A novel enzymatic tool for transferring GalNAc moiety onto challenging acceptors

https://doi.org/10.1016/j.bbapap.2019.140319Get rights and content

Highlights

  • β-N-Acetylhexosaminidase from Penicillium oxalicum glycosylates difficult acceptors.

  • Recombinant production in Pichia pastoris brings higher yield and easier purification.

  • β-N-Acetylhexosaminidase glycosylates a secondary, a tertiary hydroxyl and an aldoxime.

  • β-N-Acetylhexosaminidase cannot glycosylate aromatic hydroxyls.

  • The Penicillium oxalicum β-N-acetylhexosaminidase is very suitable for GalNAcylation.

Abstract

The β-N-acetylhexosaminidase from Penicillium oxalicum (PoHex; EC 3.2.1.52) is a fungal glycosidase with an outstandingly high GalNAcase/GlcNAcase activity ratio. It has a remarkable synthetic capability and can process carbohydrates functionalized at various positions. However, the production in the native fungal host is lengthy, unselective and purification from the fungal medium is complicated and low yielding. We present here a novel production method of this enzyme in the eukaryotic host of Pichia pastoris, followed by elegant one-step purification to homogeneity. The resulting recombinant enzyme has improved biochemical and catalytic properties compared to the fungal wild type. Its good production yield (11 mg/400 mL cultivation medium) greatly expands the scope of synthetic applications. We further demonstrate the synthetic utility and broad acceptor specificity of recombinant PoHex in the glycosylation of a series of challenging acceptors with varying structural architectures, namely secondary and tertiary hydroxyl, aldoxime and a poly-hydroxylated compound.

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.

References (23)

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    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.

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    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.

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