Characterization of an atypical, thermostable, organic solvent- and acid-tolerant 2′-deoxyribosyltransferase from Chroococcidiopsis thermalis

Abstract

In our search for thermophilic and acid-tolerant nucleoside 2'-deoxyribosyltransferases (NDTs), we found a good candidate in an enzyme encoded by Chroococcidiopsis thermalis PCC 7203 (CtNDT). Biophysical and biochemical characterization revealed CtNDT as a homotetramer endowed with good activity and stability at both high temperatures (50-100 °C) and a wide range of pH values (from 3 to 7). CtNDT recognizes purine bases and their corresponding 2'-deoxynucleosides but is also proficient using cytosine and 2'-deoxycytidine as substrates. These unusual features preclude the strict classification of CtNDT as either a type I or a type II NDT and further suggest that this simple subdivision may need to be updated in the future. Our findings also hint at a possible link between oligomeric state and NDT's substrate specificity. Interestingly from a practical perspective, CtNDT displays high activity (80-100%) in the presence of several water-miscible co-solvents in a proportion of up to 20% and was successfully employed in the enzymatic production of several therapeutic nucleosides such as didanosine, vidarabine, and cytarabine.

Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and Triphosphates

“…To investigate the effect of organic solvents on enzyme activity, phosphoribosyltransferase activity was assayed in the presence of 20% (v/v) polar protic (MeOH, EtOH, isopropanol, glycerol, ethylene glycol, and propylene glycol) and aprotic co-solvents (acetonitrile, acetone, chloroform, N,N-dimethylformamide, DMSO and ethyl acetate) (Fernández-Lucas et al, 2012;Del Arco et al, 2018d). As shown in Figure 10, there is a negligible loss of activity (less than 5%) in presence of 20% glycerol, ethylene glycol, chloroform and ethyl acetate.…”

Purine and pyrimidine salvage pathway in thermophiles: a valuable source of biocatalysts for the industrial production of nucleic acid derivatives

“…NDTs are key enzymes in the nucleobases salvage pathway, being responsible for the recovery of both purine and pyrimidine bases. In this regard, several protozoan and bacterial NDTs have been reported (Bosch et al 2006;Crespo et al 2017;Del Arco et al 2018b;Fernández-Lucas et al 2010;Fresco-Taboada et al 2013Kaminski 2002;Lawrence et al 2009;Méndez et al 2018;Miyamoto et al 2007;Steenkamp and Halbich 1992). NDTs can be classified into two classes: type I NDTs (PDTs) specific for purines (Pur↔Pur), such as adenine, guanine, or hypoxanthine, and type II NDTs (NDTs), which catalyze the transfer of 2′-deoxyribose among purines and/or pyrimidines (Pur↔Pur, Pur↔Pyr, Pyr↔Pyr) (Fresco-Taboada et al 2013;Kaminski 2002).…”

“…CtNDT is a homotetramer active on purine bases and 2′deoxynucleosides, but it also acts on cytosine and 2′deoxycytidine (Del Arco et al 2018b). All of these unique features prevent the strict classification of CtNDT into either a type I or a type II NDT and further suggest that oligomeric state and structure should complement and expand this functional classification in the future.…”

N-Ribosyltransferase From Archaeoglobus veneficus: A Novel Halotolerant and Thermostable Biocatalyst for the Synthesis of Purine Ribonucleoside Analogs

“…In this respect, several attempts have been made for tailoring NDT active sites toward new substrate specificities on the ribose moiety (Kaminski et al, 2008;Kaminski and Labesse, 2013;Del Arco et al, 2019b;Li et al, 2019). Additionally, most of the reported NDTs up to date are mesophilic enzymes, with the exception of NDTs from cold-adapted Bacillus psychrosaccharolyticus (Fresco-Taboada et al, 2014) and the extremophilic cyanobacteria Chroococcidiopsis thermalis PCC 7203 (CtNDT) (Del Arco et al, 2018a). Consequently, mesophilic enzymes usually display low stability and short lifespan under the harsh conditions usually required for industrial processes, such as extreme pH values, high temperatures or the presence of organic solvents.…”

“…The use of enzymes from microorganisms naturally living in extreme environments has proved to be a successful approach to circumventing the abovementioned drawbacks associated to industrial implementation. More specifically, the choice of enzymes from thermophiles for the synthesis of nucleosides and nucleotides offers, among other advantages, the possibility of using high-temperature reactions, resulting in higher substrate solubilization and increased overall reaction rates (Almendros et al, 2012;Del Arco et al, 2017, 2018a,b, 2019cAtalah et al, 2019;Kamel et al, 2019b;Zhou et al, 2019).…”