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Biomaterials capable of delivering therapeutic proteins are relevant in biomedicine, yet their manufacturing relies on centralized manufacturing chains that pose challenges to their remote implementation at the point of care. This study explores the viability of confined cell-free protein synthesis within porous hydrogels as biomaterials that dynamically produce and deliver proteins to in vitro and in vivo biological microenvironments. These functional biomaterials have the potential to be assembled as implants at the point of care. To this aim, we first entrap cell-free extracts (CFEs) from Escherichia coli containing the transcription-translation machinery, together with plasmid DNA encoding the super folded green fluorescence protein (sGFP) as a model protein, into hydrogels using various preparation methods. Agarose hydrogels result in the most suitable biomaterials to confine the protein synthesis system, demonstrating efficient sGFP production and diffusion from the core to the surface of the hydrogel. Freeze-drying (FD) of agarose hydrogels still allows for the synthesis and diffusion of sGFP, yielding a more attractive biomaterial for its reconstitution and implementation at the point of care. FD-agarose hydrogels are biocompatible in vitro, allowing for the colonization of cell microenvironments along with cell proliferation. Implantation assays of this biomaterial in a preclinical mouse model proved the feasibility of this protein synthesis approach in an in vivo context and indicated that the physical properties of the biomaterials influence their immune responses. This work introduces a promising avenue for biomaterial fabrication, enabling the in vivo synthesis and targeted delivery of proteins and opening new paths for advanced protein therapeutic approaches based on biocompatible biomaterials.

Enzyme immobilization is a key enabling technology for a myriad of industrial applications, yet immobilization science is still too empirical to reach highly active and robust heterogeneous biocatalysts through a general approach. Conventional protein immobilization methods lack control over how enzymes are oriented on solid carriers, resulting in negative conformational changes that drive enzyme deactivation. Site-selective enzyme immobilization through peptide tags and protein domains addresses the orientation issue, but this approach limits the possible orientations to the N- and C-termini of the target enzyme. In this work, we engineer the surface of two model dehydrogenases to introduce histidine clusters into flexible regions not involved in catalysis, through which immobilization is driven. By varying the position and the histidine density of the clusters, we create a small library of enzyme variants to be immobilized on different carriers functionalized with different densities of various metal chelates (Co 2+ , Cu 2+ , Ni 2+ , and Fe 3+ ). We first demonstrate that His-clusters can be as efficient as the conventional His-tags in immobilizing enzymes, recovering even more activity and gaining stability against some denaturing agents. Furthermore, we find that the enzyme orientation as well as the type and density of the metal chelates affect the immobilization parameters (immobilization yield and recovered activity) and the stability of the immobilized enzymes. According to proteomic studies, His-clusters enable a different enzyme orientation as compared to His-tag. Finally, these oriented heterogeneous biocatalysts are implemented in batch reactions, demonstrating that the stability achieved by an optimized orientation translates into increased operational stability.

Multidimensional kinetic analysis of immobilized enzymes is essential to understand the enzyme functionality at the interface with solid materials. However, spatiotemporal kinetic characterization of heterogeneous biocatalysts on a microscopic level and under operando conditions has been rarely approached. As a case study, we selected self-sufficient heterogeneous biocatalysts where His-tagged cofactor-dependent enzymes (dehydrogenases, transaminases, and oxidases) are co-immobilized with their corresponding phosphorylated cofactors [nicotinamide adenine dinucleotide phosphate (NAD(P)H), pyridoxal phosphate (PLP), and flavin adenine dinucleotide (FAD)] on porous agarose microbeads coated with cationic polymers. These self-sufficient systems do not require the addition of exogenous cofactors to function, thus avoiding the extensive use of expensive cofactors. To comprehend the microscopic kinetics and thermodynamics of self-sufficient systems, we performed fluorescence recovery after photobleaching measurements, time-lapse fluorescence microscopy, and image analytics at both single-particle and intraparticle levels. These studies reveal a thermodynamic equilibrium that rules out the reversible interactions between the adsorbed phosphorylated cofactors and the polycations within the pores of the carriers, enabling the confined cofactors to access the active sites of the immobilized enzymes. Furthermore, this work unveils the relationship between the apparent Michaelis-Menten kinetic parameters and the enzyme density in the confined space, eliciting a negative effect of molecular crowding on the performance of some enzymes. Finally, we demonstrate that the intraparticle apparent enzyme kinetics are significantly affected by the enzyme spatial organization. Hence, multiscale characterization of immobilized enzymes serves as an instrumental tool to better understand the in operando functionality of enzymes within confined spaces., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

The rapid demand for protein-based molecules has stimulated much research on cell-free protein synthesis (CFPS); however, there are still many challenges in terms of cost-efficiency, process intensification, and sustainability. Herein, we describe the microcompartmentalization of CFPS of superfolded green fluorescent protein (sGFP) in alginate hydrogels, which were casted into a μ-channel device. CFPS was optimized for the microcompartmentalized environment and characterized in terms of synthesis yield. To extend the scope of this technology, the use of other biocompatible materials (collagen, laponite, and agarose) was explored. In addition, the diffusion of sGFP from the hydrogel microenvironment to the bulk was demonstrated, opening a promising opportunity for concurrent synthesis and delivery of proteins. Finally, we provide an application for this system: the CFPS of enzymes. The present design of the hydrogel μ-channel device may enhance the potential application of microcompartmentalized CFPS in biosensing, bioprototyping, and therapeutic development.

The phosphatidyl- myo -inositol mannosyltransferase A (PimA) is an essential peripheral membrane glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl- myo -inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis . PimA undergoes functionally important conformational changes, including (i) α-helix-to-β-strand and β-strand-to-α-helix transitions and (ii) an "open-to-closed" motion between the two Rossmann-fold domains, a conformational change that is necessary to generate a catalytically competent active site. In previous work, we established that GDP-Man and GDP stabilize the enzyme and facilitate the switch to a more compact active state. To determine the structural contribution of the mannose ring in such an activation mechanism, we analyzed a series of chemical derivatives, including mannose phosphate (Man-P) and mannose pyrophosphate-ribose (Man-PP-RIB), and additional GDP derivatives, such as pyrophosphate ribose (PP-RIB) and GMP, by the combined use of X-ray crystallography, limited proteolysis, circular dichroism, isothermal titration calorimetry, and small angle X-ray scattering methods. Although the β-phosphate is present, we found that the mannose ring, covalently attached to neither phosphate (Man-P) nor PP-RIB (Man-PP-RIB), does promote the switch to the active compact form of the enzyme. Therefore, the nucleotide moiety of GDP-Man, and not the sugar ring, facilitates the "open-to-closed" motion, with the β-phosphate group providing the high-affinity binding to PimA. Altogether, the experimental data contribute to a better understanding of the structural determinants involved in the "open-to-closed" motion not only observed in PimA but also visualized and/or predicted in other glycosyltransfeases. In addition, the experimental data might prove to be useful for the discovery and/or development of PimA and/or glycosyltransferase inhibitors.

Spinal cord injury (SCI) limits life expectancy and causes a restriction of patient's daily activities. In the last years, robotics exoskeletons have appeared as a promising rehabilitation and assistance tool for patients with motor limitations, as people that have suffered a SCI. The usability and clinical relevance of these robotics systems could be further enhanced by brain-machine interfaces (BMIs), as they can be used to foster patients' neuroplasticity. However, there are not many studies showing the use of BMIs to control exoskeletons with patients. In this work we show a case study where one SCI patient has used a BMI based on motor imagery (MI) in order to control a lower limb exoskeleton that assists their gait.

Glycogen and starch are the major carbon and energy reserve polysaccharides in nature, providing living organisms with a survival advantage. The evolution of the enzymatic machinery responsible for the biosynthesis and degradation of such polysaccharides, led the development of mechanisms to control the assembly and disassembly rate, to store and recover glucose according to cell energy demands. The tetrameric enzyme ADP-glucose pyrophosphorylase (AGPase) catalyzes and regulates the initial step in the biosynthesis of both α-polyglucans. AGPase displays cooperativity and allosteric regulation by sensing metabolites from the cell energy flux. The understanding of the allosteric signal transduction mechanisms in AGPase arises as a long-standing challenge. In this work, we disclose the cryoEM structures of the paradigmatic homotetrameric AGPase from Escherichia coli ( Ec AGPase), in complex with either positive or negative physiological allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP respectively, both at 3.0 Å resolution. Strikingly, the structures reveal that FBP binds deeply into the allosteric cleft and overlaps the AMP site. As a consequence, FBP promotes a concerted conformational switch of a regulatory loop, RL2, from a "locked" to a "free" state, modulating ATP binding and activating the enzyme. This notion is strongly supported by our complementary biophysical and bioinformatics evidence, and a careful analysis of vast enzyme kinetics data on single-point mutants of Ec AGPase. The cryoEM structures uncover the residue interaction networks (RIN) between the allosteric and the catalytic components of the enzyme, providing unique details on how the signaling information is transmitted across the tetramer, from which cooperativity emerges. Altogether, the conformational states visualized by cryoEM reveal the regulatory mechanism of Ec AGPase, laying the foundations to understand the allosteric control of bacterial glycogen biosynthesis at the molecular level of detail., Competing Interests: The authors declare no financial conflict of interest., (© 2020 The Author(s).)

The self-assembly of styrene-type olefins into the corresponding stilbenes was conveniently performed in the Deep Eutectic Solvent ( DES ) mixture 1 ChCl /2 Gly under air and in the absence of hazardous organic co-solvents using a one-pot chemo-biocatalytic route. Here, an enzymatic decarboxylation of p -hydroxycinnamic acids sequentially followed by a ruthenium-catalyzed metathesis of olefins has been investigated in DES . Moreover, and to extend the design of chemoenzymatic processes in DESs , we also coupled the aforementioned enzymatic decarboxylation reaction to now concomitant Pd-catalyzed Heck-type C-C coupling to produce biaryl derivatives under environmentally friendly reaction conditions., (Copyright © 2020 Ríos-Lombardía, Rodríguez-Álvarez, Morís, Kourist, Comino, López-Gallego, González-Sabín and García-Álvarez.)

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., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Glycosyltransferases (GTs) play a central role in nature. They catalyze the transfer of a sugar moiety to a broad range of acceptor substrates. GTs are highly selective enzymes, allowing the recognition of subtle structural differences in the sequences and stereochemistry of their sugar and acceptor substrates. We report here a series of structural snapshots of the reaction center of the retaining glucosyl-3-phosphoglycerate synthase (GpgS). During this sequence of events, we visualize how the enzyme guides the substrates into the reaction center where the glycosyl transfer reaction takes place, and unveil the mechanism of product release, involving multiple conformational changes not only in the substrates/products but also in the enzyme. The structural data are further complemented by metadynamics free-energy calculations, revealing how the equilibrium of loop conformations is modulated along these itineraries. The information reported here represent an important contribution for the understanding of GT enzymes at the molecular level., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

ADP-glucose pyrophosphorylase (AGPase) controls bacterial glycogen and plant starch biosynthetic pathways, the most common carbon storage polysaccharides in nature. AGPase activity is allosterically regulated by a series of metabolites in the energetic flux within the cell. Very recently, we reported the first crystal structures of the paradigmatic AGPase from Escherichia coli ( Ec AGPase) in complex with its preferred physiological negative and positive allosteric regulators, adenosine 5'-monophosphate (AMP) and fructose 1,6-bisphosphate (FBP), respectively. However, understanding the molecular mechanism by which AMP and FBP allosterically modulates Ec AGPase enzymatic activity still remains enigmatic. Here we found that single point mutations of key residues in the AMP-binding site decrease its inhibitory effect but also clearly abolish the overall AMP-mediated stabilization effect in wild-type Ec AGPase. Single point mutations of key residues for FBP binding did not revert the AMP-mediated stabilization. Strikingly, an Ec AGPase-R130A mutant displayed a dramatic increase in activity when compared with wild-type Ec AGPase, and this increase correlated with a significant increment of glycogen content in vivo The crystal structure of Ec AGPase-R130A revealed unprecedented conformational changes in structural elements involved in the allosteric signal transmission. Altogether, we propose a model in which the positive and negative energy reporters regulate AGPase catalytic activity via intra- and interprotomer cross-talk, with a "sensory motif" and two loops, RL1 and RL2, flanking the ATP-binding site playing a significant role. The information reported herein provides exciting possibilities for industrial/biotechnological applications., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step of bacterial glycogen and plant starch biosynthesis, the most common carbon storage polysaccharides in nature. A major challenge is to understand how AGPase activity is regulated by metabolites in the energetic flux within the cell. Here we report crystal structures of the homotetrameric AGPase from Escherichia coli in complex with its physiological positive and negative allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP, and sucrose in the active site. FBP and AMP bind to partially overlapping sites located in a deep cleft between glycosyltransferase A-like and left-handed β helix domains of neighboring protomers, accounting for the fact that sensitivity to inhibition by AMP is modulated by the concentration of the activator FBP. We propose a model in which the energy reporters regulate EcAGPase catalytic activity by intra-protomer interactions and inter-protomer crosstalk, with a sensory motif and two regulatory loops playing a prominent role., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannoside, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of membrane-associated glycosyltransferases for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here, we determined that PimA preferentially binds to negatively charged phosphatidyl-myo-inositol substrate and non-substrate membrane model systems (small unilamellar vesicle) through its N-terminal domain, inducing an important structural reorganization of anionic phospholipids. By using a combination of single-point mutagenesis, circular dichroism, and a variety of fluorescence spectroscopy techniques, we determined that this interaction is mainly mediated by an amphipathic α-helix (α2), which undergoes a substantial conformational change and localizes in the vicinity of the negatively charged lipid headgroups and the very first carbon atoms of the acyl chains, at the PimA-phospholipid interface. Interestingly, a flexible region within the N-terminal domain, which undergoes β-strand-to-α-helix and α-helix-to-β-strand transitions during catalysis, interacts with anionic phospholipids; however, the effect is markedly less pronounced to that observed for the amphipathic α2, likely reflecting structural plasticity/variability. Altogether, we propose a model in which conformational transitions observed in PimA might reflect a molten globule state that confers to PimA, a higher affinity toward the dynamic and highly fluctuating lipid bilayer., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin-fold with a Cys(19)-Pro(20)-Trp(21)-Cys(22) motif, and an insertion consisting of a four α-helical bundle and a short α-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in α-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle x-ray scattering showed that the fully reduced form of Rv2466c adopts a "closed" compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an "open" conformation, where tertiary structural changes in the α-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical, and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Glycosyltransferases (GTs) comprise a prominent family of enzymes that play critical roles in a variety of cellular processes, including cell signaling, cell development, and host-pathogen interactions. Glycosyl transfer can proceed with either inversion or retention of the anomeric configuration with respect to the reaction substrates and products. The elucidation of the catalytic mechanism of retaining GTs remains a major challenge. A native ternary complex of a GT in a productive mode for catalysis is reported, that of the retaining glucosyl-3-phosphoglycerate synthase GpgS from M. tuberculosis in the presence of the sugar donor UDP-Glc, the acceptor substrate phosphoglycerate, and the divalent cation cofactor. Through a combination of structural, chemical, enzymatic, molecular dynamics, and quantum-mechanics/molecular-mechanics (QM/MM) calculations, the catalytic mechanism was unraveled, thereby providing a strong experimental support for a front-side substrate-assisted SN i-type reaction., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Secondary structure refolding is a key event in biology as it modulates the conformation of many proteins in the cell, generating functional or aberrant states. The crystal structures of mannosyltransferase PimA reveal an exceptional flexibility of the protein along the catalytic cycle, including β-strand-to-α-helix and α-helix-to-β-strand transitions. These structural changes modulate catalysis and are promoted by interactions of the protein with anionic phospholipids in the membrane.

The emergence of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis highlights the need to discover new antitubercular agents. Here we describe the synthesis and characterization of a new series of thienopyrimidine (TP) compounds that kill both replicating and non-replicating M. tuberculosis. The strategy to determine the mechanism of action of these TP derivatives was to generate resistant mutants to the most effective compound TP053 and to isolate the genetic mutation responsible for this phenotype. The only non-synonymous mutation found was a g83c transition in the Rv2466c gene, resulting in the replacement of tryptophan 28 by a serine. The Rv2466c overexpression increased the sensitivity of M. tuberculosis wild-type and resistant mutant strains to TP053, indicating that TP053 is a prodrug activated by Rv2466c. Biochemical studies performed with purified Rv2466c demonstrated that only the reduced form of Rv2466c can activate TP053. The 1.7 Å resolution crystal structure of the reduced form of Rv2466c, a protein whose expression is transcriptionally regulated during the oxidative stress response, revealed a unique homodimer in which a β-strand is swapped between the thioredoxin domains of each subunit. A pronounced groove harboring the unusual active-site motif CPWC might account for the uncommon reactivity profile of the protein. The mutation of Trp28Ser clearly predicts structural defects in the thioredoxin fold, including the destabilization of the dimerization core and the CPWC motif, likely impairing the activity of Rv2466c against TP053. Altogether our experimental data provide insights into the molecular mechanism underlying the anti-mycobacterial activity of TP-based compounds, paving the way for future drug development programmes.

Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of peripheral membrane-associated GTs for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here we used single molecule force spectroscopy techniques to study the mechanical and conformational properties of PimA. In our studies, we engineered a polyprotein containing PimA flanked by four copies of the well characterized I27 protein, which provides an unambiguous mechanical fingerprint. We found that PimA exhibits weak mechanical stability albeit displaying β-sheet topology expected to unfold at much higher forces. Notably, PimA unfolds following heterogeneous multiple step mechanical unfolding pathways at low force akin to molten globule states. Interestingly, the ab initio low resolution envelopes obtained from small angle x-ray scattering of the unliganded PimA and the PimA·GDP complexed forms clearly demonstrate that not only the "open" and "closed" conformations of the GT-B enzyme are largely present in solution, but in addition, PimA experiences remarkable flexibility that undoubtedly corresponds to the N-terminal "Rossmann fold" domain, which has been proved to participate in protein-membrane interactions. Based on these results and on our previous experimental data, we propose a model wherein the conformational transitions are important for the mannosyltransferase to interact with the donor and acceptor substrates/membrane.