A Cascade of Thermophilic Enzymes As an Approach to the Synthesis of Modified Nucleotides.

We propose a new approach for the synthesis of biologically important nucleotides which includes a multi-enzymatic cascade conversion of D-pentoses into purine nucleotides. The approach exploits nucleic acid exchange enzymes from thermophilic microorganisms: ribokinase, phosphoribosylpyrophosphate synthetase, and adenine phosphoribosyltransferase. We cloned the ribokinase gene from Thermus sp. 2.9, as well as two different genes of phosphoribosylpyrophosphate synthetase (PRPP-synthetase) and the adenine phosphoribosyltransferase (APR-transferase) gene from Thermus thermophilus HB27 into the expression vectors, generated high-yield E. coli producer strains, developed methods for the purification of the enzymes, and investigated enzyme substrate specificity. The enzymes were used for the conversion of D-pentoses into 5-phosphates that were further converted into 5-phospho-α-D-pentofuranose 1-pyrophosphates by means of ribokinase and PRPP-synthetases. Target nucleotides were obtained through the condensation of the pyrophosphates with adenine and its derivatives in a reaction catalyzed by APR-transferase. 2-Chloro- and 2-fluoroadenosine monophosphates were synthesized from D-ribose and appropriate heterobases in one pot using a system of thermophilic enzymes in the presence of ATP, ribokinase, PRPP-synthetase, and APR-transferase.

[Biotechnological production of recombinant analogs of hirudin-1 from Hirudo medicinalis].

Hirudin-1 is a highly selective inhibitor of thrombin secreted by salivary glands of the medicinal leech Hirudo medicinalis. This direct anticoagulant is used for the treatment and prevention of disorders in blood coagulation system. Apart from the existing recombinant analog of hirudin-1 (63-desulfatohirudin-1, desirudin) its modified analogs possessing higher activity and stability are of medical value. In this study artificial genes of hirudin and two its analogs (hirudin-1, [Leu1, Thr2]-hirudin-1 and [Leu1, Thr2]-hirudin-1/3) were synthesized and cloned in an expression vector pTWIN1 in frame with the gene of mini-intein SspDnaB from Synechocystis sp. Producing strains of the corresponding fusion proteins were constructed using E. coli strain ER2566. Biotechnological schemes for the production of 63-desulfatohirudin-1 and its analogs were developed. The scheme includes the following stages: isolation of the fusion protein after the desintegration of the cell biomass, refolding of the target peptide within the fusion protein, pH-inducible cleavage of the fusion protein, and chromatographic purification of the target product. Antithrombotic activity of the obtained peptides was determined by a standard amidolytic assay. The developed methods for the production of 63-desulfatohirudin-1, [Leu1, Thr2]-desulfatohirudin-1 [Leu1, Thr2]-desulfatohirudin-1/3 allowed to obtain these peptides with high yields (14, 25 and 24 mg per liter of cell culture respectively) and high activity (13423, 33333 and 19802 ATU/mg respectively).