Protocatechuate 4,5-dioxygenase (LigAB) is a heterodimeric enzyme that catalyzes the dioxygenation of multiple lignin derived aromatic compounds. The active site of LigAB is at the heterodimeric ...interface, with specificity conferred by the alpha subunit and catalytic residues contributed by the beta subunit. Previous research has indicated that the phenylalanine at the 103 position of the alpha subunit (F103α) controls selectivity for the C5 position of the aromatic substrates, and mutations of this residue can enhance the rate of catalysis for substrates with larger functional groups at this position. While several of the mutations to this position (Valine, V; Threonine, T; Leucine, L; and Histidine, H) were catalytically active, other mutations (Alanine, A; and Serine, S) were found to have reduced dimer interface affinity, leading to challenges in copurifing the catalytically active enzyme complex under high salt conditions. In this study, we aimed to experimentally and computationally interrogate residues at the dimer interface to discern the importance of position 103α for maintaining the integrity of the heterodimer. Molecular dynamic simulations and electrophoretic mobility assays revealed a preference for nonpolar/aromatic amino acids in this position, suggesting that while substitutions to polar amino acids may produce a dioxygenase with a useful substrate utilization profile, those considerations may be off-set by potential destabilization of the catalytically active oligomer. Understanding the dimerization of LigAB provides insight into the multimeric proteins within the largely uncharacterized superfamily and characteristics to consider when engineering proteins that can degrade lignin efficiently. These results shed light on the challenges associated with engineering proteins for broader substrate specificity.
► Advancement in food packaging materials expected to grow. ► Natural fibre/nanoclay hybrid system have potential in food packaging. ► Hybrid composites for environmentally compatible food packaging ...materials.
The increasing demand for new food packaging materials which satisfy people requirements provided thrust for advancement of nano-materials science. Inherent permeability of polymeric materials to gases and vapours; and poor barrier and mechanical properties of biopolymers have boosted interest in developing new strategies to improve these properties. Research and development in polymeric materials coupled with appropriate filler, matrix-filler interaction and new formulation strategies to develop composites have potential applications in food packaging. Advancement in food packaging materials expected to grow with the advent of cheap, renewable and sustainable materials with enhanced barrier and mechanical properties. Nanoparticles have proportionally larger surface area and significant aspect ratio than their micro-scale counterparts, which promotes the development of mechanical and barrier properties. Nanocomposites are attracting considerable interest in food packaging because of these fascinating features. On the other hand, natural fibres are susceptible to microorganisms and their biodegradability is one of the most promising aspects of their incorporation in polymeric materials. Present review article explain about different categories of nanoclay and natural fibre based composite with particular regard to its applications as packaging materials and also gives an overview of the most recent advances and emerging new aspects of nanotechnology for development of hybrid composites for environmentally compatible food packaging materials.
MshA is a GT-B glycosyltransferase catalyzing the first step in the biosynthesis of mycothiol. While many GT-B enzymes undergo an open-to-closed transition, MshA is unique because its 97° rotation is ...beyond the usual range of 10-25°. Molecular dynamics (MD) simulations were carried out for MshA in both ligand bound and unbound states to investigate the effect of ligand binding on localized protein dynamics and its conformational free energy landscape. Simulations showed that both the unliganded "opened" and liganded "closed" forms of the enzyme sample a wide degree of dihedral angles and interdomain distances with relatively low overlapping populations. Calculation of the free energy surface using replica exchange MD for the apo "opened" and an artificial generated apo "closed" structure revealed overlaps in the geometries sampled, allowing calculation of a barrier of 2 kcal/mol for the open-to-closed transition in the absence of ligands. MD simulations of fully liganded MshA revealed a smaller sampling of the dihedral angles. The localized protein fluctuation changes suggest that UDP-GlcNAc binding activates the motions of loops in the 1-l-
-inositol-1-phosphate (I1P)-binding site despite little change in the interactions with UDP-GlcNAc. Circular dichroism, intrinsic fluorescence spectroscopy, and mutagenesis studies were used to confirm the ligand-induced structural changes in MshA. The results support a proposed mechanism where UDP-GlcNAc binds with rigid interactions to the C-terminal domain of MshA and activates flexible loops in the N-terminal domain for binding and positioning of I1P. This model can be used for future structure-based drug development of inhibitors of the mycothiol biosynthetic pathway.
Understanding the dynamical motions and ligand recognition motifs of heptosyltransferase I (HepI) can be critical to discerning the behavior of other glycosyltransferase (GT) enzymes. Prior studies ...in our lab have demonstrated that GTs in the GT-B structural class, which are characterized by their connection of two Rossman-like domains by a linker region, have conserved structural fold and dynamical motions, despite low sequence homology, therefore making discoveries found in HepI transferable to other GT-B enzymes. Through molecular dynamics simulations and ligand binding free energy analysis of HepI in the apo and bound complexes (for all kinetically relevant combinations of the native substrates/products), we have determined the energetically favored enzymatic pathway for ligand binding and release. Our principal component, dynamic cross correlation, and network analyses of the simulations have revealed correlated motions involving residues within the N-terminal domain communicating with C-terminal domain residues via both proximal amino acid residues and also functional groups of the bound substrates. Analyses of the structural changes, energetics of substrate/product binding, and changes in p
have elucidated a variety of inter and intradomain interactions that are critical for enzyme catalysis. These data corroborate our experimental observations of protein conformational changes observed in both presteady state kinetic and circular dichroism analyses of HepI. These simulations provided invaluable structural insights into the regions involved in HepI conformational rearrangement upon ligand binding. Understanding the specific interactions governing conformational changes is likely to enhance our efforts to develop novel dynamics disrupting inhibitors against GT-B structural enzymes in the future.
A clinically relevant inhibitor for Heptosyltransferase I (HepI) has been sought after for many years because of its critical role in the biosynthesis of lipopolysaccharides on bacterial cell ...surfaces. While many labs have discovered or designed novel small molecule inhibitors, these compounds lacked the bioavailability and potency necessary for therapeutic use. Extensive characterization of the HepI protein has provided valuable insight into the dynamic motions necessary for catalysis that could be targeted for inhibition. Structural inspection of Kdo
-lipid A suggested aminoglycoside antibiotics as potential inhibitors for HepI. Multiple aminoglycosides have been experimentally validated to be first-in-class nanomolar inhibitors of HepI, with the best inhibitor demonstrating a K
of 600 ± 90 nM. Detailed kinetic analyses were performed to determine the mechanism of inhibition while circular dichroism spectroscopy, intrinsic tryptophan fluorescence, docking, and molecular dynamics simulations were used to corroborate kinetic experimental findings. While aminoglycosides have long been described as potent antibiotics targeting bacterial ribosomes' protein synthesis leading to disruption of the stability of bacterial cell membranes, more recently researchers have shown that they only modestly impact protein production. Our research suggests an alternative and novel mechanism of action of aminoglycosides in the inhibition of HepI, which directly leads to modification of LPS production in vivo. This finding could change our understanding of how aminoglycoside antibiotics function, with interruption of LPS biosynthesis being an additional and important mechanism of aminoglycoside action. Further research to discern the microbiological impact of aminoglycosides on cells is warranted, as inhibition of the ribosome may not be the sole and primary mechanism of action. The inhibition of HepI by aminoglycosides may dramatically alter strategies to modify the structure of aminoglycosides to improve the efficacy in fighting bacterial infections.
It has long been understood that some proteins undergo conformational transitions en route to the Michaelis Complex to allow chemistry. Examination of crystal structures of glycosyltransferase ...enzymes in the GT-B structural class reveals that the presence of ligand in the active site triggers an open-to-closed conformation transition, necessary for their catalytic functions. Herein, we describe microsecond molecular dynamics simulations of two distantly related glycosyltransferases that are part of the GT-B structural superfamily, HepI and GtfA. Simulations were performed using the open and closed conformations of these unbound proteins, respectively, and we sought to identify the major dynamical modes and communication networks that interconnect the open and closed structures. We provide the first reported evidence within the scope of our simulation parameters that the interconversion between open and closed conformations is a hierarchical multistep process which can be a conserved feature of enzymes of the same structural superfamily. Each of these motions involves of a collection of smaller molecular reorientations distributed across both domains, highlighting the complexities of protein dynamic involved in the interconversion process. Additionally, dynamic cross-correlation analysis was employed to explore the potential effect of distal residues on the catalytic efficiency of HepI. Multiple distal nonionizable residues of the C-terminal domain exhibit motions anticorrelated to positively charged residues in the active site in the
-terminal domain involved in substrate binding. Mutations of these residues resulted in a reduction in negatively correlated motions and an altered enzymatic efficiency that is dominated by lower K
values with
effectively unchanged. The findings suggest that residues with opposing conformational motions involved in the opening and closing of the bidomain HepI protein can allosterically alter the population and conformation of the "closed" state, essential to the formation of the Michaelis complex. The stabilization effects of these mutations likely equally influence the energetics of both the ground state and the transition state of the catalytic reaction, leading to the unaltered
. Our study provides new insights into the role of conformational dynamics in glycosyltransferase's function and new modality to modulate enzymatic efficiency.
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•Double mutant D11F/R122D remains catalytically active but can crystallize with an active site loop in an open conformation.•D11F/R122D causes a substrate-dependent change in ...activity, altering specificity.•Slow-timescale dynamics are perturbed in the fingers and thumb regions due to the double mutation.•Simulations show thumb opening in the double mutant is more energetically favorable vs. WT thumb opening.
While protein activity is traditionally studied with a major focus on the active site, the activity of enzymes has been hypothesized to be linked to the flexibility of adjacent regions, warranting more exploration into how the dynamics in these regions affects catalytic turnover. One such enzyme is Xylanase A (XylA), which cleaves hemicellulose xylan polymers by hydrolysis at internal β-1,4-xylosidic linkages. It contains a “thumb” region whose flexibility has been suggested to affect the activity. The double mutation D11F/R122D was previously found to affect activity and potentially bias the thumb region to a more open conformation. We find that the D11F/R122D double mutation shows substrate-dependent effects, increasing activity on the non-native substrate ONPX2 but decreasing activity on its native xylan substrate. To characterize how the double mutant causes these kinetics changes, nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations were used to probe structural and flexibility changes. NMR chemical shift perturbations revealed structural changes in the double mutant relative to the wild-type, specifically in the thumb and fingers regions. Increased slow-timescale dynamics in the fingers region was observed as intermediate-exchange line broadening. Lipari-Szabo order parameters show negligible changes in flexibility in the thumb region in the presence of the double mutation. To help understand if there is increased energetic accessibility to the open state upon mutation, alchemical free energy simulations were employed that indicated thumb opening is more favorable in the double mutant. These studies aid in further characterizing how flexibility in adjacent regions affects the function of XylA.
Bilayer hybrid composites fabricated by hand lay-up technique by impregnating oil palm empty fruit bunch and jute fibre mats with epoxy resin and cured at 100℃ for 1 h followed by post curing at ...105℃. Bilayer hybrid composites were prepared by varying the relative weight fraction of the two fibres. The mechanical, morphological and thermal properties of oil palm/jute bilayer hybrid composites were carried out. When the jute fibre loading is increased in the bilayer hybrid composites, flexural strength and modulus of the hybrid composites will be higher. The hybridization of the jute fibres with oil palm composite decreased the impact strength of the bilayer hybrid composites. Analysis of variance statistical of flexural and impact properties were also carried out; there is a statistically significant difference between the mean flexural strength, flexural modulus and impact strength from one level of composite to another at the 95.0% confidence level. Thermogravimetric analysis showed that thermal stability of oil palm composites increased with development of bilayer hybrid composites. Scanning electron micrographs of impact fracture samples are taken to study the failure mechanism, and fibre/matrix interface adhesion.
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•Lignin catabolic pathway explored for enzyme activity modulation by pathway metabolites.•Multiple lignin derived aromatic compounds inhibit DesB dioxygenase.•Protocatechuate exhibits ...mixed, competitive and non-competitive inhibition of DesB.•Computational docking revealed an evolutionarily conserved allosteric pocket in DesB.•Cross-talk within metabolic pathway should be considered for pathway engineering efforts.
Lignin is often considered an underutilized carbon source for the production of biofuels and other value-added compounds. Depolymerized lignin results in a collection of different aromatic compounds that have their own metabolic pathways for conversion into central metabolites. This investigation describes the chemical crosstalk of compounds between different metabolic pathways involved in the catabolism of lignin. Specifically, seven compounds were identified as inhibitors of the dioxygenase DesB. The most potent inhibitor, protocatechuate, moderately impacts both vmax and KM of DesB (71 s−1 and 167 μM versus 130 s−1 and 123 μM, in the presence and absence of protocatechuate, respectively) suggesting a mixed (competitive and non-competitive) inhibition profile. Molecular docking identified a putative allosteric pocket adjacent to the active site, in a position analogous to one identified previously for a related dioxygenase. This work demonstrates that lignin catabolic pathway intermediates can modulate the activity of multiple enzymes within the lignin degradation pathway and need to be considered when engineering organisms to utilize lignin for biomass conversion.
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