Cloning and Expression of the Heterodimeric Deoxyguanosine Kinase/Deoxyadenosine Kinase of Lactobacillus acidophilus R-26

Abstract

Examination of conserved motifs on the cloned subunits of the deoxyguanosine kinase/deoxyadenosine kinase (dGK/dAK) of Lactobacillus acidophilus R-26 has begun with the Asp-Arg-Ser (DRS) motif. Replacement of Asp-78 of both subunits with Glu, Ala, or Asn reduced dGK and dAK activities to less than 0.2%, whereas replacement of Arg-79 with Lys, either on both subunits in tandem (R79K), or on the dGK subunit only (R79K:dGK), yielded active but kinetically modified enzymes. These were partially purified, and their kinetic and regulatory properties were analyzed. For dAK activity, the Vmax of the R79K:dGK enzyme was increased 28-fold, with no change in the limiting Km for dAdo, but with a slightly reduced Km for MgATP. The V/K efficiency ratio of dAK was also increased 29-fold, but that of dGK was decreased to 5-10% due to a 10-fold increase in Km for dGuo and a reduced Vmax. Therefore, the R79K substitution seems to have a greater effect on dGuo binding than on that of dAdo, but dGK modification appears to produce a stimulatory conformational effect on the opposite subunit, resembling the known unidirectional activation of dAK by either dGuo or dGTP.

Cloning and expression of human mitochondrial deoxyguanosine kinase cDNA

“…Two Bacillus subtilis proteins (25.4 and 24.1 kDa, respectively) [20], one Fowl pox virus 25.9 kDa polypeptide [21] as well as a ictalurid herpes virus 1 [22] and a saimiriine herpes virus 1 thymidine kinase [23] are all part of a group of sequences that show considerably lower but still significant similarities to human dGK. The only other dGK protein sequence present in the GenBank is that of Lactobacillus acidophilus [24]. In this case the dGK gene is arranged in tandem with the deoxyadenosine kinase gene to which it shows 65% identity.…”

cis-Active Ras G2-like Sequence Implicated in the Heterotropic Activation of the Deoxyadenosine Kinase of Lactobacillus acidophilus R-26

“…7) of the GTPase superfamily (11). While within each GTPase family the amino acid sequence corresponding to the G2 region is highly conserved, some of the residues vary between families of the superfamily.…”

“…While absent from Ras, this arginine-rich region is highly conserved among all the other deoxynucleoside kinases sequenced so far, as well as in other kinases such as porcine adenylate kinase (pAK) (29,30). Since the Arg-138 residue of pAK (or, the Arg-156 of E. coli adenylate kinase) was shown to interact with the transferred phosphoryl group at the transition state (31,32), it is reasonable to assume that an arginine residue in site iii of dAK or dGK has a similar function (11). In equivalent manner, this stabilizing function is also carried out by an arginine residue within the G2 sequence among the ␣-subunits (e.g.…”

“…The chimeric dAK was still inactive until reconstituted into the heterodimer, and the extent of heterotropic activation of this revived chimera remained essentially the same as in the native heterodimer (data not shown). Apart from the first three amino acid residues at the very N terminus which distinguish the specificities of dGK and dCK (12), wild type dAK and dGK share an identical N-terminal amino acid sequence up to residue 18 (11). Therefore, the unique structural determinants of dAK which differentiate its properties from those of dCK and dGK must reside between residues 19 and 171.…”

“…The tandem dak/dgk structural genes of L. acidophilus R-26 have been cloned; the DNA insert consists of a common endogenous promoter, the dak gene preceded by its Shine-Dalgarno sequence, a 21-base pair spacer containing the Shine-Dalgarno sequence for dGK expression, the dgk gene, and a transcription termination loop (11). The derived amino acid sequences of dAK and dGK are more than 60% identical.…”