Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
A novel enzymatic tool for transferring GalNAc moiety onto challenging acceptors
Introduction
β-N-Acetylhexosaminidases (EC 3.2.1.52, GH20, http://www.cazy.org/) are exo-glycosidases, which exhibit a unique dual substrate specificity. They are able to cleave N-acetylglucosamine (GlcNAc) as well as N-acetylgalactosamine (GalNAc) residues from glycostructures. β-N-Acetylhexosaminidases are widely distributed in nature and can be found in almost all living organisms, from microorganisms to humans [1]. The importance of the enzymes and their functions vary depending on the organism, its location and the particular type of enzyme [2]. β-N-Acetylhexosaminidases from filamentous fungi are a part of the chitinolytic system in the cell wall of growing hyphae. Together with chitinases (EC 3.2.1.14), they form a binary complex produced under catabolic repression conditions to break down chitin in the cell wall [3]. Many β-N-acetylhexosaminidases from CAZY family GH20, all of them retaining enzymes, also exhibit transglycosylation activity in addition to their natural hydrolytic activity [4]. This phenomenon is not shared by all β-N-acetylhexosaminidases as exemplified in, e.g., human O-GlcNAcase [5], and most β-N-acetylhexosaminidases, especially of bacterial origin, are only aimed as hydrolytic tools [6]. Thus, in combination with their broad substrate specificity, good stability and robustness, they serve as apt tools in many synthetic applications [7].
β-N-Acetylhexosaminidase from Penicillium oxalicum (PoHex) has many characteristics common with the enzymes originating from evolutionarily related genera, such as the thoroughly studied enzyme from Aspergillus oryzae [8] but it also features some unique properties. It is mainly its remarkably high GalNAcase/GlcNAcase ratio, the highest of all known β-N-acetylhexosaminidases with transglycosylating capabilities [9]. Another unique property is an exceptionally good tolerance to chemically modified substrates, the structure of which is altered with various functional groups. These are mainly N-acyl modified substrates [10], substrates substituted with acyl groups at C-6 [11] and even 4-deoxy substrates [12]. These modified substrates are not only well cleaved but also act as donors and acceptors in transglycosylation reactions. This greatly broadens the spectrum of substrates for glycosylation; in contrast to other related enzymes, with the opportunity of efficient GalNAcylation. PoHex also has a broad pH optimum and high pH stability [13].
The functional PoHex enzyme from the fungal producer is a dimer of two monomers, each composed of a propeptide (15 kDa), and a catalytic subunit (65 kDa). Large propeptides of fungal β-N-acetylhexosaminidases (100 amino acids in PoHex) function as intracellular regulators affect secretion, and dimerization of enzyme monomers. The presence and correct association with the propeptide is conditio sine qua non for the enzyme catalytic activity. Catalytic subunits unassociated with the propeptide are not very stable, are incapable of dimerization and enzymatically inactive. This is a unique feature of fungal β-N-acetylhexosaminidases, in contrast to other fungal enzymes like proteases [14]. After expression, the enzyme is excreted into the cultivation medium. Under physiological conditions of the fungus, the propeptides are cleaved off the catalytic subunits by the action of dibasic proteases [15] and they non-covalently associate to afford a functional enzyme with the total molecular weight of 160 kDa at full glycosylation. The catalytic subunit is 501 amino acids long and contains five N-glycosylation sites, which may not always be occupied. Glycosylation supports the enzyme stability. After deglycosylation, the enzyme maintains its activity but shows reduced stability [13].
The fungal production takes ca two weeks and, due to its unselectivity and the presence of numerous other proteins in the cultivation medium, it is low yielding and the purification is complicated. Therefore, we have searched for an alternative host offering a high-yielding, fast, and selective production. Since production in E. coli is unfeasible due to the complex enzyme structure and lack of glycosylation, yeast such as Pichia pastoris have been the first choice for an efficient heterologous production of β-N-acetylhexosaminidases from filamentous fungi [16]. We have recently validated its versatility on the β-N-acetylhexosaminidase from Aspergillus versicolor [17].
In the present work we demonstrate an elegant production of a synthetically important β-N-acetylhexosaminidase from Penicillium oxalicum in Pichia pastoris. Importantly, we confirm that the recombinant enzyme not only maintained but even improved its valuable properties compared to the fungal wild-type. Thus, it represents a robust, readily available and versatile tool for glycosylations, especially with the difficult GalNAc moiety. Furthermore, we present GalNAcylation of a library of challenging acceptors, which highlights the enzyme outstanding synthetic potential.
Section snippets
General
pNP-GlcNAc and pNP-GalNAc were obtained from Gold Biotechnology (USA), GlcNAc from Acros Organics (USA), and GalNAc from GLYCON Biochemicals (D). Cyclohexanol was supplied by Spolana (CZ), myo-inositol by Serva (D), and coniferyl alcohol and pyridine-3-aldoxime by Sigma-Aldrich (USA). If not mentioned otherwise, other chemicals including solvents came from Lach:Ner and Lachema (CZ).
Gene cloning and expression
The complete gene of PoHex named Pohex (GenBank: EU189026; 1803 base pairs, encoding 601 amino acid residues) was
Heterologous expression, production and purification of PoHex
The commercially prepared gene of PoHex was cloned in the pPICZαA vector and electroporated into the Pichia pastoris competent cells KM71H. Advantageously, Pichia secrets the enzyme into the cultivation medium, similar to the fungal producer. Twelve colonies were inoculated into complex medium upon induction by methanol and screened for the presence of PoHex by SDS-PAGE and the enzyme activity assay. Out of the twelve colonies, nine of them showed the presence of PoHex. Two colonies were
Discussion
The main advantage of the heterologous production of PoHex in Pichia pastoris is unarguably the considerably shorter cultivation time – 5 days as compared to 12 days in the fungal producer. Since the fungal host produces a range of other proteins in parallel, the purification from the fungal medium is much more complicated. Ryšlavá et al. [13] reported a three-step purification procedure (hydrophobic, ion exchange and gel chromatographies) preceded by a dialysis and precipitation, with a final
Conclusion
In this work we present an outstanding enzymatic tool for introducing GalNAc moiety onto a range of acceptors with varying architecture. The recombinant PoHex can be produced in Pichia pastoris host in a good yield, with a much simpler cultivation and purification procedure. The recombinant enzyme maintained the valuable properties of its native counterpart, especially the uniquely high GalNAcase activity and broad substrate specificity, and showed additional improvement in catalytic properties
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
Support from projects LTC18038 and LTC19038 by the Ministry of Education, Youth and Sports of the Czech Republic (MEYS) is gratefully acknowledged.
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Directed evolution of a β-N-acetylhexosaminidase from Haloferula sp. for lacto-N-triose II and lacto-N-neotetraose synthesis from chitin
2023, Enzyme and Microbial TechnologyCitation Excerpt :The optimal temperature of mHaHex74 is higher than that of the β-N-acetylhexosaminidase from Pseudoalteromonas sp. (30 °C) [23], but lower than that of the enzymes from Chitinolyticbacter meiyuanensis (40 °C) [24] and Rhizomucor miehei (50 °C) [25]. Although mHaHex74 displayed relatively lower specific activities towards GlcNAc substrates than that of HaHex74, the values are still higher than that of most other β-N-acetylhexosaminidases with transglycosylation activity, such as rNag3HWLB1 (19.4 U mg1 towards pNP-GlcNAc; 0.3 U mg1 towards (GlcNAc)2) [21] and PoHex (35 U mg1 towards pNP-GlcNAc) [26]. The conditions of mHaHex74 for LNT2 synthesis were optimized to be 0.1 M (GlcNAc)2, 0.8 M β-lactose and mHaHex74 dosage of 4.0 U mL1 at pH 7.0 and 40 °C for 2.5 h with (Fig. 3).
Reprint of: Advanced glycosidases as ingenious biosynthetic instruments
2021, Biotechnology AdvancesCitation Excerpt :The broad substrate specificity for the C-4 hydroxyl of the substrate is a typical feature of the GH20 β-N-acetylhexosaminidases, enabling them to use substrates in both gluco- and galacto-configurations with the ratio of the respective activities depending on the individual enzyme source (Weignerová et al., 2003; Slámová et al., 2010b). Even though most β-N-acetylhexosaminidases prefer the gluco-configuration in their substrates, some of them favor galacto-configured substrates, such as the enzymes from Penicillium oxalicum (Nekvasilová et al., 2020a; Ryšlavá et al., 2011) and from T. flavus (Bojarová et al., 2019b). β-N-Acetylgalactosamine-containing complex carbohydrates are difficult to obtain by enzymatic synthesis, because there are no known selective transglycosylating β-N-acetylhexosaminidases, and the respective GalNAc-transferases are rare and highly acceptor-specific.
Advanced glycosidases as ingenious biosynthetic instruments
2021, Biotechnology AdvancesCitation Excerpt :The broad substrate specificity for the C-4 hydroxyl of the substrate is a typical feature of the GH20 β-N-acetylhexosaminidases, enabling them to use substrates in both gluco- and galacto-configurations with the ratio of the respective activities depending on the individual enzyme source (Weignerová et al., 2003; Slámová et al., 2010b). Even though most β-N-acetylhexosaminidases prefer the gluco-configuration in their substrates, some of them favor galacto-configured substrates, such as the enzymes from Penicillium oxalicum (Nekvasilová et al., 2020a; Ryšlavá et al., 2011) and from T. flavus (Bojarová et al., 2019b). β-N-Acetylgalactosamine-containing complex carbohydrates are difficult to obtain by enzymatic synthesis, because there are no known selective transglycosylating β-N-acetylhexosaminidases, and the respective GalNAc-transferases are rare and highly acceptor-specific.
Natural and engineered transglycosylases: Green tools for the enzyme-based synthesis of glycoproducts
2021, Current Opinion in Chemical BiologyCitation Excerpt :GH20 β-N-acetylhexosaminidases can also be used for glycoconjugate synthesis. The enzyme from Penicillium oxalicum was recently found capable of transferring GalNac from pNP-GalNAc using a broad range of acceptors, including secondary and tertiary alcohols, coniferyl alcohol, inositol, and pyridine-3-aldoxim [32]. A recently discovered endo-1,3-fucanase from the marine bacterium Wenyingzhuangia fucanilytica (GH168 family) was reported to transfer sulfated fucose to glycerol and methanol from sulfated fucan, revealing a promising synthetic activity for the synthesis of sulfated glycoconjugates [33].
Mutation Hotspot for Changing the Substrate Specificity of β-N-Acetylhexosaminidase: A Library of GlcNAcases
2022, International Journal of Molecular Sciences