Beresnev A.I., Kvach S.V., Sivets G.G.*, Zinchenko A.I.

Institute of Microbiology, National Academy of Sciences of Belarus, Minsk

*Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk

 

ENZYMATIC SYNTHESIS OF

3'-FLUORO-2',3'-DIDEOXYGUANOSINE

 

         Nowadays significant emphasis is focused on application of modified analogs of natural nucleosides and nucleotides in biochemical, molecular-biological and medical studies. Most often chemical methods are engaged to produce modified nucleosides, yet a growing number of publication describes involving microbial enzymes into the process of synthesis [1].

Nucleoside phosphorylases are major enzymes responsible for biocatalytic synthesis of modified nucleosides [2]. There are three principal types of nucleoside phosphorylases: uridine phosphorylase (UPase), thymidine phosphorylase (TPase) utilizing pyrimidine nucleosides as the substrates and purine nucleoside phosphorylase (Pur-NPase) transforming purine nucleosides [3‒5]. Frequency rate of papers devoted to synthesis of modified nucleosides mediated by pyrimidine nucleoside phosphorylase (Pyr-NPase) and based on pyrimidine nucleoside substrates, but showing wider substrate specificity in comparison with UPase and TPase [6].

Several reports indicate that chemically synthesized nucleoside – 3'-fluoro-2',3'-dideoxyguanosine (3'-F-2',3'- ddGuo) displays antiviral activity [7, 8].

Earlier we engineered strains E. coli able to express recombinant homologous UPase and TPase and strain E. coli generating recombinant heterologous Pur-NPase from K. pneumoniae. It was shown that the recombinant UPase and TPase are unable to catalyze phosphorolysis of 3'-fluoro-2',3'-dideoxythymidine (3'-F-2',3'-ddThd) generating the 3-fluoro-2,3-dideoxyribose-1-phosphate essential for synthesis of 2,6-diaminopurine-3'-fluoro-2'3'-dideoxyriboside (3'-F-2',3'-ddDAP) – intermediate of 3'-F-2',3'-ddGuo production. We assumed that Pyr-NPase of Thermus thermophilus (unlike UPase and TPase of E. coli) will show affinity to 3'-F-2',3'-ddThd.

         The present work was aimed at production and characterization of T. thermophilus  Pyr-NPase as well as enzymatic synthesis of antiviral 3'-F-2',3'-ddGuo using Pur-NPase of K. pneumoniae and Pyr-NPase of T. thermophilus.

Materials and Methods. Strain E. coli DH5α indispersable for generation and analysis of constructed recombinant vector and strain E. coli BL21 (DE3) applied for expression of engineered Pyr-NPase served as objects of studies.

Amplification of gene encoding Pyr-NPase from genomic DNA of T. thermophilus was carried out using high-precision Pfu-polymerase. The target gene was further cloned in plasmid pET42a. A novel E. coli strain derived from this vector is a superproducer of geterologous Pyr-NPase T. thermophilus.

Fermentation of bacteria was conducted at 37oC on Luria-Bertany medium containing 50 µg/ml of kanamycin. 0.5 mM IPTG was chosen as inducer of Pyr-NPase synthesis. Control of T. thermophilus Pyr-NPase synthesis in E. coli cells was performed by SDS-PAAG electrophoresis. Purification of target biocatalyst was carried out by heating bacterial lysate containing Pyr-NPase at 80oC during 1 h.

The reaction mixture comprising 15 mM 3'-F-2',3'-ddThd, 10 mM 2,6-diaminopurine, 5 mM K-phosphate buffer (pH 7.0), 5 U/ml of T. thermophilus Pyr-NPase, 50 U/ml of K. pneumoniae Pur-NPase was incubated for 3 days at 60oC. Upon termination of synthesis process the mixture containing 3'-F-2',3'-ddDAP was supplemented with 50 U/ml of recombinant E. coli adenosine deaminase [9] and incubated during 24 h at 30oC. Accumulation of 3'-F-2',3'-ddGuo was checked by HPLC.

Results and Discussion. New E. coli strain producing Pyr-NPase from T. thermophilus was obtained by genetic engineering techniques at the first stage of this work. Increased yields of target biocatalyst were achieved by IPTG induction of T. thermophilus Pyr-NPase synthesis in the course of fermentation of recombinant E. coli strain. Qualitative analysis of cell lysate including the produced Pyr-NPase is presented in fig. 1. It may be seen from electrophoresis that purification of target enzyme by heating cell lysate containing Pyr-NPase of T. thermophilus resulted in recovery of the protein with high purity grade.

    1                2                3

35 kDa
 

 


 

 

 

 

 

Fig 1. Electrophoresis of recombinant Pyr-NPase before (2) and after (3) purification.

1 – Marker proteins of standard molecular weight.

 

The ability of T. thermophilus Pyr-NPase for phosphorolysis of 3'-F-2',3'-ddThd demonstrates Fig 2.

 

 

 

 

 

 

 

 

 

 

 

Fig 2. Time course of 3'-F-2',3'-ddThd phosphorolysis by Pyr-NPase

 

Biocatalytic synthesis of 3'-F-2',3'-ddDAP as 3'-F-2',3'-ddGuo production intermediate was realized at the second stage of this work using Pyr-NPase of T. thermophilus and Pur-NPase of K. pneumoniae. The ultimate yield of the end product (3'-F-2',3'-ddDAP) reached 85±2 mol% based on 2,6-diaminopurine included into the reaction mixture. Nucleoside 3'-F-2',3'-ddGuo was derived after treatment of synthesis 3'-F-2',3'-ddDAP with adenosine deaminase catalyzing substitution of oxygen atom for NH2-moiety in the sixth position of heterocyclic base of 3'-F-2',3'-ddDAP. The final yield of the end product was calculated as 95±3.5 mol% of 3'-F-2',3'-ddDAP level in the reaction mixture.

         In toto, the performed work resulted in construction of genetically engineered E. coli strain producing recombinant Pyr-NPase T. thermophilus. Synthesis of antiviral nucleoside 3'-F-2',3'-ddGuo was originally accomplished with Pyr-NPase of T. thermophilus, Pur-NPase of K. pneumoniae and E. coli adenosine deaminase. 

 

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9. Квач С.В., Ерошевская Л.А., Зинченко А.И. // Микробные биотехнологии: фундаментальные и прикладные аспекты: сб. науч. тр. / редкол.: Э.И. Коломиец [и др.]. Минск, 2007. Т. 1. С. 125–130.