Allosteric Enzyme-Based Biosensors—Kinetic Behaviours of Immobilised L-Lysine-α-Oxidase from Trichoderma viride: pH Influence and Allosteric Properties

The present study describes the kinetics of L-lysine-α-oxidase (LO) from Trichoderma viride immobilised by co-crosslinking onto the surface of a Pt electrode. The resulting amperometric biosensor was able to analyse L-lysine, thus permitting a simple but thorough study of the kinetics of the immobilised enzyme. The kinetic study evidenced that LO behaves in an allosteric fashion and that cooperativity is strongly pH-dependent. Not less important, experimental evidence shows that cooperativity is also dependent on substrate concentration at high pH and behaves as predicted by the Monod-Wyman-Changeux model for allosteric enzymes. According to this model, the existence of two different conformational states of the enzyme was postulated, which differ in Lys species landing on LO to form the enzyme–substrate complex. Considerations about the influence of the peculiar LO kinetics on biosensor operations and extracorporeal reactor devices will be discussed as well. Not less important, the present study also shows the effectiveness of using immobilised enzymes and amperometric biosensors not only for substrate analysis, but also as a convenient tool for enzyme kinetic studies.

A shared alarmone-GTP switch underlies triggered and spontaneous persistence

SummaryPhenotypically-switched, antibiotic-refractory persisters may prevent pathogen eradication. Although how triggered persistence via starvation-induced (p)ppGpp is well characterized, generation of persisters without starvation are poorly understood. Here we visualized the formation of spontaneous persisters in a small fraction of cells from growing wild type bacteria, revealing a striking single cell rapid switch from growth to dormancy. This switch-like entrance is triggered by GTP dropping beneath a threshold due to stochastic production and self-amplification of (p)ppGpp via allosteric enzyme activation. In addition, persisters are induced by lethal and sublethal concentrations of cell wall antibiotics by inducing (p)ppGpp via cell wall stress response. Thus spontaneous, triggered and antibiotic-induced persisters can all stem from a common metabolic switch: GTP depletion by (p)ppGpp induction, and each pathway of persister formation is activated by different (p)ppGpp synthetases. These persistence pathways are likely conserved in pathogens which may be exploited to potentiate antibiotic efficacy.

Manipulation of a cation-Π sandwich reveals conformational flexibility in phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is an allosteric enzyme responsible for maintaining phenylalanine (Phe) below neurotoxic levels; its failure results in phenylketonuria. Wild type (WT) PAH equilibrates among resting-state (RS-PAH) and activated (A-PAH) conformations, whose equilibrium position depends upon allosteric Phe binding to the A-PAH conformation. The RS-PAH conformation of WT rat PAH (rPAH) contains a cation-π sandwich between Phe80, Arg123, and Arg420, which cannot exist in the A-PAH conformation. Phe80 variants F80A, F80D, F80L, and F80R were prepared; their conformational equilibrium was evaluated using native PAGE, size exclusion chromatography, ion exchange behavior, intrinsic protein fluorescence, enzyme kinetics, and limited proteolysis, each as a function of [Phe]. Like WT rPAH, F80A and F80D show allosteric activation by Phe while F80L and F80R are constitutively active. Maximal activity of all variants suggests relief of a rate-determining conformational change involving Phe80. Limited proteolysis of WT rPAH in the absence of Phe reveals facile cleavage within a C-terminal 4-helix bundle that is buried in the RS-PAH tetramer interface, reflecting dynamic dissociation of the RS-PAH conformation. This cleavage is not seen for the Phe80 variants, which all show proteolytic hypersensitivity in a linker that repositions during the RS-PAH to A-PAH conformational interchange. Hypersensitivity is corrected by addition of Phe such that all Phe80 variants become like WT rPAH and achieve the A-PAH conformation. Thus, manipulation of Phe80 perturbs the conformational space sampled by PAH, increasing the propensity to sample intermediates in the RS-PAH and A-PAH interchange, which are presumed on-pathway because they can readily achieve the A-PAH conformation by addition of Phe.

Structural basis of glycogen metabolism in bacteria

The evolution of metabolic pathways is a major force behind natural selection. In the spotlight of such process lies the structural evolution of the enzymatic machinery responsible for the central energy metabolism. Specifically, glycogen metabolism has emerged to allow organisms to save available environmental surplus of carbon and energy, using dedicated glucose polymers as a storage compartment that can be mobilized at future demand. The origins of such adaptive advantage rely on the acquisition of an enzymatic system for the biosynthesis and degradation of glycogen, along with mechanisms to balance the assembly and disassembly rate of this polysaccharide, in order to store and recover glucose according to cell energy needs. The first step in the classical bacterial glycogen biosynthetic pathway is carried out by the adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase. This allosteric enzyme synthesizes ADP-glucose and acts as a point of regulation. The second step is carried out by the glycogen synthase, an enzyme that generates linear α-(1→4)-linked glucose chains, whereas the third step catalyzed by the branching enzyme produces α-(1→6)-linked glucan branches in the polymer. Two enzymes facilitate glycogen degradation: glycogen phosphorylase, which functions as an α-(1→4)-depolymerizing enzyme, and the debranching enzyme that catalyzes the removal of α-(1→6)-linked ramifications. In this work, we rationalize the structural basis of glycogen metabolism in bacteria to the light of the current knowledge. We describe and discuss the remarkable progress made in the understanding of the molecular mechanisms of substrate recognition and product release, allosteric regulation and catalysis of all those enzymes.

Robust Determination of Protein Allosteric Signaling Pathways

To understand how protein function changes upon an allosteric perturbation, such as ligand binding and mutation, significant progress in characterizing allosteric network from molecular dynamics (MD) simulations has been made. However, determining which amino acid(s) play an essential role in the propagation of signals may prove challenging, even when the location of the source and sink is known for a protein or protein complex. This challenge is mainly due to the large fluctuations in protein dynamics that cause instability of the network topology within a single trajectory or between multiple replicas. To solve this problem, we introduce the current-flow betweenness scheme, originated from electrical network theory, to protein dynamical network analysis. To demonstrate the benefit of this new method, we chose a prototypic allosteric enzyme (IGPS or HisH-HisF dimer) as our benchmark system. Using multiple replicas of simulations and multiple network topology comparison metrics (edge ranking, path length, and node frequency), we show that the current-flow betweenness provides a significant improvement in the convergence of the allosteric networks. The improved stability of the network topology allows us to generate a delta-network between the apo and holo forms of the protein. We illustrated that the delta-network is a more rigorous way to capture the subtle changes in the networks that would otherwise be neglected by comparing node usage frequencies alone. We have also investigated the use of a linear smoothing function to improve the stability of the contact map. The methodology presented here is general and may be applied to other topology and weighting schemes. We thus conclude that, for determining protein signaling pathways between the pair(s) of source and sink, multiple MD simulation replicas are necessary and the current-flow betweenness scheme introduced here provides a more robust approach than the geodesic scheme based on correlation edge weighting.For Table of Contents Only

Eigenvector centrality for characterization of protein allosteric pathways

Determining the principal energy-transfer pathways responsible for allosteric communication in biomolecules remains challenging, partially due to the intrinsic complexity of the systems and the lack of effective characterization methods. In this work, we introduce the eigenvector centrality metric based on mutual information to elucidate allosteric mechanisms that regulate enzymatic activity. Moreover, we propose a strategy to characterize the range of correlations that underlie the allosteric processes. We use the V-type allosteric enzyme imidazole glycerol phosphate synthase (IGPS) to test the proposed methodology. The eigenvector centrality method identifies key amino acid residues of IGPS with high susceptibility to effector binding. The findings are validated by solution NMR measurements yielding important biological insights, including direct experimental evidence for interdomain motion, the central role played by helix hα1, and the short-range nature of correlations responsible for the allosteric mechanism. Beyond insights on IGPS allosteric pathways and the nature of residues that could be targeted by therapeutic drugs or site-directed mutagenesis, the reported findings demonstrate the eigenvector centrality analysis as a general cost-effective methodology to gain fundamental understanding of allosteric mechanisms at the molecular level.