Immobilized Drosophila melanogaster Deoxyribonucleoside Kinase (DmdNK) as a High Performing Biocatalyst for the Synthesis of Purine Arabinonucleotides

Abstract

Fruit fly (Drosophila melanogaster) deoxyribonucleoside kinase (DmdNK; EC: 2.7.1.145) was characterized for its substrate specificity towards natural and non-natural nucleosides, confirming its potential in the enzymatic synthesis of modified nucleotides. DmdNK was adsorbed on a solid ion exchange support (bearing primary amino groups) achieving an expressed activity > 98%. Upon cross-linking with aldehyde dextran, expressed activity was 30-40%. Both biocatalysts (adsorbed or cross-linked) were stable at pH 10 and room temperature for 24 h (about 70% of retained activity). The cross-linked DmdNK preparation was used for the preparative synthesis of arabinosyladenine monophosphate (araA-MP) and fludarabine monophosphate (FaraA-MP). Upon optimization of the reaction conditions (50 mM ammonium acetate, substrate/ATP ratio = 1:1.25, 2 mM MgCl 2 , 378C, pH 8) immobilized DmdNK afforded the title nucleotides with high conversion (> 90%), whereas with the soluble enzyme lower conversions were achieved (78-87%). Arabinosyladenine monophosphate was isolated in 95% yield and high purity (96.5%).

One-Pot Multi-Enzymatic Production of Purine Derivatives with Application in Pharmaceutical and Food Industry

“…On the contrary, the salvage pathway is composed by a group of reutilization routes by which the cell can satisfy its purine requirements from endogenous and/or exogenous sources of preformed purines. In this regard, numerous enzymes from purine salvage pathway have become valuable catalysts for mono or multi-enzymatic synthesis of nucleosides and nucleotides, such as nucleoside kinases (NKs) [12][13][14][15], phosphoribosyltransferases [7,[9][10][11], nucleoside phosphorylases [4,8,16,17], 2′-deoxyribosyltransferases [5,[18][19][20], among others.…”

Cloning, expression and biochemical characterization of xanthine and adenine phosphoribosyltransferases fromThermus thermophilusHB8

“…It often requires multiple protective group manipulations to control the regio-and stereochemistry of ribose phosphorylation and the use of chemical reagents, such as phosphoryl chloride (POCl 3 ) or phosphorus pentoxide (P 2 O 5 ) and organic solvents which are expensive and environmentally harmful (Asano 2002;Li et al 2015). Due to this, chemical synthesis of NMPs usually provide poor or moderate yields, low product purity and is also associated with harsh reaction conditions and waste disposal issues (Asano 2002;Serra et al 2014). These drawbacks increase final production costs and lead to a prohibitively expensive price of the NADs, impeding their biological trials and studies, as well as limiting their wide therapeutic application.…”

Biocatalytic approaches applied to the synthesis of nucleoside prodrugs

“…Different enzyme preparations (free, adsorbed on epoxy Sepabeads-PEI, and Sepabeads-PEI crosslinked with aldehyde dextran) were evaluated. After optimization, the cross-linked Dm-dNK preparation proved to be the best biocatalyst affording 98% yield of Ara-AMP in 12 h and 91% of F-araAMP in 4 h (Serra et al, 2014).…”