Electrochemical biosensors based on peptide-kinase interactions at the kinase docking site

Kinases are important cancer biomarkers and are conventionally detected based on their catalytic activity. Kinases regulate cellular activities by phosphorylation of motif-specific multiple substrate proteins, resulting in a lack of selectivity of activity-based kinase biosensors. We present an alternative approach of sensing kinases based on the interactions of their allosteric docking sites with a specific partner protein. The new approach was demonstrated for the ERK2 kinase and its substrate ELK-1. A peptide derived from ELK-1 was bound to a gold electrode and ERK2 sensing was performed by electrochemical impedance spectroscopy. The sensors showed a high level of target selectivity for ERK2 when compared with p38gamma kinase and BSA. ERK2 was detected in its cellular concentration range, 0.2-8.0 microM. Using the flexibility of peptide design, our method is generic for developing sensitive and substrate-specific biosensors and other disease-related enzymes based on their interactions.

Multi-site enzymes as a mechanism for bistability in reaction networks

Here, we focus on a common class of enzymes that have multiple substrate-binding sites (multi-site enzymes), and analyse their capacity to generate bistable dynamics in the reaction systems that they are embedded in. Using mathematical techniques, we show that the inherent binding and catalysis reactions arising from multiple substrate-enzyme complexes creates a potential for bistable dynamics in a reaction system. We construct a generic model of an enzyme with n substrate binding sites and derive an analytical solution for the steady state concentration of all enzyme-substrate complexes. By studying these expressions, we obtain a mechanistic understanding for bistability and derive parameter combinations that guarantee bistability and show how changing specific enzyme kinetic parameters and enzyme levels can lead to bistability in reaction systems involving mjulti-site enzymes. Thus, the presented findings provide a biochemical and mathematical basis for predicting and engineering bistability in multi-site enzymes.

Multiple substrate recognition by yeast diadenosine and diphosphoinositol polyphosphate phosphohydrolase through phosphate clamping

The yeast diadenosine and diphosphoinositol polyphosphate phosphohydrolase DDP1 is a Nudix enzyme with pyrophosphatase activity on diphosphoinositides, dinucleotides, and polyphosphates. These substrates bind to diverse protein targets and participate in signaling and metabolism, being essential for energy and phosphate homeostasis, ATPase pump regulation, or protein phosphorylation. An exhaustive structural study of DDP1 in complex with multiple ligands related to its three diverse substrate classes is reported. This allowed full characterization of the DDP1 active site depicting the molecular basis for endowing multisubstrate abilities to a Nudix enzyme, driven by phosphate anchoring following a defined path. This study, combined with multiple enzyme variants, reveals the different substrate binding modes, preferences, and selection. Our findings expand current knowledge on this important structural superfamily with implications extending beyond inositide research. This work represents a valuable tool for inhibitor/substrate design for ScDDP1 and orthologs as potential targets to address fungal infections and other health concerns.

Hydrocarbon solubilisation by oil and cellulose-degrading Chitinophaga terrae isolated from the rumen.

This study investigated the capacity of cellulose and hydrocarbon degrading bacterium isolated from the rumen of a cow to solubilise hydrocarbon. The bacterium was isolated from the rumen fluid of cow and its capacity to degrade cellulose was screened on carboxyl methyl cellulose (CMC) agar plate and the ability to degrade crude oil was carried out using Bonny Light crude. Solubilisation of hydrocarbon was determined by carrying out emulsification index (E24) using kerosene. Other bio-surfactant characteristics such as blood haemolysis, tilted slide capacity and oil displacement were tested also. The bacterium was identified based on phenotypic, biochemical and molecular characteristics. The isolate achieved 48.17% degradation of total petroleum hydrocarbon (TPH) within 14 days with emulsification index of 54.5%. The isolate also produced clear zone on agar plate containing CMC as the sole carbon source. Phylogenetic tree analyses classified the bacterial isolate as Chitinophaga terrae. The sequences have been deposited to GenBank under the accession number KJ076216.1. This study has demonstrated that the novel strain of Chitinophaga terrae used in this study not only has the capacity for multiple substrate utilization, but also has the capacity to produce bio-surfactant. Considering that the isolate was obtain from the rumen of cow it shows that rumen content may harbour bacteria with diverse economical and ecologically-friendly product, which may be utilized for bioremediation of crude oil contaminated systems.

Functional screening of a Caatinga goat (Capra hircus) rumen metagenomic library reveals a novel GH3 β-xylosidase

Functional screening of metagenomic libraries is an effective approach for identification of novel enzymes. A Caatinga biome goat rumen metagenomic library was screened using esculin as a substrate, and a gene from an unknown bacterium encoding a novel GH3 enzyme, BGL11, was identified. None of the BGL11 closely related genes have been previously characterized. Recombinant BGL11 was obtained and kinetically characterized. Substrate specificity of the purified protein was assessed using seven synthetic aryl substrates. Activity towards nitrophenyl-β-D-glucopyranoside (pNPG), 4-nitrophenyl-β-D-xylopyranoside (pNPX) and 4-nitrophenyl-β-D-cellobioside (pNPC) suggested that BGL11 is a multifunctional enzyme with β-glucosidase, β-xylosidase, and cellobiohydrolase activities. However, further testing with five natural substrates revealed that, although BGL11 has multiple substrate specificity, it is most active towards xylobiose. Thus, in its native goat rumen environment, BGL11 most likely functions as an extracellular β-xylosidase acting on hemicellulose. Biochemical characterization of BGL11 showed an optimal pH of 5.6, and an optimal temperature of 50°C. Enzyme stability, an important parameter for industrial application, was also investigated. At 40°C purified BGL11 remained active for more than 15 hours without reduction in activity, and at 50°C, after 7 hours of incubation, BGL11 remained 60% active. The enzyme kinetic parameters of Km and Vmax using xylobiose were determined to be 3.88 mM and 38.53 μmol.min-1.mg-1, respectively, and the Kcat was 57.79 s-1. In contrast to BLG11, most β-xylosidases kinetically studied belong to the GH43 family and have been characterized only using synthetic substrates. In industry, β-xylosidases can be used for plant biomass deconstruction, and the released sugars can be fermented into valuable bio-products, ranging from the biofuel ethanol to the sugar substitute xylitol.

A novel photochemical sensor based on quinoline-functionalized phenazine derivatives for multiple substrate detection

A novel photochemical sensor based on quinoline-functionalized phenazine derivatives for highly sensitive detection of multiple substrates (l-Arg, CO2, and pH) was designed and synthesized.

Simulating Multiple Substrate Binding Events by γ-Glutamyltransferase using Accelerated Molecular Dynamics

Abstractγ-glutamyltransferase (GGT) is an enzyme that uses γ-glutamyl compounds as substrate and catalyzes their transfer into a water molecule or an acceptor substrate with varied physiological-function in bacteria, plants and animals. Crystal structures of GGT are known for different species and in different states of the chemical reaction; however, structural dynamics of the substrate binding to the catalytic site of GGT is unknown. Here, we modeled Escherichia Coli GGT’s glutamine binding by using a swarm of accelerated molecular dynamics (aMD) simulations. Characterization of multiple binding events identified three structural binding motifs composed of polar residues in the binding pocket that govern glutamine binding into the active site. Simulated open and closed conformations of a lid-loop protecting the binding cavity suggests its role as a gating element by allowing or blocking substrates entry into the binding pocket. Partially open states of the lid-loop are accessible within thermal fluctuations, while the estimated free energy cost of a complete open state is 2.4 kcal/mol. Our results suggest that both specific electrostatic interactions and GGT conformational dynamics dictate the molecular recognition of substrate-GGT complexes.