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Here we describe a complex enzymatic approach to the efficient transformation of abundant waste chitin, a byproduct of the food industry, into valuable chitooligomers with a degree of polymerization (DP) ranging from 6 to 11. This method involves a three-step process: initial hydrolysis of chitin using engineered variants of a novel fungal chitinase from Talaromyces flavus to generate low-DP chitooligomers, followed by an extension to the desired DP using the high-yielding Y445N variant of ß-N-acetylhexosaminidase from Aspergillus oryzae, achieving yields of up to 57%. Subsequently, enzymatic deacetylation of chitooligomers with DP 6 and 7 was accomplished using peptidoglycan deacetylase from Bacillus subtilis BsPdaC. The innovative enzymatic procedure demonstrates a sustainable and feasible route for converting waste chitin into unavailable bioactive chitooligomers potentially applicable as natural pesticides in ecological and sustainable agriculture.
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Aspergillus oryzae , Quitina , Quitinasas , Proteínas Fúngicas , Oligosacáridos , Talaromyces , Quitina/metabolismo , Quitina/química , Quitinasas/metabolismo , Quitinasas/genética , Quitinasas/química , Talaromyces/enzimología , Talaromyces/genética , Talaromyces/química , Talaromyces/metabolismo , Oligosacáridos/metabolismo , Oligosacáridos/química , Hidrólisis , Aspergillus oryzae/enzimología , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Bacillus subtilis/genética , Bacillus subtilis/enzimología , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Biocatálisis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/químicaRESUMEN
Chitin deacetylases (CDAs) emerge as a valuable tool to produce chitosans with a nonrandom distribution of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) units. We hypothesized before that CDAs tend to bind certain sequences within the substrate matching their subsite preferences for either GlcNAc or GlcN units. Thus, they deacetylate or N-acetylate their substrates at nonrandom positions. To understand the molecular basis of these preferences, we analyzed the binding site of a CDA from Pestalotiopsis sp. (PesCDA) using a detailed activity screening of a site-saturation mutagenesis library. In addition, molecular dynamics simulations were conducted to get an in-depth view of crucial interactions along the binding site. Besides elucidating the function of several amino acids, we were able to show that only 3 residues are responsible for the highly specific binding of PesCDA to oligomeric substrates. The preference to bind a GlcNAc unit at subsite -2 and -1 can mainly be attributed to N75 and H199, respectively. Whereas an exchange of N75 at subsite -2 eliminates enzyme activity, H199 can be substituted with tyrosine to increase the GlcN acceptance at subsite -1. This change in substrate preference not only increases enzyme activity on certain substrates and changes composition of oligomeric products but also significantly changes the pattern of acetylation (PA) when N-acetylating polyglucosamine. Consequently, we could clearly show how subsite preferences influence the PA of chitosans produced with CDAs.
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Quitosano , Quitosano/química , Quitosano/metabolismo , Quitina/química , Quitina/metabolismo , Polímeros/metabolismo , Amidohidrolasas/genética , Amidohidrolasas/química , Amidohidrolasas/metabolismo , AcetilaciónRESUMEN
Bacterial ß-glycosidases are hydrolytic enzymes that depolymerize polysaccharides such as ß-cellulose, ß-glucans and ß-xylans from different sources, offering diverse biomedical and industrial uses. It has been shown that a conformational change of the substrate, from a relaxed 4 C1 conformation to a distorted 1 S3 /1,4 B conformation of the reactive sugar, is necessary for catalysis. However, the molecular determinants that stabilize the substrate's distortion are poorly understood. Here we use quantum mechanics/molecular mechanics (QM/MM)-based molecular dynamics methods to assess the impact of the interaction between the reactive sugar, i. e. the one at subsite -1, and the catalytic nucleophile (a glutamate) on substrate conformation. We show that the hydrogen bond involving the C2 exocyclic group and the nucleophile controls substrate conformation: its presence preserves sugar distortion, whereas its absence (e.g. in an enzyme mutant) knocks it out. We also show that 2-deoxy-2-fluoro derivatives, widely used to trap the reaction intermediates by X-ray crystallography, reproduce the conformation of the hydrolysable substrate at the experimental conditions. These results highlight the importance of the 2-OHâ â â nucleophile interaction in substrate recognition and catalysis in endo-glycosidases and can inform mutational campaigns aimed to search for more efficient enzymes.
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Glicósido Hidrolasas , Simulación de Dinámica Molecular , Glicósido Hidrolasas/metabolismo , Enlace de Hidrógeno , Conformación Proteica , Azúcares , Especificidad por Sustrato , Cristalografía por Rayos X , CatálisisRESUMEN
mRNA vaccination has emerged as a prominent therapy for the future of medicine. Despite the colossal advance in this technology and worldwide efficacy proof (ca. COVID vaccines), mRNA carriers still lack cell/tissue specificity, leading to possible side effects, and reduced efficacy among others. Herein we make use of the ubiquitous affinity of antigen-presenting cells (APC)s for glycosides to achieve specific targeting. To achieve this goal, we designed a new generation of α-mannosyl functionalized oligopeptide-terminated poly(ß-aminoester). Fine formulation of these polymers with mRNA resulted in nanoparticles decorated with surface-exposed α-mannoses with sizes around 180 nm and positive surface charge. Notably, these particles maintained their properties after freeze-drying and subsequent redispersion. Finally, our mRNA carriers preferentially targeted and transfected APCs in vitro and in vivo. In conclusion, we demonstrated, at a preclinical level, that the mannose functionalization enables more selective targeting of APCs and, thus, these polymer and nanoparticles are candidates for a new generation of mRNA immunotherapy vaccines.
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COVID-19 , Nanopartículas , Vacunas , Humanos , Manosa , Bazo , Células Presentadoras de Antígenos , ARN Mensajero/genéticaRESUMEN
Hydroxyalkanoyloxyalkanoates (HAA) are lipidic surfactants with a number of potential applications, but more remarkably, they are the biosynthetic precursors of rhamnolipids (RL), which are preferred biosurfactants thanks to their excellent physicochemical properties, biological activities, and environmental biodegradability. Because the natural highest producer of RLs is the pathogenic bacterium Pseudomonas aeruginosa, important efforts have been dedicated to transfer production to heterologous non-pathogenic microorganisms. Unicellular photosynthetic microalgae are emerging as important hosts for sustainable industrial biotechnology due to their ability to transform CO2 efficiently into biomass and bioproducts of interest. Here, we have explored the potential of the eukaryotic green microalgae Chlamydomonas reinhardtii as a chassis to produce RLs. Chloroplast genome engineering allowed the stable functional expression of the gene encoding RhlA acyltransferase from P. aeruginosa, an enzyme catalyzing the condensation of two 3-hydroxyacyl acid intermediaries in the fatty acid synthase cycle, to produce HAA. Four congeners of varying chain lengths were identified and quantified by UHPLC-QTOF mass spectrometry and gas chromatography, including C10-C10 and C10-C8, and the less abundant C10-C12 and C10-C6 congeners. HAA was present in the intracellular fraction, but also showed increased accumulation in the extracellular medium. Moreover, HAA production was also observed under photoautotrophic conditions based on atmospheric CO2. These results establish that RhlA is active in the chloroplast and is able to produce a new pool of HAA in a eukaryotic host. Subsequent engineering of microalgal strains should contribute to the development of an alternative clean, safe and cost-effective platform for the sustainable production of RLs.
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Chlamydomonas reinhardtii , Microalgas , Microalgas/genética , Microalgas/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Dióxido de Carbono , Cromatografía de Gases y Espectrometría de Masas , Glucolípidos/química , Cloroplastos/metabolismoRESUMEN
BACKGROUND: Vitamin B12 is a widely used compound in the feed and food, healthcare and medical industries that can only be produced by fermentation because of the complexity of its chemical synthesis. Besides, the use of Generally Recognized as Safe (GRAS) and Qualified Presumption of Safety (QPS) microorganisms, like Propionibacterium freudenreichii, especially non-GMO wild-type producers, are becoming an interesting alternative in markets where many final consumers have high health and ecological awareness. In this study, the production of vitamin B12 using the Propionibacterium freudenreichii NBRC 12391 wild-type strain was characterized and optimized in shake flasks before assessing several scale-up strategies. RESULTS: Initial results established that: (i) agitation during the early stages of the culture had an inhibitory effect on the volumetric production, (ii) 5,6-dimethylbenzimidazole (DMBI) addition was necessary for vitamin B12 production, and (iii) kinetics of vitamin B12 accumulation were dependent on the induction time when DMBI was added. When scaling up in a bioreactor, both batch and fed-batch bioprocesses proved unsuitable for obtaining high volumetric productivities mainly due to carbon source limitation and propionic acid inhibition, respectively. To overcome these drawbacks, an anaerobic single-phase continuous bioprocess strategy was developed. This culture strategy was maintained stable during more than 5 residence times in two independent cultures, resulting in 5.7-fold increase in terms of volumetric productivity compared to other scale-up strategies. CONCLUSION: Overall, compared to previously reported strategies aimed to reduce propionic acid inhibition, a less complex anaerobic single-phase continuous and more scalable bioprocess was achieved.
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Propionibacterium freudenreichii , Vitamina B 12 , Propionibacterium , Propionatos , Fermentación , VitaminasRESUMEN
Glycosyltransferases (GT) catalyse the biosynthesis of complex carbohydrates which are the most abundant group of molecules in nature. They are involved in several key mechanisms such as cell signalling, biofilm formation, host immune system invasion or cell structure and this in both prokaryotic and eukaryotic cells. As a result, research towards complete enzyme mechanisms is valuable to understand and elucidate specific structure-function relationships in this group of molecules. In a next step this knowledge could be used in GT protein engineering, not only for rational drug design but also for multiple biotechnological production processes, such as the biosynthesis of hyaluronan, cellooligosaccharides or chitooligosaccharides. Generation of these poly- and/or oligosaccharides is possible due to a common feature of several of these GTs: processivity. Enzymatic processivity has the ability to hold on to the growing polymer chain and some of these GTs can even control the number of glycosyl transfers. In a first part, recent advances in understanding the mechanism of various processive enzymes are discussed. To this end, an overview is given of possible engineering strategies for the purpose of new industrial and fundamental applications. In the second part of this review, we focused on specific chain length-controlling mechanisms, i.e., key residues or conserved regions, and this for both eukaryotic and prokaryotic enzymes.
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Carbohidratos , Glicosiltransferasas , Glicosiltransferasas/metabolismo , Glicosilación , Ingeniería de Proteínas , Células Eucariotas/metabolismoRESUMEN
Glycolipids are complex molecules involved in important cellular processes. Among them, the glycosphingolipid α-galactosylceramide has proven to be of interest in biomedicine for its immunostimulatory capabilities. Given its structural requirements, the use of ceramide glycosyltransferase enzymes capable of synthesizing this molecule under in vivo or in vitro conditions is a potential production strategy. Several GT4 enzymes from Bacteroides fragilis were considered as potential candidates in addition to the known BF9343_3149, but only this one showed glycolipid synthase activity. The enzyme was expressed as a SUMO fusion protein to produce soluble protein. It is a non-processive glycosyltransferase that prefers UDP-Gal over UDP-Glc as a donor substrate, and maximum activity was found at pH 7.3 and around 30-35 °C. It does not require metal cations for activity as other GT4 enzymes, but Zn2+ inactivates the enzyme. The reaction occurs when the ceramide lipid acceptor is solubilized with BSA (100% conversion) but not when it is presented in mixed micelles, and anionic lipids do not increase activity, as in other membrane-associated glycolipid synthases. Further protein engineering to increase stability and activity can make feasible the enzymatic synthesis of α-GalCer for biomedical applications.
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Infecciones Bacterianas , Glucolípidos , Humanos , Glucolípidos/química , Bacteroides fragilis/metabolismo , Glicosiltransferasas/metabolismo , Óxido Nítrico Sintasa , Ceramidas , Uridina DifosfatoRESUMEN
Vitamin B12 is a widely used compound in the feed and food, healthcare and medical industries that can only be produced by fermentation because of the complexity of its chemical synthesis. For this reason, finding better producer strains and optimizing their bioprocesses have been the main focus of industrial producers over the last few decades. In this review, we initially provide a historical overview of vitamin B12 research and the main biosynthetic characteristics of the two microorganism families typically used for its industrial production: several strains of Propionibacterium freudenreichii and strains related to Pseudomonas denitrificans. Later, a complete summary of the current state of vitamin B12 industrial production as well as the main advances and challenges for improving it is detailed, with a special focus on bioprocess optimization, which aims not only to increase production but also sustainability. In addition, a comprehensive list of the most important and relevant patents for the present industrial strains is provided. Finally, the potential applications of vitamin B12 in different markets are discussed.
RESUMEN
AbnA is an extracellular GH43 α-L-arabinanase from Geobacillus stearothermophilus, a key bacterial enzyme in the degradation and utilization of arabinan. We present herein its full-length crystal structure, revealing the only ultra-multimodular architecture and the largest structure to be reported so far within the GH43 family. Additionally, the structure of AbnA appears to contain two domains belonging to new uncharacterized carbohydrate-binding module (CBM) families. Three crystallographic conformational states are determined for AbnA, and this conformational flexibility is thoroughly investigated further using the "integrative structure determination" approach, integrating molecular dynamics, metadynamics, normal mode analysis, small angle X-ray scattering, dynamic light scattering, cross-linking, and kinetic experiments to reveal large functional conformational changes for AbnA, involving up to ~100 Å movement in the relative positions of its domains. The integrative structure determination approach demonstrated here may apply also to the conformational study of other ultra-multimodular proteins of diverse functions and structures.
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Glicósido Hidrolasas , Glicósido Hidrolasas/química , HumanosRESUMEN
Bifidobacterium bifidum lacto-N-biosidase (LnbB) is a critical enzyme for the degradation of human milk oligosaccharides in the gut microbiota of breast-fed infants. Guided by recent crystal structures, we unveil its molecular mechanism of catalysis using QM/MM metadynamics. We show that the oligosaccharide substrate follows 1 S 3/1,4 B â [4 E] â 4 C 1/4 H 5 and 4 C 1/4 H 5 â [4 E/4 H 5] â 1,4 B conformational itineraries for the two successive reaction steps, with reaction free energy barriers in agreement with experiments. The simulations also identify a critical histidine (His263) that switches between two orientations to modulate the pK a of the acid/base residue, facilitating catalysis. The reaction intermediate of LnbB is best depicted as an oxazolinium ion, with a minor population of neutral oxazoline. The present study sheds light on the processing of oligosaccharides of the early life microbiota and will be useful for the engineering of LnbB and similar glycosidases for biocatalysis.
RESUMEN
The bacterial cell wall peptidoglycan (PG) is a dynamic structure that is constantly synthesized, re-modeled and degraded during bacterial division and growth. Postsynthetic modifications modulate the action of endogenous autolysis during PG lysis and remodeling for growth and sporulation, but also they are a mechanism used by pathogenic bacteria to evade the host innate immune system. Modifications of the glycan backbone are limited to the C-2 amine and C-6 hydroxyl moieties of either GlcNAc or MurNAc residues. This paper reviews the functional roles and properties of peptidoglycan de-Nacetylases (distinct PG GlcNAc and MurNAc deacetylases) and recent progress through genetic studies and biochemical characterization to elucidate their mechanism of action, 3D structures, substrate specificities and biological functions. Since they are virulence factors in pathogenic bacteria, peptidoglycan deacetylases are potential targets for the design of novel antimicrobial agents.
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Antiinfecciosos , Peptidoglicano , Antiinfecciosos/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Pared Celular/química , Ácidos Murámicos/análisis , Ácidos Murámicos/química , Ácidos Murámicos/metabolismo , Peptidoglicano/metabolismoRESUMEN
Transthyretin (TTR) has a well-established role in neuroprotection in Alzheimer's Disease (AD). We have setup a drug discovery program of small-molecule compounds that act as chaperones enhancing TTR/Amyloid-beta peptide (Aß) interactions. A combination of computational drug repurposing approaches and in vitro biological assays have resulted in a set of molecules which were then screened with our in-house validated high-throughput screening ternary test. A prioritized list of chaperones was obtained and corroborated with ITC studies. Small-molecule chaperones have been discovered, among them our lead compound Iododiflunisal (IDIF), a molecule in the discovery phase; one investigational drug (luteolin); and 3 marketed drugs (sulindac, olsalazine and flufenamic), which could be directly repurposed or repositioned for clinical use. Not all TTR tetramer stabilizers behave as chaperones in vitro. These chemically diverse chaperones will be used for validating TTR as a target in vivo, and to select one repurposed drug as a candidate to enter clinical trials as AD disease-modifying drug.
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Enfermedad de Alzheimer/tratamiento farmacológico , Descubrimiento de Drogas , Chaperonas Moleculares/farmacología , Prealbúmina/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Enfermedad de Alzheimer/metabolismo , Calorimetría , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Chaperonas Moleculares/química , Estructura Molecular , Prealbúmina/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Programas Informáticos , Relación Estructura-ActividadRESUMEN
Building-up and breaking-down of carbohydrates are processes common to all forms of life. Glycoside hydrolases are a broad class of enzymes that play a central role in the cleavage of glycosidic bonds, which is fundamental to carbohydrate degradation. The large majority of substrates are five- and six-membered ring glycosides. Our interest in seven-membered ring septanose sugars has inspired the development of a way to search for septanoside hydrolase activity. Described here is a strategy for the discovery of septanoside hydrolases that uses synthetic indolyl septanosides as chromogenic substrates. Access to these tool compounds was enabled by a route where septanosyl halides act as glycosyl donors for the synthesis of the indolyl septanosides. The screening strategy leverages the known dimerization of 3-hydroxy-indoles to make colored dyes, as occurs when the ß-galactosidase substrate X-Gal is hydrolyzed. Because screens in bacterial cells would enable searches in organisms that utilize heptoses or from metagenomics libraries, we also demonstrate that septanosides are capable of entering E. coli cells through the use of a BODIPY-labeled septanoside. The modularity of the indolyl septanoside synthesis should allow the screening of a variety of substrates that mimic natural structures via this general approach.
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Escherichia coli/metabolismo , Glicósidos/biosíntesis , Hidrolasas/metabolismo , Metabolismo de los Hidratos de Carbono , Compuestos Cromogénicos/química , Escherichia coli/química , Galactósidos/biosíntesis , Galactósidos/química , Glicósido Hidrolasas/metabolismo , Glicósidos/química , Hidrólisis , Indoles/químicaRESUMEN
The health benefits of human milk oligosaccharides (HMOs) make them attractive targets as supplements for infant formula milks. However, HMO synthesis is still challenging and only two HMOs have been marketed. Engineering glycoside hydrolases into transglycosylases may provide biocatalytic routes to the synthesis of complex oligosaccharides. Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hydrolyzes lacto-N-tetraose (LNT), the core structure of the most abundant type I HMOs. Here we report a mutational study in the donor subsites of the substrate binding cleft with the aim of reducing hydrolytic activity and conferring transglycosylation activity for the synthesis of LNT from p-nitrophenyl ß-lacto-N-bioside and lactose. As compared with the wt enzyme with negligible transglycosylation activity, mutants with residual hydrolase activity within 0.05% to 1.6% of the wild-type enzyme result in transglycosylating enzymes with LNT yields in the range of 10-30%. Mutations of Trp394, located in subsite -1 next to the catalytic residues, have a large impact on the transglycosylation/hydrolysis ratio, with W394F being the best mutant as a biocatalyst producing LNT at 32% yield. It is the first reported transglycosylating LnbB enzyme variant, amenable to further engineering for practical enzymatic synthesis of LNT.
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Bifidobacterium/metabolismo , Glicósido Hidrolasas/metabolismo , Oligosacáridos/biosíntesis , Bifidobacterium/genética , Cromatografía Líquida de Alta Presión , Activación Enzimática , Glicósido Hidrolasas/genética , Glicosilación , Humanos , Hidrólisis , Cinética , Espectrometría de Masas , Leche Humana/metabolismo , Modelos Moleculares , Estructura Molecular , Mutación , Oligosacáridos/química , Conformación Proteica , Ingeniería de Proteínas , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Deacetylation of N-acetylhexosamine residues in structural polysaccharides and glycoconjugates is catalyzed by different families of carbohydrate esterases that, despite different structural folds, share a common metal-assisted acid/base mechanism with the metal cation coordinated with a conserved Asp-His-His triad. These enzymes serve diverse biological functions in the modification of cell-surface polysaccharides in bacteria and fungi as well as in the metabolism of hexosamines in the biosynthesis of cellular glycoconjugates. Focusing on carbohydrate de-N-acetylases, this article summarizes the background of the different families from a structural and functional viewpoint and covers advances in the characterization of novel enzymes over the last 2-3 years. Current research is addressed to the identification of new deacetylases and unravel their biological functions as they are candidate targets for the design of antimicrobials against pathogenic bacteria and fungi. Likewise, some families are also used as biocatalysts for the production of defined glycostructures with diverse applications.
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Acetilesterasa/metabolismo , Carbohidratos/química , Glicoconjugados/metabolismo , Polisacáridos/metabolismo , AcetilaciónRESUMEN
Depolymerization of chitin results in chitooligosaccharides (COS) that induce immunostimulatory effects and disease protective responses and have many potential applications in agriculture and medicine. Isolation of bioactive COS with degree of polymerization (DP) larger than six from chitin hydrolyzates is hampered by their water insolubility. Enzymatic synthesis by exploiting the transglycosylation activity of GH18 chitinases offers a potential strategy to access oligomers in the range of bioactive DPs. We engineered SpChiD chitinase as a glycosynthase by mutation of the assisting residue of the catalytic triad in the substrate-assisted mechanism for polymerization of an oxazoline substrate (DP5ox). The insoluble polymer containing DP10 was partially hydrolyzed due to the significant residual hydrolase activity of the mutant enzyme. Combined mutations that strongly reduce the hydrolytic activity, in which the original catalytic triad only retains the essential acid/base residue, together with neighboring mutations in the -1/+1 subsites region, render glycosynthase-like chitinases able to produce chitin oligomers with DP10 as major product in good yields.
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Quitina/análogos & derivados , Quitinasas/metabolismo , Catálisis , Dominio Catalítico , Quitina/metabolismo , Quitinasas/genética , Quitosano , Hidrólisis , Mutación , Oligosacáridos , Polimerizacion , Especificidad por SustratoRESUMEN
Human transthyretin (hTTR), a serum protein with a main role in transporting thyroid hormones and retinol through binding to the retinol-binding protein, is an amyloidogenic protein involved in familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and central nervous system selective amyloidosis. hTTR also has a neuroprotective role in Alzheimer disease, being the major Aß binding protein in human cerebrospinal fluid (CSF) that prevents amyloid-ß (Aß) aggregation with consequent abrogation of toxicity. Here we report an optimized preparative expression and purification protocol of hTTR (wt and amyloidogenic mutants) for in vitro screening assays of TTR ligands acting as amyloidogenesis inhibitors or acting as molecular chaperones to enhance the TTR:Aß interaction. Preparative yields were up to 660 mg of homogenous protein per L of culture in fed-batch bioreactor. The recombinant wt protein is mainly unmodified at Cys10, the single cysteine in the protein sequence, whereas the highly amyloidogenic Y78F variant renders mainly the S-glutathionated form, which has essentially the same amyloidogenic behavior than the reduced protein with free Cys10. The TTR production protocol has shown inter-batch reproducibility of expression and protein quality for in vitro screening assays.
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Amiloide/metabolismo , Prealbúmina/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Prealbúmina/genética , Prealbúmina/aislamiento & purificación , Proteínas Recombinantes/genéticaRESUMEN
UDP-N-acetylglucosamine (UDP-GlcNAc), the main product of the hexosamine biosynthetic pathway, is an important metabolite in protozoan parasites since its sugar moiety is incorporated into glycosylphosphatidylinositol (GPI) glycolipids and N- and O-linked glycans. Apicomplexan parasites have a hexosamine pathway comparable to other eukaryotic organisms, with the exception of the glucosamine-phosphate N-acetyltransferase (GNA1) enzymatic step that has an independent evolutionary origin and significant differences from nonapicomplexan GNA1s. By using conditional genetic engineering, we demonstrate the requirement of GNA1 for the generation of a pool of UDP-GlcNAc and for the development of intraerythrocytic asexual Plasmodium falciparum parasites. Furthermore, we present the 1.95 Å resolution structure of the GNA1 ortholog from Cryptosporidium parvum, an apicomplexan parasite which is a leading cause of diarrhea in developing countries, as a surrogate for P. falciparum GNA1. The in-depth analysis of the crystal shows the presence of specific residues relevant for GNA1 enzymatic activity that are further investigated by the creation of site-specific mutants. The experiments reveal distinct features in apicomplexan GNA1 enzymes that could be exploitable for the generation of selective inhibitors against these parasites, by targeting the hexosamine pathway. This work underscores the potential of apicomplexan GNA1 as a drug target against malaria.IMPORTANCE Apicomplexan parasites cause a major burden on global health and economy. The absence of treatments, the emergence of resistances against available therapies, and the parasite's ability to manipulate host cells and evade immune systems highlight the urgent need to characterize new drug targets to treat infections caused by these parasites. We demonstrate that glucosamine-6-phosphate N-acetyltransferase (GNA1), required for the biosynthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), is essential for P. falciparum asexual blood stage development and that the disruption of the gene encoding this enzyme quickly causes the death of the parasite within a life cycle. The high-resolution crystal structure of the GNA1 ortholog from the apicomplexan parasite C. parvum, used here as a surrogate, highlights significant differences from human GNA1. These divergences can be exploited for the design of specific inhibitors against the malaria parasite.
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Eritrocitos/parasitología , Glucosamina 6-Fosfato N-Acetiltransferasa/metabolismo , Plasmodium falciparum/enzimología , Plasmodium falciparum/crecimiento & desarrollo , Secuencia de Aminoácidos , Sitios de Unión , Vías Biosintéticas , Cryptosporidium parvum/enzimología , Cryptosporidium parvum/genética , Cristalografía por Rayos X , Ingeniería Genética , Glucosamina 6-Fosfato N-Acetiltransferasa/genética , Humanos , Malaria Falciparum/parasitología , Plasmodium falciparum/genéticaRESUMEN
In the past few years, attempts have been made to use decision criteria beyond Lipinski's guidelines (Rule of five) to guide drug discovery projects more effectively. Several variables and formulations have been proposed and investigated within the framework of multiparameter optimization methods to guide drug discovery. In this context, the combination of Ligand Efficiency Indices (LEI) has been predominantly used to map and monitor the drug discovery process in a retrospective fashion. Here we provide an example of the use of a novel application of the LEI methodology for prospective lead optimization by using the transthyretin (TTR) fibrillogenesis inhibitor iododiflunisal (IDIF) as example. Using this approach, a number of compounds with theoretical efficiencies higher than the reference compound IDIF were identified. From this group, ten compounds were selected, synthesized and biologically tested. Half of the compounds (5, 6, 7, 8 and 10) showed potencies in terms of IC50 inhibition of TTR aggregation equal or higher than the lead compound. These optimized compounds mapped within the region of more efficient candidates in the corresponding experimental nBEI-NSEI plot, matching their position in the theoretical optimization plane that was used for the prediction. Due to their upstream (North-Eastern) position in the progression lines of NPOLâ¯=â¯3 or 4 of the nBEI-NSEI plot, three of them (5, 6 and 8) are more interesting candidates than iododiflunisal because they have been optimized in the three crucial LEI variables of potency, size and polarity at the same time. This is the first example of the effectiveness of using the combined LEIs within the decision process to validate the application of the LEI formulation for the prospective optimization of lead compounds.