2′-Deoxyribosyltransferase from Bacillus psychrosaccharolyticus: A Mesophilic-Like Biocatalyst for the Synthesis of Modified Nucleosides from a Psychrotolerant Bacterium

Abstract

Structure-function relationships of a novel 2 -deoxyribosyltransferase from the psychrotolerant bacterium Bacillus psychrosaccharolyticus (BpNDT) have been exhaustively studied by biochemical and high resolution crystallographic analyses. Despite BpNDT exhibiting some structural features characteristic of cold-adapted enzymes such as localized flexibility in critical loops, its biochemical properties are typical of mesophilic enzymes. BpNDT is a highly symmetrical homohexamer with tightly associated subunits that possesses flexible and short loops bordering the active sites. The catalytic center is essentially identical to that of other mesophilic homologues. Moreover, BpNDT shows that it is a mesophilic-like enzyme since it is not heat-labile and exhibits an apparent unfolding temperature (T m ) of 49 • C, being active during 96 h at 40 and 50 • C. Finally, BpNDT synthesizes natural and modified nucleosides, with preference for purines as acceptors and pyrimidine nucleosides as donors. Remarkably, the synthesis of several therapeutic nucleosides has been efficiently carried out. In this sense, 5-hydroxymethyl-2 -deoxyuridine (5-HMdUrd), 7-deaza-6-hydroxypurine-2 -deoxyriboside (7-DHPdRib) and theophylline-2 -deoxyriboside were synthesized for the first time by an NDT enzyme, showing the biotechnological interest of BpNDT.

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

“…This superfamily encompasses the NDT and ADP-ribosyl cyclase-like families, as well as another family that includes the hypothetical protein PA1492 from Pseudomonas aeruginosa with uncharacterized function but known three-dimensional structure (PDB entry 1T1J). Within the NDT family we can distinguish two different subfamilies, i) strict N-deoxyribosyltransferases, NDTs (Kaminski, 2002;Fresco-Taboada et al, 2013) and ii) (2 ′ -deoxy)nucleoside-5'monophosphate N-glycosidases, (d)NMP glycosidases (Ghiorghi et al, 2007;Dupouy et al, 2010;Sikowitz et al, 2013;Zhao et al, 2014). Even though both types of enzymes display the canonical "NDT domain" (Pfam PF05014), they possess different substrate specificities.…”

“…Enzyme-catalyzed synthesis by whole cells or purified proteins offers several advantages, such as one-pot reactions, controlled stereo-, regio-, and enantiospecificities, and mild reaction conditions. In this regard, the most commonly used synthetic route for the synthesis of NAs is the transglycosylation reaction catalyzed by nucleoside phosphorylases or 2 ′ -deoxyribosyltransferases (Fresco-Taboada et al, 2013;Lapponi et al, 2016;Del Arco and Fernández-Lucas, 2018;Kamel et al, 2019a;Lewkowicz and Iribarren, 2019;Trelles et al, 2019).…”

“…Nucleoside-2 ′ -deoxyribosyltransferases (NDTs) are the most representative members of the N-deoxyribosyltransferase family (NDT family) which belongs to the N-(deoxy)ribosyltransferaselike superfamily (PFAM entry CL0498), also including ADPribosyl cyclase-like families. NDTs catalyze the interchange of the 2 ′ -deoxyribose moiety between a purine and/or pyrimidine 2 ′ -deoxynucleoside (donor) and a purine and/or pyrimidine base (acceptor) (Kaminski, 2002;Fernández-Lucas et al, 2010;Fresco-Taboada et al, 2013;Crespo et al, 2017;Del Arco et al, 2019a). According to their substrate specificity, NDTs are usually classified as: (i) type I NDTs (PDT), which perform this type of transglycosylation reaction between purine bases (Fresco-Taboada et al, 2013;Pérez et al, 2018) and (ii) type II NDTs (NDT), which do not discriminate between purines and pyrimidines (Kaminski, 2002;Fernández-Lucas et al, 2010;Fresco-Taboada et al, 2016).…”

“…According to their substrate specificity, NDTs are usually classified as: (i) type I NDTs (PDT), which perform this type of transglycosylation reaction between purine bases (Fresco-Taboada et al, 2013;Pérez et al, 2018) and (ii) type II NDTs (NDT), which do not discriminate between purines and pyrimidines (Kaminski, 2002;Fernández-Lucas et al, 2010;Fresco-Taboada et al, 2016). Interestingly, NDTs are highly regioselective for natural bases and a high number of nucleobase derivatives (Fresco-Taboada et al, 2013;Acosta et al, 2018). However, several examples of NDT-mediated glycosylation at multiple sites have been recently reported for several sizeexpanded purines (Ye et al, 2014) and azole derivatives (Vichier-Guerre et al, 2017).…”

Biocatalysis and Biotransformations

“…In more detail, Fresco-Taboada and coworkers investigated the synthesis of modified nucleosides by using novel 2 -deoxyribosyltransferases [13]. Acosta and coworkers reported the synthesis of purine derivatives by the sequential action of 2 -deoxyribosyltransferases and hypoxanthine-guanine-xanthine phosphoribosyltransferases [14].…”