DEOXYCYTIDINE KINASE AND DEOXYGUANOSINE KINASE OF LACTOBACILLUS-ACIDOPHILUS R-26 ARE COLINEAR PRODUCTS OF A SINGLE-GENE

Three of the four deoxynucleoside kinases required for growth of Lacto bacillus acidophilus R-26 exist as heterodimeric pairs specific for de oxyadenosine (dAK) and deoxycytidine (dCK) or dAK and deoxyguanosine ( dGK), However, only two tandem genes, dak/dgk, are found, and are expr essed only as dAK/dGK in transformed Escherichria coli, Sequencing pep tides spanning 63% of the native dCK subunit revealed a sequence ident ical to that deduced from dgk (beginning MTVIVL ...), except that dCK lacks residues 2 and 3 (dCK is M .. IVL; dGK is . TVIVL). Also, mass s pectrometry indicates that native dCK and dGK subunits are identical i n mass adjusted for the first three residues, Furthermore, the native enzymes have identical isoelectric pH values, indicating an equal numb er of charged residues. To enable E. coli to express peptide having th e native dCK sequence, codons 2 and 3 were deleted from the dgk portio n of the tandem genes, resulting in expression of protein having the s pecificities and regulatory properties of native dAK/dCK, including he terotropic stimulation of dAK activity by deoxycytidine or dCTP (not d eoxyguanosine or dGTP) and end-product inhibition of the respective ac tivities by dATP and dCTP. Subcloning normal and mutant dgk yielded ho modimeric dGK and dCK, respectively. The dCK homodimer strongly resemb les human dCK, with a low K-m for deoxycytidine, the ability to phosph orylate deoxyadenosine and deoxgguanosine at much higher K-m values, a nd end-product inhibition by dCTP. Thus two distinct and specific enzy mes evidently are derived from a single Lactobacillus gene, The mechan ism by which this occurs in live has yet to be elucidated.