ABSTRACT

Two uniquely paired deoxynucleoside kinases, deoxycytidine kinase/deoxyadenosine kinase (dCK/dAK) and deoxyguanosine kinase/deoxyadenosine kinase (dGK/dAK) are required, together with thymidine kinase (TK), for deoxynucleotide synthesis in Lactobacillus acidophilus R-26. Using polymerase chain reaction-generated probes based on N-terminal amino acid sequences, we have cloned tandem genes for 25- and 26-kDa polypeptides, whose derived amino acid sequences and size correspond to wild-type Lactobacillus enzyme subunits. Expression in Escherichia coli uses a single endogenous promoter and yields active dGK/dAK (3% of extracted protein) closely resembling wild-type dGK/dAK in specificity, kinetics, heterotropic activation, and end product inhibition. Alignment of cloned genes reveals 65% identity in their DNA sequences and 61% identity in derived amino acid sequences. Comparison with herpesviral TKs reveals three conserved regions: glycine- and arginine-rich ATP-binding motifs and a D/E-R-S/H motif at the putative TK deoxynucleoside site. Greater homology, however, is seen upon multiple alignment of dGK with mammalian deoxycytidine kinases, yielding the consensus sequence -D/E-R-S-I/V-Y-x-D-. dGK also shares a sequence (-Y-D-P-T-I/L-E-D-S/Y-Y-) required for GTP hydrolysis by p21.

FOOTNOTES

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This work was supported in part by National Institutes of Health Grants CA-47828 and GM49635. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U01881[GenBank].

§

Present address: Dept. of Biochemistry, Molecular and Cellular Biology, Northwestern University, Evanston, IL 60208-3520.

¶

Present address: Dept. of Biochemistry, State University of New York at Buffalo, Buffalo, NY 14214.

**

To whom correspondence should be addressed: Dept. of Biochemistry, The Ohio State University, 484 W. 12th Ave., Columbus, OH 43210-1292. Tel.: 614-292-0485; Fax: 614-292-6773.

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The abbreviations used are: TK, thymidine kinase; dCK, deoxycytidine kinase; dAK, deoxyadenosine kinase; dGK, deoxyguanosine kinase; PCR, polymerase chain reaction; ORF, open reading frame; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; bp, base pair(s); kb, kilobase pairs (kb).

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S. Ikeda, personal communication.

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S. Ikeda, personal communication.

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