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 Lactobacillus acidophilus R-26 exist as heterodimeric pairs specific for deoxyadenosine (dAK) and deoxycytidine (dCK) or dAK and deoxyguanosine (dGK). However, only two tandem genes, dak/dgk, are found, and are expressed only as dAK/dGK in transformed Escherichia coli. Sequencing peptides spanning 63% of the native dCK subunit revealed a sequence identical to that deduced from dgk (beginning MTVIVL···), except that dCK lacks residues 2 and 3 (dCK is M··IVL; dGK is ·TVIVL). Also, mass spectrometry indicates that native dCK and dGK subunits are identical in mass adjusted for the first three residues. Furthermore, the native enzymes have identical isoelectric pH values, indicating an equal number of charged residues. To enable E. coli to express peptide having the native dCK sequence, codons 2 and 3 were deleted from the dgk portion of the tandem genes, resulting in expression of protein having the specificities and regulatory properties of native dAK/dCK, including heterotropic stimulation of dAK activity by deoxycytidine or dCTP (not deoxyguanosine or dGTP) and end-product inhibition of the respective activities by dATP and dCTP. Subcloning normal and mutant dgk yielded homodimeric dGK and dCK, respectively. The dCK homodimer strongly resembles human dCK, with a low Km for deoxycytidine, the ability to phosphorylate deoxyadenosine and deoxyguanosine at much higher Km values, and end-product inhibition by dCTP. Thus two distinct and specific enzymes evidently are derived from a single Lactobacillus gene. The mechanism by which this occurs in vivo has yet to be elucidated.

Structural homologies with ATP- and folate-binding enzymes in the crystal structure of folylpolyglutamate synthetase

Folylpolyglutamate synthetase, which is responsible for the addition of a polyglutamate tail to folate and folate derivatives, is an ATP-dependent enzyme isolated from eukaryotic and bacterial sources, where it plays a key role in the retention of the intracellular folate pool. Here, we report the 2.4-Å resolution crystal structure of the MgATP complex of the enzyme from Lactobacillus casei. The structural analysis reveals that folylpolyglutamate synthetase is a modular protein consisting of two domains, one with a typical mononucleotide-binding fold and the other strikingly similar to the folate-binding enzyme dihydrofolate reductase. We have located the active site of the enzyme in a large interdomain cleft adjacent to an ATP-binding P-loop motif. Opposite this site, in the C domain, a cavity likely to be the folate binding site has been identified, and inspection of this cavity and the surrounding protein structure suggests that the glutamate tail of the substrate may project into the active site. A further feature of the structure is a well defined Ω loop, which contributes both to the active site and to interdomain interactions. The determination of the structure of this enzyme represents the first step toward the elucidation of the molecular mechanism of polyglutamylation of folates and antifolates.