Alteration Of Substrate Specificity Of Nucleoside Deoxyribosyltransferase ? And Establishment Of A High-throughput Screening System

Nucleoside analogues have been widely used in the medicine field as antiviral or antitumor agents.Nucleoside analogues can be synthesized by chemical or enzymatic methods.Nucleoside deoxyribosyltransferase is one of the important enzymes for enzymatic synthesis of nucleoside analogues.Nucleoside deoxyribosyltransferase II(NDT)catalyzes the transglicosylation reaction of 2'-deoxyribose moiety between purine and/or pyrimidine bases,and has been used in the synthesis of nucleoside analogues.The high specificity for 2'-deoxyribose limits the application of NDT.Due to 2'C-and/or 3'C-modified nucleosides have been widely used as antiviral or antitumor agents,the improvement of NDT's activity towards these modified nucleosides by protein engineering should be very interesting for pharmaceutical industry.In order to obtain NDT mutants with 2',3'-dideoxynucleoside or arabinoside activity,the mutation sites of nucleoside deoxyribosyltransferase II from Lactobacillus helveticus(Lh NDT)were chosen by semi-rational design.Mutant libraries of Lh NDT Gly10,Ala11 and Leu96 were constructed by site-saturation mutagenesis.In this study,a high-throughput screening system was established by enzyme coexpression,indophenol colorimetric assay and whole-cell catalysis.The screening system was set up with low background,high sensitivity and high accuracy and could be applied to detect NDT-specific activity for a variety of cytidine analogues,such as 5-aza-2'-deoxycytidine,2',3'-dideoxycytidine,cytosine-?-D-arabinofuranoside.A G10 S mutant with 2',3'-dideoxynucleoside catalytic activity was screened out.The conversion rate of 2',3'-dideoxycytidine was 5.2 times higher than that of wild-type NDT catalyzed reaction when incubating 2 ?L purified enzyme with 10 m M 2',3'-dideoxyinosine and 15 m M cytosine in 50 m M,p H 6.0 potassium phosphate buffer for 12 h.At present,there are few reports about NDT engineering.This study broadened the recognition range of Lh NDT for glycosyl modification.NDT engineering is hindered by a lack of effective screening methods.In this study,a high throughput screening system for NDT mutants was established for the first time,which laid a foundation for further engineering and property optimization of NDT.