Identification of Orthosteric and Allosteric Pharmacological Chaperones for Mucopolysaccharidosis type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal inherited disease caused by mutations in gene encoding the lysosomal enzyme N-acetyl-alpha-glucosaminidase (NAGLU). These mutations result in reduced NAGLU activity, preventing it from catalyzing the hydrolysis of the glycosaminoglycan heparan sulfate (HS). There are currently no approved treatments for MPS IIIB. A novel approach in the treatment of lysosomal storage diseases is the use of pharmacological chaperones (PC). In this study, we used a drug repurposing approach to identify and characterize novel potential PCs for NAGLU enzyme. We modeled the interaction of natural and artificial substrates within the active cavity of NAGLU (orthosteric site) and predicted potential allosteric sites. We performed a virtual screening for both the orthosteric and the predicted allosteric site against a curated database of human tested molecules. Considering the binding affinity and predicted blood-brain barrier permeability and gastrointestinal absorption, we selected atovaquone and piperaquine as orthosteric and allosteric PCs. The PCs were evaluated by their capacity to bind NAGLU and the ability to restore the enzymatic activity in human MPS IIIB fibroblasts These results represent novel PCs described for MPS IIIB and demonstrate the potential to develop novel therapeutic alternatives for this and other protein deficiency diseases.

Metabolic impact of infant formulas in young infants. An outlook from the urine metabolome.

Introduction: Although breast milk is the ideal food source for newborns during the first six months of life, a high percentage of children receive infant formulas. There is evidence that specific diet habits may influence individual metabolic profile. Therefore, in newborns, such profile can be influenced by the use of infantile formulas given the composition differences that display compared to human milk. Up to now, there are no reports in the literature that address this issue. Objectives: this work aims to compare the metabolic profile of full-term newborns that were feed with either breast milk (n = 32) or infantile formulas (n = 21). Methods: Metabolic profile was established based on urine analysis through gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (H-NMR). Results: our results evidenced a more gluconeogenic profile in breast-fed infants characterized by elevation of Kreb's cycle intermediaries like fumaric, succinic and ketoglutaric acids compared to infants receiving infant formula. In addition, infant formula fed infants presented urinary excretion of metabolites derived from specific compounds present in this type of diet that were not observed in breast-fed infants, for instance D-glucitol, and 4-deoxytetronic. Moreover, in infant formula fed infants there was excretion of basal levels of metabolites of clinical relevance like 3-hydroxy-3-methyl-glutaric, 2-methyl-3-keto-valeric and 3,4-dihydroxybutyric. Conclusion: These results show the importance of understanding the metabolic impact of diet in newborn population in normal and pathological contexts.

Engineering a heterologously expressed fructosyltransferase from Aspergillus oryzae N74 in Komagataella phaffii (Pichia pastoris) for kestose production.

Fructo-oligosaccharides (FOS) are one of the most well-studied and commercialized prebiotics. FOS can be obtained either by controlled hydrolysis of inulin or by sucrose transfructosylation. FOS produced from sucrose are typically classified as short-chain FOS (scFOS), of which the best known are 1-kestotriose (GF2), 1,1-kestotetraose (GF3), and 1,1,1-kestopentaose (GF4), produced by fructosyltransferases (FTases) or ß-fructofuranosidases. In previous work, FOS production was studied using the Aspergillus oryzae N74 strain, its ftase gene was heterologously expressed in Komagataella phaffii (Pichia pastoris), and the enzyme's tertiary structure modeled. More recently, residues that may be involved in protein-substrate interactions were predicted. In this study, the aim was to experimentally validate previous in silico results by independently producing recombinant wild-type A. oryzae N74 FTase and three single-point mutations in Komagataella phaffii (Pichia pastoris). The R163A mutation virtually abolished the transfructosylating activity, indicating a requirement for the positively charged arginine residue in the catalytic domain D. In contrast, transfructosylating activity was improved by introducing the mutations V242E or F254H, with V242E resulting in higher production of GF2 without affecting that of GF3. Interestingly, initial sucrose concentration, reaction temperature and the presence of metal cofactors did not affect the enhanced activity of mutant V242E. Overall, these results shed light on the mechanism of transfructosylation of the FTase from A. oryzae and expand considerations regarding the design of biotechnological processes for specific FOS production.

The prevalence of Chlamydia psittaci in confiscated Psittacidae in Colombia.

Chlamydia psittaci is a highly zoonotic bacteria distributed worldwide; it is responsible for psittacosis, one of the most important infectious diseases affecting the Psittacidae, mostly parrots. This work was aimed at determining C. psittaci prevalence and genotype in 177 parrots confiscated in Colombia; cloacal swab (166) and faecal (177) samples were analysed from birds confiscated and housed in a Temporary Wildlife Reception Centre (Centro de Reception de Fauna Temporal). Conventional PCR was run on the samples for amplifying the MOMP gene and then the ompA gene. The C. psittaci genotype A was found in 81.3 % (144/177) of the birds analysed. Cloacal swabs accounted for 129/166 (77.7 %) positive samples and faecal matter for 53/177 (29.9 %), 38 birds proving positive for both types of sample; there was an 8.15 times greater probability of detection for cloacal swabs compared to faecal swabs (p < 0.05). Clinical examination findings were correlated with the animals' positivity for cloacal swabs, faecal matter or both, finding a statistically significant relationship with low respiratory rate (p < 0.05) and broken plumage for cloacal swab sample results (p < 0.1). Even though 85 % seroprevalence has previously been reported in Colombia using indirect ELISA, this study reports for the first time C. psittaci genotype A endemicity in psittacines in captivity in Colombia using molecular techniques, considering the zoonotic risk involved in having these birds as pets.

Assessment of Adaptive Performance and its Role as a Moderator of the Relationship Between Person-Organization Fit and Organizational Citizenship Behaviors.

BACKGROUND: Adaptive performance is of central interest for today's organizations, insofar as employees increasingly need to be able to adjust their behaviors to dynamic, changing work situations. The aims of this study were to develop a Spanish adaptation of Charbonnier-Voirin and Roussel's (2012) scale for measuring adaptive performance, and to examine whether the dimensions of adaptive performance moderate the relationship between person-organization fit (PO fit) and organizational citizenship behaviors (OCBs). We hypothesized that the relationship between PO fit and OCBs would be stronger in individuals with a higher level of adaptive performance. METHOD: The sample comprised 678 employees (65% women) in the public sector in the Basque Country. They ranged from 21 to 63 years old (M = 44.63; SD = 7.66). RESULTS: The Spanish version of the scale has good psychometric properties. Furthermore, the Interpersonal Adaptability dimension of adaptive performance moderates the relationship between PO fit and OCBs directed both at the organization and at individuals, following the expected direction. The relationship between PO fit and OCBs directed at individuals was also moderated by the Reactivity in the Face of Emergencies dimension. CONCLUSIONS: We discuss the practical implications of these results in the field of personnel selection.

Assessment of Adaptive Performance and its Role as a Moderator of the Relationship Between Person - Organization Fit and Organizational Citizenship Behaviors / Evaluación del Desempeño Adaptativo y su Rol Moderador en la Relación Entre el Ajuste Persona - Organización y la Ciudadanía Organizacional

Background: Adaptive performance is of central interest for today’sorganizations, insofar as employees increasingly need to be able to adjusttheir behaviors to dynamic, changing work situations. The aims of thisstudy were to develop a Spanish adaptation of Charbonnier-Voirin andRoussel’s (2012) scale for measuring adaptive performance, and to examinewhether the dimensions of adaptive performance moderate the relationshipbetween person-organization fit (PO fit) and organizational citizenshipbehaviors (OCBs). We hypothesized that the relationship between POfit and OCBs would be stronger in individuals with a higher level ofadaptive performance. Method: The sample comprised 678 employees(65% women) in the public sector in the Basque Country. They rangedfrom 21 to 63 years old (M = 44.63; SD = 7.66). Results: The Spanishversion of the scale has good psychometric properties. Furthermore, theInterpersonal Adaptability dimension of adaptive performance moderatesthe relationship between PO fit and OCBs directed both at the organizationand at individuals, following the expected direction. The relationshipbetween PO fit and OCBs directed at individuals was also moderated bythe Reactivity in the Face of Emergencies dimension. Conclusions: Wediscuss the practical implications of these results in the field of personnelselection

Antecedentes: el desempeño adaptativo es una variable crucial enlas organizaciones actuales dado que la flexibilidad y la capacidad deadaptación son necesarias en un entorno laboral tan dinámico y cambiantecomo el actual. Los objetivos de este estudio fueron adaptar al castellanoel instrumento de Charbonnier-Voirin y Roussel (2012), que mide eldesempeño adaptativo y examinar si las dimensiones que configuranel desempeño adaptativo moderan la relación entre el ajuste persona-organización (PO fit) y las conductas de ciudadanía organizacional(OCBs). Se pronosticó que la relación entre PO fit y OCBs sería másintensa en aquellas personas con mayor desempeño adaptativo. Método: la muestra estuvo compuesta por 678 empleados/as (65% mujeres) delsector público del País Vasco, con edades comprendidas entre 21 y 63años (M = 44.63; SD = 7.66). Resultados: los resultados muestran quela versión española de la escala posee buenas propiedades psicométricas.Además, la dimensión Adaptabilidad Interpersonal del desempeñoadaptativo modera la relación entre el PO fit y las OCBs. En la predicciónde las OCBs dirigidas a otros individuos, la Reactividad ante emergenciastambién modera la relación. Conclusiones: se discuten las implicacionesprácticas que pueden tener en el ámbito de la selección de personal losresultados derivados del estudio.

1H-Nuclear Magnetic Resonance Analysis of Urine as Diagnostic Tool for Organic Acidemias and Aminoacidopathies.

The utility of low-resolution 1H-NMR analysis for the identification of biomarkers provided evidence for rapid biochemical diagnoses of organic acidemia and aminoacidopathy. 1H-NMR, with a sensitivity expected for a field strength of 400 MHz at 64 scans was used to establish the metabolomic urine sample profiles of an infant population diagnosed with small molecule Inborn Errors of Metabolism (smIEM) compared to unaffected individuals. A qualitative differentiation of the 1H-NMR spectral profiles of urine samples obtained from individuals affected by different organic acidemias and aminoacidopathies was achieved in combination with GC-MS. The smIEM disorders investigated in this study included phenylalanine metabolism; isovaleric, propionic, 3-methylglutaconicm and glutaric type I acidemia; and deficiencies in medium chain acyl-coenzyme and holocarboxylase synthase. The observed metabolites were comparable and similar to those reported in the literature, as well as to those detected with higher-resolution NMR. In this study, diagnostic marker metabolites were identified for the smIEM disorders. In some cases, changes in metabolite profiles differentiated post-treatments and follow-ups while allowing for the establishment of different clinical states of a biochemical disorder. In addition, for the first time, a 1H-NMR-based biomarker profile was established for holocarboxylase synthase deficiency spectrum.

Assessment of Personality in Basque Public Sector Employees and Its Role in Predicting Organizational Citizenship Behaviors in Selection Processes.

Organizational citizenship behaviors (OCBs) are an important aspect of job performance as they enhance the effectiveness of organizations. Research has shown that personality is a moderate predictor of job performance. This study, involving a sample of 678 public sector employees in the Basque Country (northern Spain), pursued two aims: First, to develop and validate a Basque-language version of the Overall Personality Assessment Scale (OPERAS), a scale designed to assess the Big Five personality factors in a wide range of settings; and second, to examine whether person-organization fit (PO fit) and adaptive performance improve the capacity of personality to predict OCBs. The results indicated that the adapted scale was a suitable instrument for assessing personality in the Basque-speaking population. Furthermore, PO fit and adaptive performance improved the capacity of personality to predict OCBs. Based on these results, we propose a new predictive model that may enhance the efficiency of personnel selection processes.

Use of a neuron-glia genome-scale metabolic reconstruction to model the metabolic consequences of the Arylsulphatase a deficiency through a systems biology approach.

Metachromatic leukodystrophy (MLD) is a human neurodegenerative disorder characterized by progressive damage on the myelin band in the nervous system. MLD is caused by the impaired function of the lysosomal enzyme Arylsulphatase A (ARSA). The physiopathology mechanisms and the biochemical consequences in the brain of ARSA deficiency are not entirely understood. In recent years, the use of genome-scale metabolic (GEM) models has been explored as a tool for the study of the biochemical alterations in MLD. Previously, we modeled the metabolic consequences of different lysosomal storage diseases using single GEMs. In the case of MLD, using a glia GEM, we previously predicted that the metabolism of glycosphingolipids and neurotransmitters was altered. The results also suggested that mitochondrial metabolism and amino acid transport were the main reactions affected. In this study, we extended the modeling of the metabolic consequences of ARSA deficiency through the integration of neuron and glial cell metabolic models. Cell-specific models were generated from Recon2, and these were used to create a neuron-glial bi-cellular model. We propose a workflow for the integration of this type of model and its subsequent study. The results predicted the impairment pathways involved in the transport of amino acids, lipids metabolism, and catabolism of purines and pyrimidines. The use of this neuron-glial GEM metabolic reconstruction allowed to improve the prediction capacity of the metabolic consequences of ARSA deficiency, which might pave the way for the modeling of the biochemical alterations of other inborn errors of metabolism with central nervous system involvement.

Human recombinant lysosomal ß-Hexosaminidases produced in Pichia pastoris efficiently reduced lipid accumulation in Tay-Sachs fibroblasts.

GM2 gangliosidosis, Tay-Sachs and Sandhoff diseases, are lysosomal storage disorders characterized by the lysosomal accumulation of GM2 gangliosides. This accumulation is due to deficiency in the activity of the ß-hexosaminidases Hex-A or Hex-B, which are dimeric hydrolases formed by αß or ßß subunits, respectively. These disorders show similar clinical manifestations that range from mild systemic symptoms to neurological damage and premature death. There is still no effective therapy for GM2 gangliosidoses, but some therapeutic alternatives, as enzyme replacement therapy, have being evaluated. Previously, we reported the production of active human recombinant ß-hexosaminidases (rhHex-A and rhHex-B) in the methylotrophic yeast Pichia pastoris. In this study, we evaluated in vitro the cellular uptake, intracellular delivery to lysosome, and reduction of stored substrates. Both enzymes were taken-up via endocytic pathway mediated by mannose and mannose-6-phosphate receptors and delivered to lysosomes. Noteworthy, rhHex-A diminished the levels of stored lipids and lysosome mass in fibroblasts from Tay-Sachs patients. Overall, these results confirm the potential of P. pastoris as host to produce recombinant ß-hexosaminidases intended to be used in the treatment of GM2 gangliosidosis.

Bromocriptine as a Novel Pharmacological Chaperone for Mucopolysaccharidosis IV A.

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the gene encoding for the enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to lysosomal accumulation of keratan sulfate (KS) and chondroitin-6-sulfate. In this study, we identified and characterized bromocriptine (BC) as a novel PC for MPS IVA. BC was identified through virtual screening and predicted to be docked within the active cavity of GALNS in a similar conformation to that observed for KS. BC interacted with similar residues to those predicted for natural GALNS substrates. In vitro inhibitory assay showed that BC at 50 µM reduced GALNS activity up to 30%. However, the activity of hrGALNS produced in HEK293 cells was increased up to 1.48-fold. BC increased GALNS activity and reduced lysosomal mass in MPS IVA fibroblasts in a mutation-dependent manner. Overall, these results show the potential of BC as a novel PC for MPS IVA and contribute to the consolidation of PCs as a potential therapy for this disease.

Media improvement for 10 L bioreactor production of rPOXA 1B laccase by P. pastoris.

In this work, we statistically improved culture media for rPOXA 1B laccase production, expressed in Pichia pastoris containing pGAPZαA-LaccPost-Stop construct and assayed at 10 L bioreactor production scale (6 L effective work volume). The concentrated enzyme was evaluated for temperature and pH stability and kinetic parameter, characterized by monitoring oxidation of different ABTS [2, 20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] substrate concentrations. Plackett-Burman experimental design (PBED) implementation improved previous work results by 3.05-fold, obtaining a laccase activity of 1373.72 ± 0.37 U L-1 at 168 h of culture in a 500 mL shake flask. In contrast, one factor experimental design (OFED) applied after PBED improved by threefold the previous study, additionally increasing the C/N ratio. Employing OFED media at 10 L bioreactor scale was capable of producing 3159.93 ± 498.90 U L-1 at 192 h, representing a 2.4-fold increase. rPOXA 1B concentrate remained stable between 10 and 50 °C and retained over 70% residual enzymatic activity at 60 °C and 50% at 70 °C. Concerning pH stability, the enzyme was stable at pH 4.0 ± 0.2 with a residual activity greater than 90%. The lowest residual activity (60%) was obtained at pH 10.0 ± 0.2. Furthermore, the apparent kinetic parameters were V max of 3.163 × 10-2 mM min-1 and K m of 1.716 mM. Collectively, regarding enzyme stability our data provide possibilities for applications involving a wide range of pH and temperatures.

Identification of Ezetimibe and Pranlukast as Pharmacological Chaperones for the Treatment of the Rare Disease Mucopolysaccharidosis Type IVA.

Mucopolysaccharidosis type IVA (MPS IVA) is a rare disease caused by mutations in the gene encoding the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). We report here two GALNS pharmacological chaperones, ezetimibe and pranlukast, identified by molecular docking-based virtual screening. These compounds bound to the active cavity of GALNS and increased its thermal stability as well as the production of recombinant GALNS in bacteria, yeast, and HEK293 cells. MPS IVA fibroblasts treated with these chaperones exhibited increases in GALNS protein and enzyme activity and reduced the size of enlarged lysosomes. Abnormalities in autophagy markers p62 and LC3B-II were alleviated by ezetimibe and pranlukast. Combined treatment of recombinant GALNS with ezetimibe or pranlukast produced an additive effect. Altogether, the results demonstrate that ezetimibe and pranlukast can increase the yield of recombinant GALNS and be used as a monotherapy or combination therapy to improve the therapeutic efficacy of MPS IVA enzyme replacement therapy.

Identification of the iduronate-2-sulfatase proteome in wild-type mouse brain.

Iduronate-2-sulfatase (IDS) is a lysosomal enzyme involved in the metabolism of the glycosaminoglycans heparan (HS) and dermatan (DS) sulfate. Mutations on IDS gene produce mucopolysaccharidosis II (MPS II), characterized by the lysosomal accumulation of HS and DS, leading to severe damage of the central nervous system (CNS) and other tissues. In this study, we used a neurochemistry and proteomic approaches to identify the brain distribution of IDS and its interacting proteins on wild-type mouse brain. IDS immunoreactivity showed a robust staining throughout the entire brain, suggesting an intracellular reactivity in nerve cells and astrocytes. By using affinity purification and mass spectrometry we identified 187 putative IDS partners-proteins, mainly hydrolases, cytoskeletal proteins, transporters, transferases, oxidoreductases, nucleic acid binding proteins, membrane traffic proteins, chaperons and enzyme modulators, among others. The interactions with some of these proteins were predicted by using bioinformatics tools and confirmed by co-immunoprecipitation analysis and Blue Native PAGE. In addition, we identified cytosolic IDS-complexes containing proteins from predicted highly connected nodes (hubs), with molecular functions including catalytic activity, redox balance, binding, transport, receptor activity and structural molecule activity. The proteins identified in this study would provide new insights about IDS physiological role into the CNS and its potential role in the brain-specific protein networks.

Characterization of Human Recombinant N-Acetylgalactosamine-6-Sulfate Sulfatase Produced in Pichia pastoris as Potential Enzyme for Mucopolysaccharidosis IVA Treatment.

Mucopolysaccharidosis IVA (MPS IVA or Morquio A syndrome) is a lysosomal storage disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to lysosomal storage of keratan sulfate and chondroitin-6-sulfate. Currently, enzyme replacement therapy using an enzyme produced in CHO cells represents the main treatment option for MPS IVA patients. As an alternative, we reported the production of an active GALNS enzyme produced in the yeast Pichia pastoris (prGALNS), which showed internalization by cultured cells through a potential receptor-mediated process and similar post-translational processing as human enzyme. In this study, we further studied the therapeutic potential of prGALNS through the characterization of the N-glycosylation structure, in vitro cell uptake and keratan sulfate reduction, and in vivo biodistribution and generation of anti-prGALNS antibodies. Taken together, these results represent an important step in the development of a P. pastoris-based platform for production of a therapeutic GALNS for MPS IVA enzyme replacement therapy.

Neural stem cells for disease modeling and evaluation of therapeutics for Tay-Sachs disease.

BACKGROUND: Tay-Sachs disease (TSD) is a rare neurodegenerative disorder caused by autosomal recessive mutations in the HEXA gene on chromosome 15 that encodes ß-hexosaminidase. Deficiency in HEXA results in accumulation of GM2 ganglioside, a glycosphingolipid, in lysosomes. Currently, there is no effective treatment for TSD. RESULTS: We generated induced pluripotent stem cells (iPSCs) from two TSD patient dermal fibroblast lines and further differentiated them into neural stem cells (NSCs). The TSD neural stem cells exhibited a disease phenotype of lysosomal lipid accumulation. The Tay-Sachs disease NSCs were then used to evaluate the therapeutic effects of enzyme replacement therapy (ERT) with recombinant human Hex A protein and two small molecular compounds: hydroxypropyl-ß-cyclodextrin (HPßCD) and δ-tocopherol. Using this disease model, we observed reduction of lipid accumulation by employing enzyme replacement therapy as well as by the use of HPßCD and δ-tocopherol. CONCLUSION: Our results demonstrate that the Tay-Sachs disease NSCs possess the characteristic phenotype to serve as a cell-based disease model for study of the disease pathogenesis and evaluation of drug efficacy. The enzyme replacement therapy with recombinant Hex A protein and two small molecules (cyclodextrin and tocopherol) significantly ameliorated lipid accumulation in the Tay-Sachs disease cell model.

Research, diagnosis and education in inborn errors of metabolism in Colombia: 20 years' experience from a reference center.

The use of specialized centers has been the main alternative for an appropriate diagnosis, management and follow up of patients affected by inborn errors of metabolism (IEM). These centers facilitate the training of different professionals, as well as the research at basic, translational and clinical levels. Nevertheless, few reports have described the experience of these centers and their local and/or global impact in the study of IEM. In this paper, we describe the experience of a Colombian reference center for the research, diagnosis, training and education on IEM. During the last 20 years, important advances have been achieved in the clinical knowledge of these disorders, as well as in the local availability of several diagnosis tests. Organic acidurias have been the most frequently detected diseases, followed by aminoacidopathies and peroxisomal disorders. Research efforts have been focused in the production of recombinant proteins in microorganisms towards the development of new enzyme replacement therapies, the design of gene therapy vectors and the use of bioinformatics tools for the understanding of IEM. In addition, this center has participated in the education and training of a large number professionals at different levels, which has contributed to increase the knowledge and divulgation of these disorders along the country. Noteworthy, in close collaboration with patient advocacy groups, we have participated in the discussion and construction of initiatives for the inclusion of diagnosis tests and treatments in the health system.

Production and characterization of a human lysosomal recombinant iduronate-2-sulfatase produced in Pichia pastoris.

Hunter syndrome (Mucopolysaccharidosis II, MPS II) is an X-linked lysosomal storage disease produced by the deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). Currently, MPS II patients are mainly treated with enzyme replacement therapy (ERT) using recombinant enzymes produced in mammalian cells. As an alternative, several studies have shown the production of active and therapeutic forms of lysosomal proteins in microorganisms. In this paper, we report the production and characterization of a recombinant IDS produced in the yeast Pichia pastoris (prIDS). We evaluated the effect of culture conditions and gene sequence optimization on prIDS production. The results showed that the highest production of prIDS was obtained at oxygen-limited conditions using a codon-optimized IDS cDNA. The purified enzyme showed a final activity of 12.45 nmol mg-1 H-1 and an apparent molecular mass of about 90 kDa. The highest stability was achieved at pH 6.0, and prIDS also showed high stability in human serum. Noteworthy, the enzyme was taken up by culture cells in a dose-dependent manner through mannose receptors, which allowed the delivery of the enzyme to the lysosome. In summary, these results show the potential of Pichia pastoris as a host to produce an IDS intended for a MPS II ERT.

In Silico Analysis of the Structure of Fungal Fructooligosaccharides-Synthesizing Enzymes.

Fructooligosaccharides (FOS) are prebiotics commonly manufactured using fungal fructosyltransferases (FTases) or ß-fructofuranosidases. Several reports have attempted to optimize FOS production by changing operational conditions. Nevertheless, there is a lack of information related to the molecular enzyme-substrate interaction. In this study, we present an in silico evaluation of the interactions between substrates (i.e., glucose, sucrose, GF2, GF3, and GF4) and native FOS-synthesizing enzymes from fungi, with reported FOS production yield. In addition, a molecular dynamic simulation was conducted to assess the stability of these interactions. Six fungal enzymes with reported data of FOS production were selected: sucrose-sucrose 1-fructosyltransferase from A. foetidus (GenBank No. CAA04131); intracellular invertase from A. niger (GenBank No. ABB59679); extracellular invertase from A. niger (GenBank No. ABB59678); ß-fructofuranidase from A. japonicus ATCC 20611 (GenBank No. BAB67771); fructosyltransferase from A. oryzae N74 (GenBank No. ACZ48670); and fructosyltransferase from A. japonicus (PDB ID 3LF7). These enzymes shared an identity between 15 and 96 %, but have a highly conserved folding, and the characteristic FTases domains. Docking results showed that these enzymes also share a similar protein-ligand interaction profile. It was observed that the production yield of total FOS correlated with the sum of affinity energies for GF2, GF3, and GF4. Finally, we present the first molecular dynamic simulation for FOS and fungal FOS-synthesizing enzymes, showing that the protein-ligand interaction does not induce significant changes on the enzyme stability. Overall, these results represent valuable information to continue understanding the FOS synthesis process by fungal FOS-synthesizing enzymes, and they can have a significant impact toward the improvement in their catalytic properties and the synthesis of specific FOS.

Anaerobic sulfatase maturase AslB from Escherichia coli activates human recombinant iduronate-2-sulfate sulfatase (IDS) and N-acetylgalactosamine-6-sulfate sulfatase (GALNS).

Maturation of type I sulfatases requires the conversion of the cysteine (Cys) or serine (Ser) present in the active site to formylglycine (FGly). This activation represents a limiting step during the production of recombinant sulfatases in bacteria and eukaryotic hosts. AslB, YdeM and YidF have been proposed to participate in the activation of sulfatases in Escherichia coli. In this study, we combined in-silico and experimental approaches to study the interaction between Escherichia coli BL21(DE3) AslB and human sulfatases, more specifically iduronate-2-sulfate sulfatase (IDS) and N-acetylgalactosamine-6-sulfate sulfatase (GALNS). In-silico results show that AslB has a higher affinity for the residual motif of GALNS (-9.4kcalmol-1), Cys- and Ser-type, than for the one of IDS (-8.0kcalmol-1). However, the distance between the AslB active residue and the target motif favors the interaction with IDS (4.4Å) more than with GALNS (5.5Å). Experimental observations supported in-silico results where the co-expression of AslB with GALNS Cys- and Ser-type presented an activity increment of 2.0- and 1.5-fold compared to the control cultures, lacking overexpressed AslB. Similarly, IDS activity was increased in 4.6-fold when co-expressed with AslB. The higher sulfatase activity of AslB-IDS suggests that the distance between the AslB active residue and the motif target is a key parameter for the in-silico search of potential sulfatase activators. In conclusion, our results suggest that AslB is involve in the maturation of heterologous human sulfatases in E. coli BL21(DE3), and that it can have important implications in the production of recombinant sulfatases for therapeutic purposes and research.