Glucose Binding Site Research Articles

Inhibition mechanism understanding from molecular dynamics simulation of the interactions between several flavonoids and proton-dependent glucose transporter

Proton-dependent glucose transporters as important drug targets can have different protonation states and adjust their conformational state under different pHs. So based on this character, research on its inhibition mechanism is a significant work. In this article, to study its inhibitory mechanism, we performed the molecular dynamics of several classical flavonoid molecules (Three inhibitors Phloretin, Naringenin, Resveratrol. Two non-inhibitors Isoliquiritigenin, Butein) with glucose transporters under two distinct environmental pHs. The results show inhibitors occupy glucose binding sites (GLN137, ILE255, ASN256) and have strong hydrophobic interactions with proteins through core moiety (C6-Cn-C6). In addition, inhibitors had better inhibitory effects in protonation state. In contrast, non-inhibitors can not occupy glucose binding sites (GLN137, ILE255, ASN256), thus they do not have intense interactions with the protein. It is suggested that favorable inhibitors should effectively take up the glucose-binding site (GLN137, ILE255, ASN256) and limit the protein conformational changes.Communicated by Ramaswamy H. Sarma

Emergence of Cooperative Glucose-Binding Networks in Adaptive Peptide Systems.

Minimalistic peptide-based systems that bind sugars in water are challenging to design due to the weakness of interactions and required cooperative contributions from specific amino-acid side chains. Here, we used a bottom-up approach to create peptide-based adaptive glucose-binding networks by mixing glucose with selected sets of input dipeptides (up to 4) in the presence of an amidase to enable in situ reversible peptide elongation, forming mixtures of up to 16 dynamically interacting tetrapeptides. The choice of input dipeptides was based on amino-acid abundance in glucose-binding sites found in the protein data bank, with side chains that can support hydrogen bonding and CH-π interactions. Tetrapeptide sequence amplification patterns, determined through LC-MS analysis, served as a readout for collective interactions and led to the identification of optimized binding networks. Systematic variation of dipeptide input revealed the emergence of two networks of non-covalent hydrogen bonding and CH-π interactions that can co-exist, are cooperative and context-dependent. A cooperative binding mode was determined by studying the binding of the most amplified tetrapeptide (AWAD) with glucose in isolation. Overall, these results demonstrate that the bottom-up design of complex systems can recreate emergent behaviors driven by covalent and non-covalent self-organization that are not observed in reductionist designs and lead to the identification of system-level cooperative binding motifs.

Genomic evolution towards azole resistance in Candida glabrata clinical isolates unveils the importance of CgHxt4/6/7 in azole accumulation

The increasing prevalence of candidosis caused by Candida glabrata is related to its ability to acquire azole resistance. Although azole resistance mechanisms are well known, the mechanisms for azole import into fungal cells have remained obscure. In this work, we have characterized two hexose transporters in C. glabrata and further investigate their role as potential azole importers. Three azole susceptible C. glabrata clinical isolates were evolved towards azole resistance and the acquired resistance phenotype was found to be independent of CgPDR1 or CgERG11 mutations. Through whole-genome sequencing, CgHXT4/6/7 was found to be mutated in the three evolved strains, when compared to their susceptible parents. CgHxt4/6/7 and the 96% identical CgHxt6/7 were found to confer azole susceptibility and increase azole accumulation in C. glabrata cells, strikingly rescuing the susceptibility phenotype imposed by CgPDR1 deletion, while the identified loss-of-function mutation in CgHXT4/6/7, leads to increased azole resistance. In silico docking analysis shows that azoles display a strong predicted affinity for the glucose binding site of CgHxt4/6/7. Altogether, we hypothesize that hexose transporters, such as CgHxt4/6/7 and CgHxt6/7, may constitute a family of azole importers, involved in clinical drug resistance in fungal pathogens, and constituting promising targets for improved antifungal therapy.

“Mango in all her majesty”—the potential of mangiferin and its analogues in the inhibition of Eimeria tenella hexokinase—a computational approach

The potential of natural products in mitigating infections and diseases are being considered lately. Herein, via in silico methods, we report the possible molecular mechanism of mangiferin (isolated from the fruit, peel, bark and leaves of mango tree) and its derivatives in inhibiting Eimeria tenella hexokinase. We evaluated the binding affinity of these inhibitors to the glucose binding site of EtHK and thereafter proceeded to molecular dynamics simulation. The Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) reveals that three of the derivatives (CPAMM, MxPAMM and NAMM) had better total binding free energy than mangiferin. The ADMET and physicochemical properties assessed shows that inhibitors also hold a potential to be drug-likely. Finally, in mediating their inhibitory potentials, the ligands stabilize both the global and local structures of the protein. This study provides a theoretical premise on which the anti-coccidial propensities of mangiferin most especially its derivatives can be investigate in vitro and in vivo. Communicated by Ramaswamy H. Sarma

Carbohydrate-Functionalized Triazolylidene Iridium Complexes: Hydrogenation Catalysis in Water with Asymmetric Induction.

Two sets of carbohydrate‐NHC hybrid iridium complexes were synthesised in order to combine properties of carbohydrates and triazolylidene (trz) ligands in organometallic catalysis. One set features a direct trz linkage to the anomeric carbohydrate carbon, while the second set is comprised of an ethyl linker between the two functional units. Deprotection of the carbohydrate afforded hybrid complexes that efficiently catalyse the direct hydrogenation of ketones in water. The catalytic activity of the hybrid complexes was influenced by the pH of the aqueous medium and surpassed the activity of carbohydrate‐free or acetyl‐protected analogues (>90 % vs 13 % yield). While no enantiomeric induction was observed for the ethyl‐linked hybrids, a moderate enantiomeric excess (ee) was induced by the directly linked systems. Moreover, these carbohydrate‐trz hybrid complexes displayed mixed inhibitory activity towards a glycosidase from H. orenii that contain a glucose binding site.

A Lead-Based Fragment Library Screening of the Glycosyltransferase WaaG from Escherichia coli.

Glucosyl transferase I (WaaG) in E. coli catalyzes the transfer of an α-d-glucosyl group to the inner core of the lipopolysaccharide (LPS) and plays an important role in the biogenesis of the outer membrane. If its activity could be inhibited, the integrity of the outer membrane would be compromised and the bacterium would be susceptible to antibiotics that are normally prevented from entering the cell. Herein, three libraries of molecules (A, B and C) were docked in the binding pocket of WaaG, utilizing the docking binding affinity as a filter to select fragment-based compounds for further investigations. From the results of the docking procedure, a selection of compounds was investigated by molecular dynamics (MD) simulations to obtain binding free energy (BFE) and KD values for ligands as an evaluation for the binding to WaaG. Derivatives of 1,3-thiazoles (A7 and A4) from library A and 1,3,4-thiadiazole (B33) from library B displayed a promising profile of BFE, with KD < mM, viz., 0.11, 0.62 and 0.04 mM, respectively. Further root-mean-square-deviation (RMSD), electrostatic/van der Waals contribution to the binding and H-bond interactions displayed a favorable profile for ligands A4 and B33. Mannose and/or heptose-containing disaccharides C1–C4, representing sub-structures of the inner core of the LPS, were also investigated by MD simulations, and compound C42− showed a calculated KD = 0.4 µM. In the presence of UDP-Glc2−, the best-docked pose of disaccharide C42− is proximate to the glucose-binding site of WaaG. A study of the variation in angle and distance was performed on the different portions of WaaG (N-, the C- domains and the hinge region). The Spearman correlation coefficient between the two variables was close to unity, where both variables increase in the same way, suggesting a conformational rearrangement of the protein during the MD simulation, revealing molecular motions of the enzyme that may be part of the catalytic cycle. Selected compounds were also analyzed by Saturation Transfer Difference (STD) NMR experiments. STD effects were notable for the 1,3-thiazole derivatives A4, A8 and A15 with the apo form of the protein as well as in the presence of UDP for A4.

Role of Conformational Change and Glucose Binding Sites in the Enhanced Glucose Tolerance of Agrobacterium tumefaciens 5A GH1 β-Glucosidase Mutants.

β-Glucosidases are often inhibited by their reaction product glucose and a barrier to the efficient lignocellulosic biomass hydrolysis to glucose. We had previously reported the mutants, C174V, and H229S, with a nearly 2-fold increased glucose tolerance over the wild type (WT), H0HC94, encoded in Agrobacterium tumefaciens 5A (apparent Ki,Glc = 686 mM). We report our steady-state and time-resolved intrinsic fluorescence spectroscopy, circular dichroism, and isothermal titration calorimetry (ITC) studies to further understand increased glucose tolerance. Changes in the mutants' emission intensity and the differential change in quenching rate in the absence and presence of glucose reflect changes in protein conformation by glucose. Time-resolved lifetime and anisotropy measurements further indicated the microenvironment differences across solvent-exposed tryptophan residues and a higher hydrodynamic radius due to glucose binding, respectively. ITC measurements confirmed the increase of glucose binding sites in the mutants. The experiment results were supported by molecular dynamics simulations, which revealed significant variations in the glucose-protein hydrogen-bonding profiles. Protein structure network analysis of the simulated structures further indicates the mutants' conformation change than the WT. Computational studies also indicated additional glucose binding sites in mutants. Our results indicate the role of glucose binding in modulating the enzyme response to glucose.

Tyrosine Kinase Inhibitors Reduce Glucose Uptake by Binding to an Exofacial Site on hGLUT-1: Influence on 18 F-FDG PET Uptake.

Positron emission tomography (PET) using 2‐deoxy‐2‐[18F]fluoro‐d‐glucose ([18F]FDG), a marker of energy metabolism and cell proliferation, is routinely used in the clinic to assess patient response to chemotherapy and to monitor tumor growth. Treatment with some tyrosine kinase inhibitors (TKIs) causes changes in blood glucose levels in both nondiabetic and diabetic patients. We evaluated the interaction of several classes of TKIs with human glucose transporter‐1 (hGLUT‐1) in FaDu and GIST‐1 cells by measuring [3H]2‐deoxy‐d‐glucose ([3H]2‐DG) and [3H]FDG uptake. Uptake of both was inhibited to varying extents by the TKIs, and representative TKIs from each class showed competitive inhibition of [3H]2‐DG uptake. In GIST‐1 cells, [3H]FDG uptake inhibition by temsirolimus and nilotinib was irreversible, whereas inhibition by imatinib, gefitinib, and pazopanib was reversible. Molecular modeling studies showed that TKIs form multiple hydrogen bonds with polar residues of the sugar binding site (i.e., Q161, Q282, Q283, N288, N317, and W388), and van der Waals interactions with the H‐pocket site. Our results showed interaction of TKIs with amino acid residues at the glucose binding site to inhibit glucose uptake by hGLUT‐1. We hypothesize that inhibition of hGLUT‐1 by TKIs could alter glucose levels in patients treated with TKIs, leading to hypoglycemia and fatigue, although further studies are required to evaluate roles of other SLC2 and SLC5 members. In addition, TKIs could affect tumor [18F]FDG uptake, increasingly used as a marker of tumor response. The hGLUT‐1 inhibition by TKIs may have implications for routine [18F]FDG‐PET monitoring of tumor response in patients.

Molecular modeling, docking and simulation dynamics of β-glucosidase reveals high-efficiency, thermo-stable, glucose tolerant enzyme in Paenibacillus lautus BHU3 strain

The enzyme β-glucosidase mediates the rate limiting step of conversion of cellobiose to glucose and thus plays a vital role in the process of cellulose degradation. The present study deals with analysis of the effective novel strain of Paenibacillus lautus BHU3 for identifying high-efficiency thermostable, glucose tolerant β-glucosidases. Seven counterparts with elevated Tm values ranging from 64.6 to 75.8 °C with high thermo-stability, were revealed through this analysis. The blind molecular docking of the model enzymes structures with cellobiose and pNPG gave high negative interaction energies ranging from −11.33 to −13.29 and −6.43 to −9.054 (kcal mol−1), respectively. The enzyme WP_096774744.1 effectively formed 5 hydrogen bonds with the highest interaction energy (−13.29 kcal mol−1) with cellobiose at its catalytic site. Molecular dynamics simulation analysis performed for the WP_096774744.1-pNPG complex predicted Glu5, Arg7, Lue68, Gly69 and Phe325 as the major contributing residues for accomplishing hydrolysis of β-1-4-linkage. Further, the molecular docking of WP_096774744.1 enzyme with glucose revealed a distinct glucose-binding site distant from the substrate-binding site, thus confirming the deficient competitive inhibition by glucose. Hence, WP_096774744.1 β-glucosidase appears to be an efficient enzyme with enhanced activity to biodegrade the cellulosic materials and highly relevant for waste management and various industrial applications.

Fine-Tuning of Neuronal Ion Channels-Mapping of Residues Involved in Glucose Sensitivity of Recombinant Human Glycine Receptors.

The inhibitory glycine receptor (GlyR) mediates synaptic inhibition in the spinal cord, brain stem, and other regions of the mammalian central nervous system. Glucose was shown to potentiate α1 GlyRs by interacting with K143. Here, additional amino acids involved in glucose modulation were identified using a structure-based approach of site-directed mutagenesis followed by whole-cell patch-clamp analysis. We identified two additional lysine residues in the α1 GlyR extracellular domain, K16 and K281, that were involved in glucose modulation. Mutation of either residue to alanine abolished glucose potentiation. Residue K281 is located in the same pocket as K143 and could thus contribute to glucose binding. The double mutant K143A-K281A showed a 6-fold increase of EC50, while EC50 of both single mutants K143A and K281A was only slightly increased (1.7- and 1.3-fold, respectively). K16 is located at an analgesic binding site that is distant from the agonist or glucose sites, and the K16A mutation may generate a receptor species that is not potentiated. GlyR position α1-S267 is close to the postulated glucose binding site and known for interactions with ethanol and anesthetics. In the presence of glucose, GlyR α1 mutants S267A, S267I, and S267R showed potentiation, no effect, and reduction of current responses, respectively. This pattern follows that of ethanol modulation and suggests that the interaction sites of glucose and ethanol are identical or located close to each other. Our results support the presence of a distinct binding site for glucose on the glycine receptor, overlapping with the ivermectin/ethanol binding pocket near the transmembrane region and the TM2-3 loop.

Ultrasensitive Poly(boric acid) Hydrogel-Coated Quartz Crystal Microbalance Sensor by Using UV Pressing-Assisted Polymerization for Saliva Glucose Monitoring.

Quartz crystal microbalance (QCM) has attracted extensive attention in the field of biological analysis and detection because of its high sensitivity, fast response, real-time measurement, good operability, and low-cost production. However, to detect the trace amounts of small molecules, such as low-concentration saliva glucose under physiological conditions, is still a major challenge. Herein, the surface of a QCM chip was coated with a poly(boric acid)-based hydrogel using UV pressing-assisted polymerization to obtain a simple device for glucose detection. The designed QCM sensor shows a record-low detection limit of glucose (3 mg/L at pH 7.5), which is ∼30 times lower than that of sensors fabricated by conventional surface initiation-spin coating. The outperformance of the poly(boric acid) hydrogel-coated QCM sensor is probably due to the uniform and compact microstructure, as well as the presence of sufficient glucose-binding sites resulting from the hydrogel coating generated by UV pressing-assisted polymerization. This method provides an important solution to detect the trace amounts of small organic molecules or ions and has the potential to push forward the practical applications of QCM sensors.

Engineering of β-Glucosidase Bgl15 with Simultaneously Enhanced Glucose Tolerance and Thermostability To Improve Its Performance in High-Solid Cellulose Hydrolysis.

In this study, a Petri-dish-based double-layer high-throughput screening method was established to improve glucose tolerance of β-glucosidase Bgl15. Two beneficial mutations were identified, and the joint mutant 2R1 improved the half-maximal inhibitory concentration of glucose from 0.04 to 2.1 M. The crystal structure of 2R1 was subsequently determined at 2.7 Å. Structure analysis revealed that enhancement of glucose tolerance may be due to improved transglycosylation activity made possible by a hydrophobic binding site for glucose as an acceptor and more stringent control of a putative water channel. To further ameliorate the application potential of the enzyme, it was engineered to increase the half-life at 50 °C from 0.8 h (Bgl15) to 180 h (mutant 5R1). Furthermore, supplementation of 5R1 to the cellulase cocktail significantly improved glucose production from pretreated sugar cane bagasse by 38%. Consequently, this study provided an efficient approach to enhance glucose tolerance and generated a promising catalyst for cellulose saccharification.

Molecular insight into conformational transformation of human glucokinase: conventional and targeted molecular dynamics

Glucokinase (GK) plays a key role in the regulation of hepatic glucose metabolism. An unusual mechanism of positive cooperativity of monomeric GK containing only a single binding site for glucose is very interesting and still unclear. The activation process of GK is associated with a large-scale conformational change from the inactive to the active state. Here, conventional and targeted molecular dynamics simulations were used to study the conformational dynamics of GK in the stable configurations and in the transition from active to inactive state. Three phases of the structural reorganization of GK were detected. The first step is a transformation of GK from the active state to the intermediate structure, where the cleft between the domains is open, but alpha helix 13 is still inside the small domain. From this point, there are two alternative paths. One path leads to the inactive state through the release of helix 13 from the inside of small domain to the outside. Other path goes back to the active state. Simulation results reveal the critical role of helix 13 in the transformation of GK from the open state to inactive one and the influence of the loop 2 on the protein transformation between the open and the closed active states. Principal component analysis and covariance matrix analysis were carried out to analyze the dynamics of protein. Importance of hydrogen bonds in the stability of the closed conformation is shown. Overall, our simulations provide new information about the dynamics of GK and its structural transformation. Communicated by Ramaswamy H. Sarma

Design, synthesis and biological evaluation of 6-deoxy O-spiroketal C-arylglucosides as novel renal sodium-dependent glucose cotransporter 2 (SGLT2) inhibitors for the treatment of type 2 diabetes

In this work, aiming at finding a novel, potent, and selective sodium-dependent glucose cotransporter 2 (SGLT2) inhibitor with good pharmacokinetic profiles for the treatment of diabetes, we focus on modifying the sugar moiety of SGLT2 inhibitors, which dominates the binding with glucose binding site of hSGLT, via removing the C-6 hydroxy group to adjust the physicochemical properties and target-recognition manners of SGLT2 inhibitors. In addition, tofogliflozin containing a special O-spiroketal C-arylglucoside scaffold, displayed good efficacy and bioavailability both in animals and in humans. Therefore, a series of 6-deoxy O-spiroketal C-arylglucosides as novel SGLT2 inhibitors were designed, synthesized, and evaluated in this work. The structure-activity relationship (SAR) research on this novel series and a comprehensive in vitro and in vivo biological evaluation afforded compound 39 with high in vitro hSGLT2 inhibitory activity (IC50 = 4.5 nM), good pharmacokinetic profiles, and more remarkable efficacy in C57BL/6J mice and Sprague-Dawley rats than marketed drug tofogliflozin.

Mechanistic insights into the host-microbe interaction and pathogen exclusion mediated by the Mucus-binding protein of Lactobacillus plantarum

Surface adhesins of pathogens and probiotics strains are implicated in mediating the binding of microbes to host. Mucus-binding protein (Mub) is unique to gut inhabiting lactic acid bacteria; however, the precise role of Mub proteins or its structural domains in host-microbial interaction is not well understood. Last two domains (Mubs5s6) of the six mucus-binding domains arranged in tandem at the C-terminus of the Lp_1643 protein of Lactobacillus plantarum was expressed in E. coli. Mubs5s6 showed binding with the rat intestinal mucus, pig gastric mucins and human intestinal tissues. Preincubation of Mubs5s6 with the Caco-2 and HT-29 cell lines inhibited the binding of pathogenic enterotoxigenic E. coli cells to the enterocytes by 68% and 81%, respectively. Pull-down assay suggested Mubs5s6 binding to the host mucosa components like cytokeratins, Hsp90 and Laminin. Mubs5s6 was predicted to possess calcium and glucose binding sites. Binding of Mubs5s6 with these ligands was also experimentally observed. These ligands are known to be associated with pathogenesis suggesting Mub might negotiate pathogens in multiple ways. To study the feasibility of Mubs5s6 delivery in the gut, it was encapsulated in chitosan-sodium tripolyphosphate microspheres with an efficiency of 65% and release up to 85% in near neutral pH zone over a period of 20 hours. Our results show that Mub plays an important role in the host-microbial cross-talk and possesses the potential for pathogen exclusion to a greater extent than mediated by L. plantarum cells. The functional and technological characteristics of Mubs5s6 make it suitable for breaking the host-pathogen interaction.

Nitric Oxide Activates β-Cell Glucokinase by Promoting Formation of the "Glucose-Activated" State.

The release of insulin from the pancreas is tightly controlled by glucokinase (GCK) activity that couples β-cell metabolism to changes in blood sugar. Despite having only a single glucose-binding site, GCK displays positive glucose cooperativity. Ex vivo structural studies have identified several potential protein conformations with varying levels of enzymatic activity, yet it is unclear how living cells regulate GCK cooperativity. To better understand the cellular regulation of GCK activation, we developed a homotransfer Förster resonance energy transfer (FRET) GCK biosensor and used polarization microscopy to eliminate fluorescence crosstalk from FRET quantification and improve the signal-to-noise ratio. This approach enhanced sensor contrast compared to that seen with the heterotransfer FRET GCK reporter and allowed observation of individual GCK states using an automated method to analyze FRET data at the pixel level. Mutations known to activate and inhibit GCK activity produced distinct anisotropy distributions, suggesting that at least two conformational states exist in living cells. A high glucose level activated the biosensor in a manner consistent with GCK's enzymology. Interestingly, glucose-free conditions did not affect GCK biosensor FRET, indicating that there is a single low-activity state, which is counter to proposed structural models of GCK cooperativity. Under low-glucose conditions, application of chemical NO donors efficiently shifted GCK to the more active conformation. Notably, GCK activation by mutation, a high glucose level, a pharmacological GCK activator, or S-nitrosylation all shared the same FRET distribution. These data suggest a simplified model for GCK activation in living cells, where post-translational modification of GCK by S-nitrosylation facilitates a single conformational transition that enhances GCK enzymatic activity.

Novel thiosemicarbazide derivatives with 4-nitrophenyl group as multi-target drugs: α-glucosidase inhibitors with antibacterial and antiproliferative activity

A series of thiosemicarbazides with 4-nitrophenyl group was obtained in the reaction of carboxylic acid hydrazides with isothiocyanates. All compounds were checked for their antibacterial and antiproliferative activity. Our results have shown that derivatives 6-8 possessed antibacterial activity against S. aureus, S. epidermidis, S. mutans and S. sanguinis, moderate cytotoxicity and good therapeutic safety in vitro. Additionally, compounds 1 and 4 significantly inhibited A549, HepG2 and MCF-7 cell division. Moreover, PASS software indicated that newly obtained compounds are potential α-glucosidase inhibitors. This was confirmed by in vitro studies. To investigate the mode of interaction with the molecular target compounds were docked to glucose binding site of the enzyme and exhibited a similar binding mode as glucose.

Understanding the role of residues around the active site tunnel towards generating a glucose-tolerant β-glucosidase from Agrobacterium tumefaciens 5A.

Most β-glucosidases are subjected to inhibition by the final hydrolysis product glucose resulting in the accumulation of cellobiose and oligosaccharides. This accumulated cellobiose and oligosaccharides further inhibit the activities of endoglucanase and cellobiohydrolases, resulting in the inhibition of cellulose degradation and a more expensive biofuel. To elucidate the mechanism(s) of glucose tolerance, we designed and characterised six mutations of a moderately glucose-tolerant β-glucosidase (H0HC94) from the mesophilic bacterium Agrobacterium tumefaciens 5A. The hydrophobicity and steric were varied across non-conserved residues in specific regions of the active site tunnel. In contrast to the uncompetitive inhibition of WT enzyme by glucose, C174V and H229S are competitively inhibited pointing towards a possible glucose-binding site in the protein at these positions. Increasing hydrophobicity at the +1 subsite and increasing hydrophobicity and steric at +2 subsites seemed to be critical for glucose tolerance for this BG. Additionally, in L178E, specific activity was 1.8 times higher on the natural substrate cellobiose while both W127F and L178E mutants showed an enhancement in thermostability. The kinetic stability of W127F, V176A, L178A and L178E also increased between 2- and 3-folds compared to WT. Our results indicate that while the structure between subsites +1 and +2 is critical for the glucose tolerance, the specific residues may not be identical across such enzymes.

Structures of a glucose-tolerant β-glucosidase provide insights into its mechanism.

Cellulose can be converted to ethanol via the fermentation of glucose, which is considered as a promising green alternative for transportation fuels. The conversion of cellulose to glucose needs three enzymes, in which β-glucosidase (BGL) plays an essential role. However, BGL is inhibited by its own product glucose, greatly limiting its applications in industry. We previously obtained a novel BGL named Bgl6 with a high glucose tolerance. Further engineering through random mutagenesis produced a triple mutant M3 with improved thermostability. This enzyme shows promising properties for wide applications but the structural basis of the unusual properties of Bgl6 is not clear. In this study, we determined the crystal structures of Bgl6 and variants at high resolution, which provide insights into its glucose-tolerant mechanism and thermostability. Particularly, Bgl6 forms an extra channel that could be used as a secondary binding site for glucose, which may contribute to glucose tolerance. Additionally, the triple mutations could strengthen the hydrophobic interactions within the enzyme and may be responsible for the enhanced thermostability exhibited by M3, which was further confirmed by dynamic light scattering data. Lastly, structural comparison to other orthologs allows us to formulate new strategies on how to improve the catalytic efficiency of Bgl6.

Regulation of Glucokinase in Pancreatic Beta Cells

Glucokinase (GCK) is the main glucose sensor in pancreatic β cells and controls insulin secretion. Because of this, GCK is an important target for type 2 diabetes mellitus therapies. The mechanism of how GCK, a monomeric enzyme with a single binding site for glucose, exhibits cooperativity is still unknown although there are many proposed models. Our lab has shown that nitric oxide (NO) enhances GCK activity, but the underlying mechanism of activation and the relationship between GCK S-nitrosylation and cooperativity are unclear. To further investigate GCK regulation, we created a novel single-color GCK reporter. This Forster resonance energy transfer (FRET) biosensor allows for more precise and quantitative measurements than traditional two-color FRET reporters. Here we show that our single-color GCK sensor accurately measures changes in GCK conformation in response to increasing concentrations of glucose. 7.5mM glucose is representative of post-prandial blood glucose levels and also corresponds to half-maximal GCK activation. We show that 7.5mM also has the highest degree of variability further highlighting the need to elucidate the mechanism of GCK activation. Introducing known activating and inactivating GCK point mutations into our GCK FRET sensor provide evidence for two conformations of GCK. We measured distinct FRET efficiencies for the both the active and inactive states that converged on precise anisotropy values. Addition of the NO donor, SNAP, stimulates formation of the more active GCK conformation. Together, these findings provide support for a two state GCK model with NO facilitating formation of the more active GCK state. Learning how GCK is regulated is important for understanding the pathology of diabetes.