xylanases produced from diverse sources. In this study, a bioinformatics and molecular modeling approach was adopted to explore properties and structure of xylanases. Physico-chemical properties were predicted and prediction of motifs, disulfide bridges and secondary structure was performed for functional characterization. Apart from these analyses, three dimensional structures were constructed and stereo-chemical quality was evaluated by different structure validation tools. Comparative catalytic site analysis and assessment was performed to extract information about the important residues. Asn72 was found to be the common residue in the active sites of the proteins P35809 and Q12603.
| S-EPMC2737497 | biostudies-literature
Project description:We present an experimental and computational pipeline for the generation of kinetic models of metabolism, and demonstrate its application to glycolysis in Saccharomyces cerevisiae. Starting from an approximate mathematical model, we employ a "cycle of knowledge" strategy, identifying the steps with most control over flux. Kinetic parameters of the individual isoenzymes within these steps are measured experimentally under a standardised set of conditions. Experimental strategies are applied to establish a set of in vivo concentrations for isoenzymes and metabolites. The data are integrated into a mathematical model that is used to predict a new set of metabolite concentrations and reevaluate the control properties of the system. This bottom-up modelling study reveals that control over the metabolic network most directly involved in yeast glycolysis is more widely distributed than previously thought.
| S-EPMC3764422 | biostudies-literature
Project description:Generalized microscopic reversibility implies that the apparent rate of any catalytic process in a complex mechanism is paralleled by substrate desorption in such a way that this ratio is held constant within the reaction mechanism [Whitehead (1976) Biochem. J. 159, 449--456]. The physical and evolutionary significances of this concept, for both polymeric and monomeric enzymes, are discussed. For polymeric enzymes, generalized microscopic reversibility of necessity occurs if, within the same reaction sequence, the substrate stabilizes one type of conformation of the active site only. Generalized microscopic reversibility suppresses the kinetic co-operativity of the slow transition model [Ainslie, Shill & Neet (1972) J. Biol. Chem. 247, 7088--7096]. This situation is obtained if the free-energy difference between the corresponding transition states of the two enzyme forms is held constant along the reaction co-ordinate. This situation implies that the 'extra costs' of energy (required to pass each energy barrier) that are not covered by the corresponding binding energies of the transition states vary in a similar way along the two reaction co-ordinates. The regulatory behaviour of monomeric enzymes is discussed in the light of the concept of 'catalytic perfection' proposed by Albery & Knowles [(1976) Biochemistry 15, 5631--5640]. These authors claim that an enzyme will be catalytically 'perfect' when its catalytic efficiency is maximum. If this situation occurs for a monomeric enzyme obeying either the slow transition or the mnemonical model, it can be shown that the kinetic co-operativity disappears. In other words, kinetic co-operativity of a monomeric enzyme is 'paid for' at the expense of catalytic efficiency, and the monomeric enzyme cannot be simultaneously co-operative and catalytically very efficient. This is precisely what has been found experimentally in a number of cases.
| S-EPMC1186138 | biostudies-other
Project description:Cereals high in amylose content (AC) and resistant starch (RS) offer potential health benefits. Previous studies using chemical mutagenesis or RNA interference have demonstrated that starch branching enzyme (SBE) plays a major role in determining the fine structure and physical properties of starch. However, it remains a challenge to control starch branching in commercial lines. Here, we use CRISPR/Cas9 technology to generate targeted mutagenesis in SBEI and SBEIIb in rice. The frequencies of obtained homozygous or bi-allelic mutant lines with indels in SBEI and SBEIIb in T0 generation were from 26.7 to 40%. Mutations in the homozygous T0 lines stably transmitted to the T1 generation and those in the bi-allelic lines segregated in a Mendelian fashion. Transgene-free plants carrying only the frame-shifted mutagenesis were recovered in T1 generation following segregation. Whereas no obvious differences were observed between the sbeI mutants and wild type, sbeII mutants showed higher proportion of long chains presented in debranched amylopectin, significantly increased AC and RS content to as higher as 25.0 and 9.8%, respectively, and thus altered fine structure and nutritional properties of starch. Taken together, our results demonstrated for the first time the feasibility to create high-amylose rice through CRISPR/Cas9-mediated editing of SBEIIb.
| S-EPMC5339335 | biostudies-literature