N. Ma et al., DEOXYCYTIDINE KINASE AND DEOXYGUANOSINE KINASE OF LACTOBACILLUS-ACIDOPHILUS R-26 ARE COLINEAR PRODUCTS OF A SINGLE-GENE, Proceedings of the National Academy of Sciences of the United Statesof America, 93(25), 1996, pp. 14385-14390
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.