Elsevier

Bioresource Technology

Volume 276, March 2019, Pages 244-252
Bioresource Technology

Structural and functional characterization of thermostable biocatalysts for the synthesis of 6-aminopurine nucleoside-5′-monophospate analogues

https://doi.org/10.1016/j.biortech.2018.12.120Get rights and content

Highlights

  • Structural characterization of adenine phosphoribosyltransferase 2 from Thermus thermophilus.

  • TtAPRT2 was immobilized onto glutaraldehyde activated magnetic microspheres (MTtAPRT2)

  • MTtAPRT2 derivatives displayed higher thermostability than soluble enzyme.

  • MTtAPRT2F was biochemically characterized, and reused up to 10 times in the synthesis of AMP.

  • MTtAPRT2F was employed in the one-pot, one-step production of NMP analogues.

Abstract

The present work describes the functional and structural characterization of adenine phosphoribosyltransferase 2 from Thermus thermophilus HB8 (TtAPRT2). The combination of structural and substrate specificity data provided valuable information for immobilization studies. Dimeric TtAPRT2 was immobilized onto glutaraldehyde-activated MagReSyn®Amine magnetic iron oxide porous microparticles by two different strategies: a) an enzyme immobilization at pH 8.5 to encourage the immobilization process by N-termini (MTtAPRT2A, MTtAPRT2B, MTtAPRT2C) or b) an enzyme immobilization at pH 10.0 to encourage the immobilization process through surface exposed lysine residues (MTtAPRT2D, MTtAPRT2E, MTtAPRT2F). According to catalyst load experiments, MTtAPRT2B (activity: 480 IU g−1biocatalyst, activity recovery: 52%) and MTtAPRT2F (activity: 507 IU g−1biocatalyst, activity recovery: 44%) were chosen as optimal derivatives. The biochemical characterization studies demonstrated that immobilization process improved the thermostability of TtAPRT2. Moreover, the potential reusability of MTtAPRT2B and MTtAPRT2F was also tested. Finally, MTtAPRT2F was employed in the synthesis of nucleoside-5′-monophosphate analogues.

Introduction

Nucleic acid components (nucleosides, nucleotides and nucleobases) are essential metabolites in numerous biochemical processes. For this reason, the use of nucleic acid derivatives as therapeutic molecules for the treatment of many different diseases, such as cancer, acquired immune deficiency syndrome (AIDS) or hepatitis B, among others, has been extensively reported during last decades (De Clercq, 2005, Galmarini et al., 2002, Parker, 2009). As consequence, the development of efficient synthetic processes for these molecules is the main goal for pharmaceutical industry.

The enzymatic synthesis of nucleoside-5′-monophosphates (NMPs) offers many different advantages compared with multi-step, expensive and environmentally harmful chemical methods, such as the possibility of one-pot reactions under mild conditions, high chemo-, regio- and stereoselectivity, and an ecofriendly technology (Del Arco and Fernández-Lucas, 2017, Del Arco et al., 2018a, Li et al., 2017, Liu et al., 2012, Serra et al., 2014, Scism et al., 2007, Zou et al., 2008). However, despite the great potential of enzymes as a biocatalyst, their use in industry is often hindered by several factors, including: i) the high cost of recombinant enzymes, ii) the low stability and short lifespan of the mesophilic enzymes subjected to the rather harsh conditions often needed in some industrial processes (extreme pH values, high temperatures, and the presence of organic solvents), and iii) the difficulty in isolating the biocatalyst from the reaction medium for its reutilization (Chapman et al., 2018, Jemli et al., 2016, Mateo et al., 2007). In fact, these constraints require careful consideration in biocatalytic process development.

One of the most frequently used strategies to circumvent the abovementioned scale-up problems is the use of enzymes from thermophiles. The use of thermozymes as biocatalysts offers several advantages, such as the possibility of using high-temperature reactions, resulting in a higher solubilization of substrates and a diminution of medium viscosity. This increases substrate diffusion coefficients, leading to higher overall reaction rates (Del Arco and Fernández-Lucas, 2018, Dumorne et al., 2017, Elleuche et al., 2014, Raddadi et al., 2015, Rathinam and Sani, 2018, Shrestha et al., 2018).

Moreover, enzyme immobilization may help to overcome these problems derived from the scale-up, by enhancing protein stability, facilitating separation of products and allowing the reuse of biocatalyst (Del Arco et al., 2018b, Mateo et al., 2007), but also may enhance activity, selectivity, specificity, and resistance to inhibitors or chemical reagents, even purity (Barbosa et al., 2015, Mateo et al., 2007, Rodrigues et al., 2013). Among the different materials used in enzyme immobilization, porous materials have attracted much attention due to their high specific surface area, good biocompatibility, chemically modifiable surface and good reusability (Zhao et al., 2006). However, the use of small-size matrixes as carriers for enzyme immobilization usually requires complex and costly separation processes (e.g. ultrafiltration or ultracentrifugation) for the isolation and reutilization of biocatalysts, which increases overall costs. Because this, during the last decade, the immobilization of enzymes onto magnetic porous supports has appeared as an alternative immobilization methodology which enables an easy recovery of biocatalyst by applying a magnetic field (Del Arco et al., 2018b, Pérez et al., 2018).

The covalent immobilization of proteins usually proceeds via the nucleophilic attack of primary amino groups (especially the ε-NH2 of lysine residues) on activated supports. In this respect, the activation of primary amino groups in the support by reaction with glutaraldehyde is one of the most versatile methods for support activation. However, since glutaraldehyde is a heterofunctional support the coupling mechanism between protein and glutaraldehyde is not well defined yet, and it seems that it is not limited to just one mechanism (Barbosa et al., 2014, Zucca and Sanjust, 2014).

Adenine phosphoribosyltransferase, APRT (EC 2.4.2.7) catalyzes the transfer of the 5-phosphoribosyl group from 5-phospho-α-D-ribosyl-1-pyrophosphate (PRPP) to N9 in 6-aminopurines, such as adenine or 6-aminopurine derivatives (e.g. 2,6-diaminopurine, 6-methylpurine, 2-fluorodenine, among others), in presence of Mg2+, to obtain the corresponding NMPs (Del Arco and Fernández-Lucas, 2017, Del Arco et al., 2018c). In a previous work (Del Arco et al., 2018c), adenine phosphoribosyltransferase 2 from Thermus thermophilus HB8 (TtAPRT2) was cloned, expressed and purified. The present work aims to explore the potential application of TtAPRT2 as a biocatalyst for the synthesis of NMP analogues. For this purpose, the structural and biochemical characterization of TtAPRT2 was studied. The complete crystal structure of TtAPRT2 was reported, providing valuable structural information for further immobilization studies. The thermal stability, as well as the substrate specificity, of TtAPRT2 was also tested. The complete biochemical characterization of TtAPRT2 allowed to select the most effective immobilization procedures for the covalent immobilization of TtAPRT2 onto glutaraldehyde activated magnetic microspheres (MTtAPRT2 derivatives). In addition, the effect of pH and temperature on the activity and stability of MTtAPRT2 derivatives, as well as their reusability, was also tested. Taking into account the previous experimental work, derivative MTtAPRT2F was chosen as the most suitable biocatalyst and was successfully employed in the enzymatic synthesis of several different 6-aminopurine NMPs.

Section snippets

Materials

Cell culture medium reagents were purchased from Difco (St. Louis, MO). Trimethyl ammonium acetate buffer and 5-phospho-α-D-ribosyl-1-pyrophosphate were acquired from Sigma-Aldrich. All other reagents and organic solvents were provided by Scharlab (Barcelona, Spain) and Symta (Madrid, Spain). All natural and non-natural purine bases used in this work were purchased from Carbosynth Ltd. (Compton, United Kingdom).

Enzyme cloning, expression and purification

Two different protocols were used in this study, the first one for biochemical

Structural characterization of APRT2 from Thermus thermophilus HB8

In a previous work, Del Arco et al. (2018c) described the cloning, production and purification of adenine phosphoribosyltransferase 2 (APRT, EC 2.4.2.7) from Thermus thermophilus HB8 as biocatalyst for the synthesis of NMPs. The present work continues to focus on this matter and seeks to further explore the full structural and biochemical characterization of TtAPRT2.

The crystal structure of APRT2 from Thermus thermophilus HB8 was determined (TtAPRT2; PDB ID 5ZGO) at 2.6 Å resolution (See

Conclusions

Herein, the structural and functional characterization of soluble and immobilized TtAPRT2, and their application as biocatalyst for the synthesis of NMP analogues, is reported. Among the produced derivatives, MTtAPRT2F was selected as optimal biocatalyst (activity: 507 IU g−1biocatalyst; 44% retained activity). MTtAPRT2F outperformed soluble TtAPRT2 in thermal stability (any loss of activity after 15 h at 70 °C) and was reused for up to 10 cycles. Finally, MTtAPRT2F was successfully employed in

Conflict of interest statement

The authors declare no commercial or financial conflict of interest.

Acknowledgments

This work was supported by the Santander Foundation [SAN151610] and the Universidad Europea de Madrid [2016/UEM08]. The study on the crystal structure was done by the RIKEN group. Authors also thank to Peter Bonney for their continued support and enthusiasm for the project.

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