Kristýna Slámová

β-N-acetylhexosaminidase: what's in a name…?

β-N-acetylhexosaminidases (EC 3.2.1.52, belonging to CAZy GH families 3, 20 and 84) have recently gained a lot of attention, not only due to their implication in human physiology and disease, but also due to their great potential in the enzymatic synthesis of carbohydrates and glycomimetics. GH family 20 β-N-acetylhexosaminidases, and GH family 3 and 84 β-N-acetylglucosaminidases from all kinds of organisms have been intensively studied from the point of view of their physiological roles, reaction mechanisms, structure and inhibition. Thanks to their outstanding substrate promiscuity, extracellular β-N-acetylhexosaminidases from filamentous fungi are able to cleave and transfer substrates bearing various functionalities, ranging from carboxylates, sulfates, acylations to azides, and even 4-deoxy glycosides. Thus, they have proved to be versatile biosynthetic tools for the preparation of both natural and modified hexosaminides under mild conditions with good yields.

Synthesis and biological activity of glycosyl-1H-1,2,3-triazoles

Glycosyl 1,2,3-triazoles with alpha-D-gluco, beta-D-gluco, alpha-D-galacto, beta-D-galacto and beta-2-acetamido-2-deoxygluco (GlcNAc) stereochemistry were prepared by reaction of the corresponding azides with vinyl acetate under microwave irradiation. The deprotected glucosyl and galactosyl triazoles did not display inhibitory activity against the tested glycosidases at 1 mM. Of the four fungal glycosidases evaluated, GlcNAc-triazole was found to be hydrolyzed by Talaromyces flavus CCF 2686 beta-N-acetylhexosaminidase. Beta-GlcNAc-triazole was furthermore established to act as a strong ligand of rat and human natural killer cell activating receptors.

Heterologous expression, purification and characterization of nitrilase from Aspergillus niger K10

BackgroundNitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult.ResultsA nitrilase gene was amplified by PCR from the cDNA library of Aspergillus niger K10. The PCR product was ligated into expression vectors pET-30(+) and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec) expressed in Escherichia coli BL21-Gold(DE3)(pOK101/pTf16) was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT), indicating post-translational cleavage in the enzymes native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val327 - Asn356) was present in Nit-ANigRec but missing in Nit-ANigWT and Asp298-Val313 peptide was shortened to Asp298-Arg310 in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an arrangement of dimers into helical segments as a plausible structural solution.ConclusionsThe nitrilase from Aspergillus niger K10 is highly homologous (≥86%) with proteins deduced from gene sequencing in Aspergillus and Penicillium genera. As the first of these proteins, it was shown to exhibit nitrilase activity towards organic nitriles. The comparison of the Nit-ANigRec and Nit-ANigWT suggested that the catalytic properties of nitrilases may be changed due to missing posttranslational cleavage of the former enzyme. Nit-ANigRec exhibits a lower tendency to form filaments and, moreover, the sample homogeneity can be further improved by in vitro protein refolding. The homogeneous protein species consisting of short spirals is expected to be more suitable for structural studies.

Enzymatic characterization and molecular modeling of an evolutionarily interesting fungal β‐N‐acetylhexosaminidase

Fungal β‐N‐acetylhexosaminidases are inducible extracellular enzymes with many biotechnological applications. The enzyme from Penicillium oxalicum has unique enzymatic properties despite its close evolutionary relationship with other fungal hexosaminidases. It has high GalNAcase activity, tolerates substrates with the modified N‐acyl group better and has some other unusual catalytic properties. In order to understand these features, we performed isolation, biochemical and enzymological characterization, molecular cloning and molecular modelling. The native enzyme is composed of two catalytic units (65 kDa each) and two propeptides (15 kDa each), yielding a molecular weight of 160 kDa. Enzyme deglycosylated by endoglycosidase H had comparable activity, but reduced stability. We have cloned and sequenced the gene coding for the entire hexosaminidase from P. oxalicum. Sufficient sequence identity of this hexosaminidase with the structurally solved enzymes from bacteria and humans with complete conservation of all catalytic residues allowed us to construct a molecular model of the enzyme. Results from molecular dynamics simulations and substrate docking supported the experimental kinetic and substrate specificity data and provided a molecular explanation for why the hexosaminidase from P. oxalicum is unique among the family of fungal hexosaminidases.

Enzymatic synthesis of dimeric glycomimetic ligands of NK cell activation receptors.

This work reveals new structural relationships in the complex process of the interaction between activation receptors of natural killer cells (rat NKR-P1, human CD69) and novel bivalent carbohydrate glycomimetics. The length, glycosylation pattern and linker structure of receptor ligands were examined with respect to their ability to precipitate the receptor protein from solution, which simulates the in vivo process of receptor aggregation during NK cell activation. It was found that di-LacdiNAc triazole compounds show optimal performance, reaching up to 100% precipitation of the present protein receptors, and achieving high immunostimulatory activities without any tendency to trigger activation-induced apoptosis. In the synthesis of the compounds tested, two enzymatic approaches were applied. Whereas a β-N-acetylhexosaminidase could only glycosylate one of the two acceptor sites available with yields below 10%, the Y284L mutant of human placental β1,4-galactosyltransferase-1 worked as a perfect synthetic tool, accomplishing even quantitative glycosylation at both acceptor sites and with absolute regioselectivity for the C-4 position. This work insinuates new directions for further ligand structure optimisation and demonstrates the strong synthetic potential of the mutant human placental β1,4-galactosyltransferase-1 in the synthesis of multivalent glycomimetics and glycomaterials.

Sequencing, cloning and high-yield expression of a fungal β-N-acetylhexosaminidase in Pichia pastoris.

The β-N-acetylhexosaminidase from Talaromyces flavus has a remarkable synthetic ability, processing even carbohydrates with various functionalities. Its broader use is partially hampered by low-yield production in the native fungus. Here, we present an optimized 3-day production of this enzyme in the eukaryotic host of Pichia pastoris, in ca 10-fold higher volume activity (10 U/ml) and close-to-perfect purity (one chromatographic step needed). Importantly, the recombinant enzyme features the same biochemical and catalytic properties, including the syntheses with derivatized carbohydrate substrates. This is the first example of the overexpression of a fungal β-N-acetylhexosaminidase by a single-cell producer in liquid medium. It represents a promising solution for wider biotechnological applications of this outstanding enzyme.

Expression, characterization and homology modeling of a novel eukaryotic GH84 β-N-acetylglucosaminidase from Penicillium chrysogenum

β-N-acetylglucosaminidases from the family 84 of glycoside hydrolases form a small group of glycosidases in eukaryotes responsible for the modification of nuclear and cytosolic proteins with O-GlcNAc, thus they are involved in a number of important cell processes. Here, the first fungal β-N-acetylglucosaminidase from Penicillium chrysogenum was expressed in Pichia pastoris and secreted into the media, purified and characterized. Moreover, homology modeling and substrate and inhibitor docking were performed to obtain structural information on this new member of the GH84 family. Surprisingly, we found that this fungal β-N-acetylglucosaminidase with its sequence and structure perfectly fitting to the GH84 family displays biochemical properties rather resembling the β-N-acetylhexosaminidases from the family 20 of glycoside hydrolases. This work helped to increase the knowledge on the scarcely studied glycosidase family and revealed a new type of eukaryotic β-N-acetylglucosaminidase.

Engineered N-acetylhexosamine-active enzymes in glycoscience

BACKGROUND In recent years, enzymes modifying N-acetylhexosamine substrates have emerged in numerous theoretical studies as well as practical applications from biology, biomedicine, and biotechnology. Advanced enzyme engineering techniques converted them into potent synthetic instruments affording a variety of valuable glycosides. SCOPE OF REVIEW This review presents the diversity of engineered enzymes active with N-acetylhexosamine carbohydrates: from popular glycoside hydrolases and glycosyltransferases to less known oxidases, epimerases, kinases, sulfotransferases, and acetylases. Though hydrolases in natura, engineered chitinases, β-N-acetylhexosaminidases, and endo-β-N-acetylglucosaminidases were successfully employed in the synthesis of defined natural and derivatized chitooligomers and in the remodeling of N-glycosylation patterns of therapeutic antibodies. The genes of various N-acetylhexosaminyltransferases were cloned into metabolically engineered microorganisms for producing human milk oligosaccharides, Lewis X structures, and human-like glycoproteins. Moreover, mutant N-acetylhexosamine-active glycosyltransferases were applied, e.g., in the construction of glycomimetics and complex glycostructures, industrial production of low-lactose milk, and metabolic labeling of glycans. In the synthesis of biotechnologically important compounds, several innovative glycoengineered systems are presented for an efficient bioproduction of GlcNAc, UDP-GlcNAc, N-acetylneuraminic acid, and of defined glycosaminoglycans. MAJOR CONCLUSIONS The above examples demonstrate that engineering of N-acetylhexosamine-active enzymes was able to solve complex issues such as synthesis of tailored human-like glycoproteins or industrial-scale production of desired oligosaccharides. Due to the specific catalytic mechanism, mutagenesis of these catalysts was often realized through rational solutions. GENERAL SIGNIFICANCE Specific N-acetylhexosamine glycosylation is crucial in biological, biomedical and biotechnological applications and a good understanding of its details opens new possibilities in this fast developing area of glycoscience.

The α-galactosidase type A gene aglA from Aspergillus niger encodes a fully functional α-N-acetylgalactosaminidase

Two genes in the genome of Aspergillus niger, aglA and aglB, have been assigned to encode for α-d-galactosidases variant A and B. However, analyses of primary and 3D structures based on structural models of these two enzymes revealed significant differences in their active centers suggesting important differences in their specificity for the hydrolyzed carbohydrates. To test this unexpected finding, a large screening of libraries from 42 strains of filamentous fungi succeeded in identifying an enzyme from A. niger CCIM K2 that exhibited both α-galactosidase and α-N-acetylgalactosaminidase activities, with the latter activity predominating. The enzyme protein was sequenced, and its amino acid sequence could be unequivocally assigned to the enzyme encoded the aglA gene. Enzyme activity measurements and substrate docking clearly demonstrated the preference of the identified enzyme for α-N-acetyl-d-galactosaminide over α-d-galactoside. Thus, we provide evidence that the α-galactosidase type A gene aglA from A. niger in fact encodes a fully functional α-N-acetylgalactosaminidase using a retaining mechanism.

Computational study of β-N-acetylhexosaminidase from Talaromyces flavus, a glycosidase with high substrate flexibility

Backgroundβ-N-Acetylhexosaminidase (GH20) from the filamentous fungus Talaromyces flavus, previously identified as a prominent enzyme in the biosynthesis of modified glycosides, lacks a high resolution three-dimensional structure so far. Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification. Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest.ResultsComputational methods including homology modeling and molecular dynamics simulations were applied to shad light on the structure-activity relationship in the enzyme. Primary sequence analysis revealed some variable regions able to influence difference in substrate affinity of hexosaminidases. Moreover, docking in combination with consequent molecular dynamics simulations of C-6 modified glycosides enabled us to identify the structural features required for accommodation and processing of these bulky substrates in the active site of hexosaminidase from T. flavus. To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model.ConclusionsThe main variable regions in β-N-acetylhexosaminidases determining difference in modified substrate affinity are located close to the active site entrance and engage two loops. Differences in primary sequence and the spatial arrangement of these loops and their interplay with active site amino acids, reflected by interaction energies and dynamics, account for the different catalytic activity and substrate specificity of the various fungal and bacterial β-N-acetylhexosaminidases.