Encapsulation of enzymes allows to preserve their biological activities in various environmental conditions, such as exposure to elevated temperature or to proteases. This is particularly relevant for in vivo applications, where proteases represent a severe obstacle to maintaining the activity of enzymes. Polyelectrolyte multilayer capsules are suitable for enzyme encapsulation, where CaCO3 particles and temperature-dependent capsule formation are the best templates and the most adequate method, respectively. In this work, these two areas are combined and, ALP (alkaline phosphatase), which is a robust and therapeutically relevant enzyme, is encapsulated into thermally shrunk polyelectrolyte multilayer (PDADMAC/PSS)4 capsules templated on calcium carbonate particles (original average diameter: ≈3.5 µm). The activity of the encapsulated enzyme and the optimal temperature range for encapsulation are investigated. The enzymatic activity is almost four times higher upon encapsulation when the temperature range for encapsulation is situated just above the glass transition temperature (40 °C), while its optimal conditions are dictated, on the one hand, by the enzyme activity (better at lower temperatures) and, on the other hand, by the size and mechanical properties of capsules (better at higher temperatures).

Composite bioceramic and hydrogel-based containers harboring alkaline phosphatase are generated through encapsulation of this enzyme by its immobilization into CaCO3-based bioceramic materials in combination with a hydrogel assembly technique and subsequent gelification. A refined way of synthesis and modification allows preparing the enzyme delivery system with functionalized protection layers. The particles are characterized by electron microscopy, Fourier transform infrared (FTIR) spectroscopy, and enzyme activity measurements. Loading efficiency and loading capacity are investigated depending on particle size, time of enzyme loading, and various container compositions and enzyme concentrations. Our results reveal that the size of particles influences their morphology and this, in turn, affects the activity of the encapsulated enzymes. Various functionalizations of the surfaces, including protection by the hydrogel layer, formation of hollow silver alginate, or calcium alginate encapsulation, decrease the enzymatic activity. The presence of a good therapeutic effect on osteoblastic cells coupled with a relatively high loading capacity, biocompatibility, and ease of fabrication suggests that the developed carriers are promising candidates for efficient drug delivery, especially in the field of bone reconstruction.

We investigated melting transitions in biological membranes in their native state that include their membrane proteins. These membranes originated from \textit{E. coli}, \textit{B. subtilis}, lung surfactant and nerve tissue from the spinal cord of several mammals. For some preparations, we studied the pressure, pH and ionic strength dependence of the transition. For porcine spine, we compared the transition of the native membrane to that of the extracted lipids. All preparations displayed melting transitions of 10-20 degrees below physiological or growth temperature, independent of the organism of origin and the respective cell type. The position of transitions in \textit{E. coli} membranes depends on the growth temperature. We discuss these findings in the context of the thermodynamic theory of membrane fluctuations that leads to largely altered elastic constants, an increase in fluctuation lifetime and in membrane permeability associated with the transitions. We also discuss how to distinguish lipid transitions from protein unfolding transitions. Since the feature of a transition slightly below physiological temperature is conserved even when growth conditions change, we conclude that the transitions are likely to be of major biological importance for the survival and the function of the cell.

Human guanylate kinase (hGMPK) is the only known enzyme responsible for cellular GDP production, making it essential for cellular viability and proliferation. Moreover, hGMPK has been assigned a critical role in metabolic activation of antiviral and antineoplastic nucleoside-analog prodrugs. Given that hGMPK is indispensable for producing the nucleotide building blocks of DNA, RNA, and cGMP and that cancer cells possess elevated GTP levels, it is surprising that a detailed structural and functional characterization of hGMPK is lacking. Here, we present the first high-resolution structure of hGMPK in the apo form, determined with NMR spectroscopy. The structure revealed that hGMPK consists of three distinct regions designated as the LID, GMP-binding (GMP-BD), and CORE domains and is in an open configuration that is nucleotide binding-competent. We also demonstrate that nonsynonymous single-nucleotide variants (nsSNVs) of the hGMPK CORE domain distant from the nucleotide-binding site of this domain modulate enzymatic activity without significantly affecting hGMPK's structure. Finally, we show that knocking down the hGMPK gene in lung adenocarcinoma cell lines decreases cellular viability, proliferation, and clonogenic potential while not altering the proliferation of immortalized, noncancerous human peripheral airway cells. Taken together, our results provide an important step toward establishing hGMPK as a potential biomolecular target, from both an orthosteric (ligand-binding sites) and allosteric (location of CORE domain-located nsSNVs) standpoint.

Microorganisms are highly efficient biocatalysts. Yet making use of their capabilities for chemical transformations requiring synergistic interactions between different microbes is challenging as the competition for resources might reduce the diversity and ultimately disrupt the synergies. Here, a new method is proposed for constructing microbial consortia for the integration of multistep transformations. Bacteria are successively grown and trapped within semipermeable inorganic foams produced as millimeter-sized beads. The beads function as efficient living biocatalysts is demonstrated. These living heterogeneous biocatalysts are manipulated to perform cycles of biochemical reactions and furthermore assembled to perform preprogrammed sequences of reactions. This new family of living advanced biocatalysts should find applications in a wide range of basic research and industrial systems where complex tasks have to be performed by controlled consortia of microorganisms.

5-Aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR, or acadesine) is a precursor of the monophosphate derivative 5-amino-4-imidazole carboxamide ribonucleoside 5′-phosphate (ZMP), an intermediate in de novo purine biosynthesis. AICAR proved to have promising anti-proliferative properties, although the molecular basis of its toxicity is poorly understood. To exert cytotoxicity, AICAR needs to be metabolized, but the AICAR-derived toxic metabolite was not identified. Here, we show that ZMP is the major toxic derivative of AICAR in yeast and establish that its metabolization to succinyl-ZMP, ZDP, or ZTP (di- and triphosphate derivatives of AICAR) strongly reduced its toxicity. Affinity chromatography identified 74 ZMP-binding proteins, including 41 that were found neither as AMP nor as AICAR or succinyl-ZMP binders. Overexpression of karyopherin-β Kap123, one of the ZMP-specific binders, partially rescued AICAR toxicity. Quantitative proteomic analyses revealed 57 proteins significantly less abundant on nuclei-enriched fractions from AICAR-fed cells, this effect being compensated by overexpression of KAP123 for 15 of them. These results reveal nuclear protein trafficking as a function affected by AICAR.

L-asparaginase (L-ASNase) in an enzyme which catalyzes the hydrolysis of L-asparagine (L-Asn) to L-aspartic acid (L-Asp) and ammonia. E. coli L-ASNase is used in the treatment of acute lymphoblastic leukemia but induce strong immune responses [1]. However, three asparaginases are produced by the human genome and could be used as alternative therapeutic candidates with minimum immuno-response. One strategy is to improve the human asparaginase 3 ( ̃ 2000 times less active) using directed evolution. The human asparaginase 3 belonging to the Ntn-family hydrolase requires an intramolecular cleavage to become active [2]. This proteolysis is promoted by glycine and occurs between residues G167 and T168 resulting in two subunits α and β [3]. In this study, we developed a protocol permitting to promote autoproteolysis directly in bacterial culture. An expression system (APEx4b) has been implemented permitting to display the hASNase3 in the periplasm of bacteria. In order to detect asparaginase activity, we used a three-step coupled enzyme assay using the natural substrate of the enzyme, L-asparagine [4]. In this enzymatic cascade, L-asparaginase catalyzes the hydrolysis of L-asparagine into aspartate and ammonia. Then aspartate is oxidized by aspartate oxidase and flavin adenine dinucleotide into oxaloacetate and hydrogen peroxide. This hydrogen peroxide is finally used by horseradish peroxidase to oxidize Amplex Ultra Red into fluorescent resorufin. We successfully promoted the self-cleavage of hASNase3 localized in the periplasm using a high glycine concentration within the culture media. Currently, we plan on enhancing the catalytic activity of hASNase3 by directed evolution. Finally, the libraries obtained will be submitted to high throughput screening and sorted using the microfluidic platform developed in our team. Droplet-based microfluidic technology consists in controlling monodisperse water in oil (w/o) droplets at the single droplet level [5]. It can be used for single cell analysis and sorting and allows HTS of librairies [6,7]. References M. Strullu et al., Leuk Lymphoma. 51(8), 1464 (2010) K. Michalska et al., J Biol Chem. 283, 13388 (2008) Y. Su et al., Chem. Biol. 20, 533 (2013) ∗Speaker sciencesconf.org:biosynsys2017:169660 C. Karamitros et al., Anal. Biochem, 445, 20 (2014). R. Seemann et al., Rep. Prog. Phys, 75, 016601 (2011) J-C. Baret et al., Lab on a Chip, 9, 1850 (2009). J.J. Agresti et al., Proc. Natl. Acad. Sci. USA, 107, 4404 (2010).

Human guanylate kinase (hGMPK) is a critical enzyme that, in addition to phosphorylating its physiological substrate (d)GMP, catalyzes the second phosphorylation step in the conversion of anti-viral and anti-cancer nucleoside analogs to their corresponding active nucleoside analog triphosphates. Until now, a high-resolution structure of hGMPK is unavailable and thus, we studied free hGMPK by NMR and assigned the chemical shift resonances of backbone and side chain 1H, 13C, and 15N nuclei as a first step towards the enzyme’s structural and mechanistic analysis with atomic resolution.

Immunogenicity is one of the most common complications occurring during therapy making use of protein drugs of nonhuman origin. A notable example of such a case is bacterial l-asparaginases (L-ASNases) used for the treatment of acute lymphoblastic leukemia (ALL). The replacement of the bacterial enzymes by human ones is thought to set the basis for a major improvement of antileukemic therapy. Recently, we solved the crystal structure of a human enzyme possessing L-ASNase activity, designated hASNase-3. This enzyme is expressed as an inactive precursor protein and post-translationally undergoes intramolecular processing leading to the generation of two subunits which remain noncovalently, yet tightly associated and constitute the catalytically active form of the enzyme. We discovered that this intramolecular processing can be drastically and selectively accelerated by the free amino acid glycine. In the present study, we report on the molecular engineering of hASNase-3 aiming at the improvement of its catalytic properties. We created a fluorescence-activated cell sorting (FACS)-based high-throughput screening system for the characterization of rationally designed mutant libraries, capitalizing on the finding that free glycine promotes autoproteolytic cleavage, which activates the mutant proteins expressed in an E. coli strain devoid of aspartate biosynthesis. Successive screening rounds led to the isolation of catalytically improved variants showing up to 6-fold better catalytic efficiency as compared to the wild-type enzyme. Our work establishes a powerful strategy for further exploitation of the human asparaginase sequence space to facilitate the identification of in vitro-evolved enzyme species that will lay the basis for improved ALL therapy.

A new method of fabrication of calcium carbonate microparticles of ellipsoidal, rhomboidal, and spherical geometries is reported by adjusting the relative concentration ratios of the initial salt solutions and/or the ethylene glycol content in the reaction medium. Morphology, porosity, crystallinity, and loading capacity of synthesized CaCO3 templates were characterized in detail. Particles harboring dextran or the enzyme guanylate kinase were obtained through encapsulation of these macromolecules using the layer-by-layer assembly technique to deposit positively and negatively charged polymers on these differently shaped CaCO3 templates and were characterized by confocal laser scanning fluorescence microscopy, fluorometric techniques, and enzyme activity measurements. The enzymatic activity, an important application of such porous particles and containers, has been analyzed in comparison with the loading capacity and geometry. Our results reveal that the particles' shape influences morphology of particles and that, as a result, affects the activity of the encapsulated enzymes, in addition to the earlier reported influence on cellular uptake. These particles are promising candidates for efficient drug delivery due to their relatively high loading capacity, biocompatibility, and easy fabrication and handling.

Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the ckβ gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme's activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis.

Our work aims to ameliorate catalytic properties of L-asparaginase (L-ASNase) which is a protein drug used in antileukemic therapy. Bacterial L-ASNases are FDA-approved therapeutic enzymes for use in the treatment of various blood cancers to deplete serum L-Asn. Their therapeutic efficacy is based on the fact that several hematological malignancies, in particular Acute Lymphoblastic Leukemia (ALL), depend for growth on the extracellular supply of L-Asn. To avoid various side reactions inherent to the bacterial enzymes, it would be beneficial to substitute them with human L-ASNases. In order to find variants of improved specific activities in mutated enzyme libraries, we developed a droplet-based microfluidic platform for high throughput and miniaturization of kinetic assays that can be performed not only on purified enzymes, but also in single cells. When analyzing enzyme activity in bacterial cells, critical assumptions are: (i) protein expression levels are uniform in the population, and (ii) the reaction rate is not limited by the diffusion of small-molecule substrates/products to/from the enzyme. We demonstrate here a three-step coupled-enzyme assay for measuring ASNase activity on its natural substrate Asn, using water-in-oil droplets of nL to pL reaction volumes, at throughput rates of up to 104 per sec. Catalytic rates were found to vary by a factor of less than two within a population of more than 108 bacterial cells, following the same kinetics as the purified enzyme free in solution. To increase serum half-life of the enzyme, we loaded L-ASNases onto calcium carbonate nanoparticles serving as core templates, and manufactured biocompatible microcapsules under mild conditions to preserve catalytic activity, thereby improving protein stability and preventing exposure of the enzyme to the immune system. Encapsulated and free enzymes were able to kill leukemic cells with similar efficacy.

Bio-catalysis is the outcome of a subtle interplay between internal motions in enzymes and chemical kinetics. Small-angle X-ray scattering (SAXS) investigation of an enzyme's internal motions during catalysis offers an integral view of the protein's structural plasticity, dynamics, and function, which is useful for understanding allosteric effects and developing novel medicines. Guanylate kinase (GMPK) is an essential enzyme involved in the guanine nucleotide metabolism of unicellular and multicellular organisms. It is also required for the intracellular activation of numerous antiviral and anticancer purine nucleoside analog prodrugs. Catalytically active recombinant human GMPK (hGMPK) was purified for the first time and changes in the size and shape of open/closed hGMPK were tracked by SAXS. The binding of substrates (GMP + AMPPNP or Ap5G or GMP + ADP) resulted in the compaction of size and shape of hGMPK. The structural changes between open and completely closed hGMPK conformation were confirmed by observing differences in the hGMPK secondary structures with circular dichroism spectroscopy.

Droplet-based microfluidic technologies are powerful tools for applications requiring high-throughput, for example, in biochemistry or material sciences. Several systems have been proposed for the high-throughput production of monodisperse emulsions by parallelizing multiple droplet makers. However, these systems have two main limitations: (1) they allow the use of only a single disperse phase; (2) they are based on multiple layer microfabrication techniques. We present here a pipette-and-play solution offering the possibility of manipulating simultaneously 10 different disperse phases on a single layer device. This system allows high-throughput emulsion production using aqueous flow rates of up to 26 ml/h (>110 000 drops/s) leading to emulsions with user-defined complex chemical composition. We demonstrate the multiplex capabilities of our system by measuring the kinetics of β-galactosidase in droplets using nine different concentrations of a fluorogenic substrate.

A photo-electrochemical sensor for the specific detection of guanosine monophosphate (GMP) is demonstrated, based on three enzymes combined in a coupled reaction assay. The first reaction involves the adenosine triphosphate (ATP)-dependent conversion of GMP to guanosine diphosphate (GDP) by guanylate kinase, which warrants substrate specificity. The reaction products ADP and GDPare co-substrates for the enzymatic conversion of phosphoenolpyruvate to pyruvate in a second reaction mediated by pyruvate kinase. Pyruvate in turn is the co-substrate for lactate dehydrogenase that generates lactate via oxidation of nicotinamide adenine dinucleotide (reduced form) NADH to NAD(+). This third enzymatic reaction is electrochemically detected. For this purpose a CdS/ZnS quantum dot (QD) electrode is illuminated and the photocurrent response under fixed potential conditions is evaluated. The sequential enzyme reactions are first evaluated in solution. Subsequently, a sensor for GMP is constructed using polyelectrolytes for enzyme immobilization.

The structural and functional characterization of human enzymes that are of potential medical and therapeutic interest is of prime significance for translational research. One of the most notable examples of a therapeutic enzyme is L-asparaginase, which has been established as an antileukemic protein drug for more than four decades. Up until now, only bacterial enzymes have been used in therapy despite a plethora of undesired side effects mainly attributed to the bacterial origins of these enzymes. Therefore, the replacement of the currently approved bacterial drugs by human homologs aiming at the elimination of adverse effects is of great importance. Recently, we structurally and biochemically characterized the enzyme human L-asparaginase 3 (hASNase3), which possesses L-asparaginase activity and belongs to the N-terminal nucleophile superfamily of enzymes. Inspired by the necessity for the development of a protein drug of human origin, in the present study, we focused on the characterization of another human L-asparaginase, termed hASNase1. This bacterial-type cytoplasmic L-asparaginase resides in the N-terminal subdomain of an overall 573-residue protein previously reported to function as a lysophospholipase. Our kinetic, mutagenesis, structural modeling, and fluorescence labeling data highlight allosteric features of hASNase1 that are similar to those of its Escherichia coli homolog, EcASNase1. Differential scanning fluorometry and urea denaturation experiments demonstrate the impact of particular mutations on the structural and functional integrity of the L-asparaginase domain and provide a direct comparison of sites critical for the conformational stability of the human and E. coli enzymes.

The limitations of the ganciclovir (GCV)/herpes simplex virus thymidine kinase (HSV1 TK: EC 2.7.1.21) system as a suicide gene therapy approach have been extensively studied over the years. In our study, we focused on improving the cytotoxic profile of the GCV/equine herpes virus-4 thymidine kinase (EHV4 TK: EC 2.7.1.21) system. Our approach involved the structure-guided mutagenesis of EHV4 TK in order to switch its ability to preferentially phosphorylate the natural substrate deoxythymidine (dT) to that of GCV. We performed steady-state kinetic analysis, genetic complementation in a thymidine kinase-deficient Escherichia coli strain, isothermal titration calorimetry, and analysis of GCV-induced cell killing through generation of HEK 293 stable cell-lines expressing EHV4 TK mutants and wild-type EHV4 TK. We found that the EHV4 TK S144H-GFP mutant preferentially phosphorylates GCV and confers increased GCV-induced cytotoxicity compared to wild-type EHV4 TK.

l-asparaginases hydrolyze l-asparagine to l-aspartic acid and ammonia. Enzymes of bacterial origin are used as therapeutic agents for the treatment of acute lymphoblastic leukemia. Recently, the structure of a human homolog, hASNase3, which possesses l-asparaginase activity, was solved setting the basis for the development of an anti-leukemic protein drug of human origin. Being an N-terminal hydrolase, hASNase3 undergoes intramolecular self-cleavage generating two protomers (subunits α and β) which remain non-covalently associated and constitute the catalytically active form of the enzyme. However, recombinant expression of full-length hASNase3 in Escherichiacoli results in only partial processing towards the active enzyme. We developed a co-expression system for the two subunits that allowed production of the β-subunit complexed to the α-subunit such that the N-terminal methionine is removed by endogenous methionine aminopeptidase to expose the catalytically essential threonine residue at the N-terminus of the β-subunit. The enzyme produced by this co-expression strategy is fully active, thus obviating the necessity of self-activation by slow autoproteolytic cleavage.