CLONING AND EXPRESSION OF THE HETERODIMERIC DEOXYGUANOSINE KINASE DEOXYADENOSINE KINASE OF LACTOBACILLUS-ACIDOPHILUS R-26

Two uniquely paired deoxynucleoside kinases, deoxycytidine kinase/deox yadenosine kinase (dCK/dAK) and deoxyguanosine kinase/deoxyadenosine k inase (dGK/dAK) are required, together with thymidine kinase (TK), for deoxynucleotide synthesis in Lactobacillus acidophilus R-26. Using po lymerase chain reaction-generated probes based on N-terminal amino aci d sequences, me have cloned tandem genes for 25- and 26-kDa polypeptid es, whose derived amino acid sequences and size correspond to wild-typ e Lactobacillus enzyme subunits. Expression in Escherichia coli uses a single endogenous promoter and yields active dGK/dAK (similar to 3% o f extracted protein) closely resembling wild-type dGK/dAK in specifici ty, kinetics, heterotropic activation, and end product inhibition. Ali gnment of cloned genes reveals 65% identity in their DNA sequences and 61% identity in derived amino acid sequences. Comparison with herpes- viral TKs reveals three conserved regions: glycine- and arginine-rich ATP-binding motifs and a D/E-R-S/H motif at the putative TK deoxynucle oside site. Greater homology, however, is seen upon multiple alignment of dGK with mammalian deoxycytidine kinases, yielding the consensus s equence -D/E-R-S-I/V-Y-x-D-, dGK also shares a sequence (-Y-D-P-T-I/L- E-D-S/Y-Y-) required for GTP hydrolysis by p21(ras).