Heterodimeric deoxyguanosine/deoxyadenosine kinase from Lactobacillus acidophilus R-26: Affinity protein purification, molecular cloning, sequence of the genes, and expression in Escherichia coli.

摘要

A new affinity medium, dAp{dollar}sb4{dollar}-Sepharose, for deoxyadenosine (dAdo) kinase was constructed. This dAp4-Sepharose column has increased the specific activities of both deoxyguanosine (dGuo) kinase (2150 units/mg) and dAdo kinase (280 units/mg) by 2,700-fold, and efficiently purified the enzyme to homogeneity. The enzyme appeared to be a heterodimer with similar subunit molecular weights of 26,000 daltons. The N-terminal protein sequences of the two subunits reflected high homology to each other, and to the deoxycytidine (dCyd) kinase/deoxyadenosine (dAdo) kinase, except for the initial amino acids.; Based on the known N-terminal protein sequence of dCyd kinase/dAdo kinase, highly selective cloning probes specific for the dCyd kinase gene and the dGuo kinase gene was constructed by employing the DNA amplification method of the polymerase chain reaction (PCR). A clone containing an intact dAdo kinase gene and a dGuo kinase gene was identified by the PCR probe, from a Kpn I restricted partial genomic library of L. acidophilus R-26, constructed in the pBluescript vector. The DNA sequence revealed two tandem genes, which were separated by a 21 bp spacer, consisting of sequence homologies with the known N-terminal amino acid sequence of dAdo kinase/dCyd (or dGuo) kinase. The upstream gene (later identified as the dAdo kinase gene) and the downstream gene (later identified as the dGuo kinase gene), respectively, encoded a 25 KDa polypeptide and a 26 KDa polypeptide, which agree with the subunit molecular weights of the purified Lactobacillus protein. Comparison of the two genes revealed 65% overall homology in DNA sequence, and 60.9% identity in the derived amino acid sequence. Consensus sequences of promoter, ribosome binding site and transcription terminator have been identified. Codon biases of the dAdo kinase/dGuo kinase genes of L. acidophilus were compared with those of other Lactobacillus genes.; Expression of the genes, using their single endogenous promoter, in E. coli results in dAdo and dGuo phosphorylation specific activities (in crude extract) 1000 to 2000-fold higher than background, and 10-fold higher than L. acidophilus. Patterns of substrate phosphorylation, allosteric interaction, and end-product inhibition confirm the identities of the gene products as the dAdo kinase/dGuo kinase of L. acidophilus R-26.