Cloning of deoxynucleoside monophosphate kinase genes and biosynthesis of deoxynucleoside diphosphates.
The genes encoding four deoxynucleoside monophosphate kinase (dNMP kinase) enzymes, including ADK1 for deoxyadenylate monophosphate kinase (AK), GUK1 for deoxyguanylate monophosphate kinase (GK), URA6 for deoxycytidylate monophosphate kinase (CK), and CDC8 for deoxythymidylate monophosphate kinase (TK), were isolated from the genome of Saccharomyces cerevisiae ATCC 2610 strain and cloned into E. coli strain BL21(DE3). Four recombinant plasmids, pET17b-JB1 containing ADK1, pET17b-JB2 containing GUK1, pET17b-JB3 containing URA6, and pET17b-JB4 containing CDC8, were constructed and transformed into E. coli strain for over-expression of AK, GK, CK, and TK. The amino acid sequences of these enzymes were analyzed and a putative conserved peptide sequence for the ATP active site was proposed. The four deoxynucleoside diphosphates (dNDP) including deoxyadenosine diphosphate (dADP), deoxyguanosine diphosphate (dGDP), deoxycytidine diphosphate (dCDP), and deoxythymidine diphosphate (dTDP), were synthesized from the corresponding deoxynucleoside monophosphates (dNMP) using the purified AK, GK, CK, and TK, respectively. The effects of pH and magnesium ion concentration on the dNDP biosynthesis were found to be important. A kinetic model for the synthetic reactions of dNDP was developed based on the Bi-Bi random rapid equilibrium mechanism. The kinetic parameters including the maximum reaction velocity and Michaelis-Menten constants were experimentally determined. The study on dNDP biosynthesis reported in this article are important to the proposed bioprocess for production of deoxynucleoside triphosphates (dNTP) that are used as precursors for in vitro DNA synthesis. There is a significant advantage of using enzymatic biosyntheses of dNDP as compared to the chemical method that has been in commercial use.
Bao
Jie
J
Biochemical Engineering Program, University of California, Davis, 95616, USA.
Ryu
Dewey D Y
DD
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000096:biosynthesis / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000235:genetics
D004269:DNA, Bacterial; D003854:Deoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D017856:Phosphotransferases (Phosphate Group Acceptor); C020015:(deoxy)nucleoside-phosphate kinase; D008274:Magnesium