©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Expression of the Heterodimeric Deoxyguanosine Kinase/Deoxyadenosine Kinase of Lactobacillus acidophilus R-26 (*)

(Received for publication, November 9, 1994; and in revised form, January 23, 1995)

Grace T. Ma (§) Young Soo Hong (¶) David H. Ives (**)

From the Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210-1292

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

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.


INTRODUCTION

Lactobacillus acidophilus R-26 requires a source of deoxyribose (1) and is dependent upon four deoxynucleoside kinases to supply its DNA precursor nucleotides, having no functional ribonucleotide reductase(2) . While most bacteria have thymidine kinase (TK), (^1)few appear to have any of the other three deoxynucleoside kinases. Deoxycytidine kinase (dCK) has been detected in extracts of Bacillus megaterium KM(3) , Bacillus subtilis(4) , and a number of species of the class Mollicutes(5) , but none of the latter exhibited ATP-dependent deoxyadenosine kinase (dAK), and only one (Spiroplasma ciri) contained deoxyguanosine kinase (dGK). Extracts of pneumococci phosphorylate dAdo and dGuo, but apparently not dCyd(6) . The only other bacterium clearly shown to contain all four deoxynucleoside kinases, so far as we are aware, is B. subtilis(7) in which dCyd and dAdo compete for a broadly specific common active site. A spontaneous B. subtilis mutant lacking dCK and dAK activities continues to grow, however.

Thus, the presence of all four kinases as obligatory activities in L. acidophilus may well be unique among the bacteria. Its thymidine kinase closely resembles the E. coli enzyme (8) in its kinetic and regulatory behavior, (^2)whereas the other three deoxynucleoside kinase activities are uniquely organized into heterodimeric proteins containing subunits highly specific for the phosphorylation of dCK/dAK or dGK/dAK, respectively. The subunits of each pair have very recently been separated, identified functionally, and partially sequenced(9) ; the N-terminal sequences of all of these subunits appear to be very similar but not identical.

While it is unclear what advantage is conferred by the separate expression of two subunits specific for deoxyadenosine, it is known that the total activities toward deoxyadenosine, deoxycytidine, and deoxyguanosine are about equal when both of the deoxyadenosine kinases are allosterically activated by half-saturation of the opposing subunit with deoxycytidine and deoxyguanosine, respectively(10, 11) . In addition, each activity is controlled by means of end product inhibition by its respective deoxynucleoside triphosphate, which apparently binds to both the nucleoside and triphosphate sites in the manner of a bisubstrate analog, thereby achieving feedback control in a nonallosteric fashion(9) , as has also been proposed for human deoxycytidine kinase(12) . So that the mechanisms of these effects on the enzyme proteins can be studied by chemical and physical means, it is clearly desirable to clone their genes and to express greater quantities of these proteins. It is also of great interest to learn how these genes are organized for expression by the bacterial genome.

Molecular cloning of the deoxyguanosine/deoxyadenosine kinase has now been achieved and the DNA sequences determined. Development of a cloning probe was complicated by the presence of the highly conserved glycine-rich ATP-binding site very near the N terminus (9, 13) of each subunit, as well as a high degree of codon degeneracy, but the polymerase chain reaction was used to provide the necessary selection specificity. The enzyme has been expressed efficiently in an E. coli host, utilizing the Lactobacillus promoter.


EXPERIMENTAL PROCEDURES

Materials

The Photogene nucleic acid detection kit and biotin-7-dATP were purchased from Life Technologies, Inc. The Sequenase kit with modified T7 DNA polymerase was from U. S. Biochemical Corp. [alpha-S]dATP was purchased from Amersham Corp. Restriction endonucleases and DNA modifying enzymes were obtained from Boehringer Mannheim or Life Technologies, Inc. Taq polymerase was purchased from Amersham, Promega, or Perkin-Elmer. Deoxynucleotides were from Perkin-Elmer. Plasmid DNA purification cartridges were purchased from Qiagen. Phagemid pBluescript(+)KS was from Stratagene. NENSORB-PREP oligonucleotide purification cartridges were purchased from DuPont. Oligonucleotide primers were synthesized in the Biochemical Instrument Center of The Ohio State University.

Bacterial Strains and Plasmids

L. acidophilus R26 (ATCC 11506, recently designated Lactobacillus sp. johnsonii) was used as the source of genomic DNA for the recombinant library. The bacteria were cultured as described previously (14, 15) . The phagemid pBluescript(+)KS was used for library construction and subcloning. Libraries were constructed from purified size-selected restriction fragments of genomic DNA.

Construction of a Biotinylated Cloning Probe by PCR and Colony Hybridization

A DNA probe representing the coding sequences of the N-terminal 26 amino acids was amplified by PCR with thermostable Taq polymerase(16) . Genomic DNA (1 µg) was subjected to PCR for 30 cycles in a total volume of 50 µl containing 3 and 5 µM degenerate sense and antisense primers, respectively, and 200 µM each of dNTPs (dATP, dCTP, dGTP, and dTTP), 1 times buffer (50 mM KCl, 10 mM Tris-HCl at pH 8.4, and 2.5 mM MgCl(2)), and 1.25 units of Taq polymerase. The DNA samples were denatured at 94 °C for 1 min, annealed at 37 °C for the first five cycles and at 50 °C for the subsequent 25 cycles for 1 min, and extended at 72 °C for 30 s. The desired 93-bp product was extracted from agarose electrophoresis gel and reamplified by PCR under the conditions described above, except for annealing at 50 °C throughout all 30 cycles. Finally, using the reamplified PCR 93-bp product (1 ng) as a template, PCR was repeated using 150 µM dATP and 50 µM biotin-7-dATP, for 20-30 cycles of amplification.

Colony hybridization was performed by established procedures(17) , but signals were detected nonisotopically by the Photogene nucleic acid detection system (Life Technologies, Inc.), as described in the manual.

Genomic Library Screening by PCR

Colonies grown to 2-3-mm diameter on LB plates containing ampicillin were resuspended in 50 µl of TTE buffer (1% Triton X-100, 20 mM Tris-HCl, pH 8.5, and 2 mM EDTA). Pooled suspensions of 10-20 colonies were screened by the PCR reaction. After boiling for 5 min to lyse the cells and centrifuging, 5 µl of the supernatant fraction were used directly to provide templates for PCR, amplifying for 35 cycles (see ``Results'' for primers used). In this manner, about 200 colonies could easily be screened in 10 reactions. Each colony of the 20-clone pool giving a positive PCR reaction was screened individually by PCR and a clone selected.

DNA Sequencing

Plasmid DNA templates used for double-stranded sequencing were purified on a Qiagen tip-20 column. Restriction fragments of the positive clones were subcloned into pBluescript, and DNA sequences were determined using the dideoxynucleotide chain termination method of Sanger et al.(18) , labeling the fragments with [P]dATP or dATP-alpha-[S]thiophosphate.

Asymmetrical PCR was employed to generate single-stranded enriched DNA (19, 20, 21) so that the probe could be sequenced directly. Sense and antisense primers were at a 1 to 20 molar ratio, or vice versa. The lower concentration primer was set at 10 nM so it would be depleted after 10-15 cycles, and 1-10 ng of template DNA were used. Other reaction components and concentrations were the same as for the regular PCR described above. In the last cycle, the reaction was extended at 72 °C for 7 min. Single-strand enriched PCR products were purified on a Qiagen tip-5 column, and DNA sequencing was performed, with the lower concentration PCR primer as the sequencing primer.

Extraction and Purification of Enzymes from E. coli Clones-Transformed E. coli XL1-Blue cells were grown at 37 °C in LB broth containing 75 µg/ml ampicillin to an A of about 1.0, harvested by centrifugation, resuspended in extraction buffer (0.1 M Tris-HCl, pH 8.0, 3 mM EDTA, 20% glycerol) containing 2 mM phenylmethylsulfonyl fluoride, and opened by sonication, on ice. Crude extracts were further purified by streptomycin fractionation, ammonium sulfate fractionation, and dATP-Sepharose affinity chromatography(15, 22) .


RESULTS

Construction of Cloning Probe by PCR

At the outset of this work, the only amino acid sequence information available on the Lactobacillus deoxynucleoside kinases was that of the N-terminal 28 residues of the the combined dCK/dAK subunits (13) from enzyme purified to homogeneity, in very small quantities, by dCTP-Sepharose affinity chromatography. Several factors prevented the successful application of mixed oligonucleotide probes, including highly degenerate codons, a conserved ATP-binding site near the N terminus, and finding two different amino acids at several residue positions in the sequence. By employing the PCR, a cloning probe with most of the sequence degeneracy eliminated (and therefore one with a higher degree of specificity) has been constructed on the basis of that sequence. The sense primer for the PCR reaction, 5`-tgctctagATGATXGTN(CT)TN(AT)(GC)NGG-3` (a 17-mer, degeneracy of 1536), comprised the coding sequence of amino acids 1-6 plus an XbaI restriction site added to the 5` end (N denotes T+C+A+G, X denotes T+C+A). The antisense primer 5`-cggaattcT(TG)NGTNCCNA(GA)(GA)TA-(TC)TT-3` (a 17-mer, degeneracy of 1024), contained the complementary coding sequence of amino acids 26 to 21, with an EcoRI site added to its 5` end. Thus, the putative template sequence in the genomic DNA should be 77 bp, and the amplified PCR product, including the added restriction sites, should be 93 bp. Nucleotide sequence analysis of the PCR product showed that the sequence between the primers correlated precisely with the amino acid sequences of dCK/dAK and was nondegenerate. Within the primer regions, however, the sequence was occasionally unclear, indicating utilization of multiple oligonucleotides from the mixture by the PCR reaction. More recent results, in which N-terminal peptide sequences of the separate subunits have been determined and functional identities assigned, make it clear that this probe could hybridize with the genes of any of the four subunits of dCK/dAK or dGK/dAK since their N-terminal amino acid sequences are nearly identical(9) . The 93-bp PCR product was then labeled nonisotopically with biotin by incorporating biotin-dATP during PCR amplification, as described under ``Experimental Procedures.''

Identification of Clones Containing Tandem Genes

The biotinylated 93-mer PCR product was used as a probe in the identification and isolation of one or more of the Lactobacillus deoxynucleoside genes. A 3.4-kb XbaI-digested genomic fragment hybridized strongly to the probe. Colony hybridization revealed two positive clones out of more than a thousand recombinants from an XbaI library, which was constructed in the pBluescript vector in E. coli XL1-Blue cells. Both clones were found to contain a 3.4-kb XbaI insert that hybridized to the probe, and the nucleotide sequences revealed apparently tandem genes, but with one-third of the 3` end of the putative second gene missing from the XbaI fragment. No activity could be detected toward any deoxynucleoside substrate, so we could not determine which enzymes were represented by these partial clones.

Because the 3` end of one of the paired genes was missing, a new library had to be constructed. To be certain of cloning the same genes, a more specific probe was prepared by PCR. A new sense primer, 5`-ACTAGTTAACGAATAGAAGG-3`, a 20-mer reflecting the short noncoding sequence between the tandem genes, was used, keeping the original antisense primer. This new probe was therefore a 117-mer specific for the beginning of the fragmentary gene. Knowing that the 5` to 3` orientation of the genes was from the KpnI to XbaI, the 2.5-kb KpnI genomic fragment that hybridized to the probe on the Southern blot could be expected to include all of the downstream gene. Therefore, a partial genomic library was constructed from the 2-4-kb region of a KpnI digest, also in pBluescript, and transformed into the E. coli XL1-Blue cells. After initially screening more than a thousand recombinants by colony hybridization with the new probe, a secondary PCR screening of groups of 20 colonies was carried out, using the primers employed in constructing the probe. Then, each colony of the pooled group which gave rise to the expected 117-bp fragment was screened individually by PCR. A positive clone, GTM-K48, was identified and was found to contain nucleotide sequences encoding the N-terminal amino acids of dAK and dCK (or dGK).

Nucleotide Sequence Analysis

The coding and the complementary strands of the entire 2.5-kb KpnI insert have been sequenced. Sequencing strategies involved subcloning and ``gene-walking,'' using additional sequence-specific primers. The nucleotide sequence revealed two open reading frames (ORF). The nucleotide and derived amino acid sequences of the two genes are shown in Fig. 1. The first ORF is 648 bp in length, with the 215 codons encoding a peptide with a predicted size of 25 kDa. The N-terminal sequence encoded in this gene exactly matches the known amino acid sequence of residues 2-28 of wild-type dAK(9) . This gene is terminated by a single translation stop codon (TAG) and is separated from the next ORF by a 21-bp spacer. The next 675-bp ORF encodes a peptide of 224 amino acid residues with a predicted molecular mass of 26 kDa. The N-terminal sequence inferred from the DNA of this gene is identical to that determined for the purified peptide of dGK, except for the initial methionine, but it also matches the sequence of the dCK subunit residues 2-28(9, 13) . However, from a determination of the activities expressed (see below) we find that we have cloned the genes for the dGK/dAK pair.


Figure 1: Sequence of the dAdo kinase and dGuo kinase genes from L. acidophilus. Lowercase letters indicate the nucleotide sequence; uppercase letters indicate the amino acid sequence. Promoter elements (-35, -10, etc.) and ribosome binding sites (S/D) are underlined and indicated as marked. The transcription terminator is marked by opposing arrows.



Weak ``-10'' and ``-35'' promoter elements precede the dAK gene, with the sequences TACACT and TTGTTT beginning at nucleotide positions -39 and -63, respectively. Additional promoter elements known to be conserved among Gram-positive bacteria (23) are also found. The first is the ``-45'' A cluster (AAAAA) beginning at nucleotide -72; the second is the T sequence at nucleotide -75; and finally the TG sequence at nucleotides -44. The sequence GAAAGA, 6 bases upstream from the translation start codon of the dAK gene, is a variant of the consensus ribosome-binding site.

Preceding the second gene (the dGK gene), sequences homologous to the ``-10'' and ``-35'' promoter elements are positioned at 141, 259, 331, and 355 bases upstream from the initiation codon of the second gene and overlap the coding region of the first gene. However, since none of the conserved Gram-positive promoter elements precedes the second ORF and also since possible promoter elements are not close to the gene, it is likely that only one promoter functions in the transcription of both genes. There is also a putative ribosome-binding site 6 bases upstream from the initiation codon of the second gene, with the sequence GAAGGA.

Just 4 bases downstream from the translational termination codon (TAA) of the second gene, a sequence having the characteristics of a transcription terminator (24) is seen. A GC-rich region of dyad symmetry, AAAACTGCGGTCCA and TGGATCGCAGTTTT (marked by arrows in Fig. 1), which in the transcript may form a stem and loop hairpin structure, is followed by a stretch of thymidines (positions 1531-1536), another feature of transcription terminators. No such characteristic sequence is observed near the stop codon of the dAdo kinase gene, so it seems likely that the two subunits are transcribed as a polycistronic mRNA.

Expression in E. coli

Lysates of E. coli XL1-Blue cells containing the pBluescript vector with or without insert (control) were assayed for all three deoxynucleoside kinase activities: dCK, dGK, and dAK. Enzyme activities of dGK and dAK, but not dCK, were observed in the crude extract of the KpnI clone. Addition of isopropyl-1-thio-beta-D-galactopyranoside to activate the lac promoter of the vector did not further increase the kinase activities, indicating that the dAK/dGK was expressed by means of its endogenous promoter. Specific activities for both dAdo phosphorylation and dGuo phosphorylation in the crude extract of the KpnI clone were more than 10-fold higher than in protein extracted from wild-type Lactobacillus.

To verify that this cloned dGK/dAK is similar to the wild-type Lactobacillus dGK/dAK, the enzyme activities were tested for allosteric interactions and end product inhibition. Like the wild-type enzyme, the cloned dAK in crude extract was activated about 5-fold by 0.1 mM dGuo, and also was inhibited 90% by 0.5 mM dATP; dGK was also potently inhibited by 0.5 mM dGTP, with only 5% activity remaining. However, dCyd has no stimulating effect on dAdo phosphorylation, again indicating that the dAK cloned is the one associated with the dGK, not with the dCK subunit.

The cloned dGK/dAK could be purified to apparent homogeneity by dATP-Sepharose affinity chromatography in exactly the same fashion as the wild-type enzyme(22) . The specific activities of the pure enzyme were 1000 and 65 units/mg for dGK and dAK, respectively, compared with 2000 and 250 units/mg for the wild-type enzyme. We suspected that these differences might be due to differences in the processing mechanisms in Lactobacillus versus the E. coli host. Comparing the inferred N-terminal amino acid sequences with sequences of wild-type dGK and dAK subunits, obtained previously(9) , reveals two discrepancies (Fig. 2). The cloned dAK gene has codons for two additional amino acids (Thr-Val) immediately following the initial methionine, and the initiating methionine encoded in the cloned dGK gene is missing from the wild-type dGK protein sequence. While it is not uncommon to have the initial methionine processed off, the Lactobacillus strain seems to possess some sort of a mechanism to remove the Thr and Val residues, while leaving the initial Met.


Figure 2: Comparisons of N-terminal amino acid sequences determined for wild-type dGK and dAK subunits (9) with sequences inferred from cloned DNA sequences.



Preliminary N-terminal sequence analysis was performed on the purified dimeric expressed protein, anticipating two residues per cycle if the cloned enzyme were identical to wild type. However, each cycle yielded only one amino acid residue, in order: Thr, Val, Ile, Val, Leu, and Ser, exactly as found in the wild-type dGK subunit. The absence of N-terminal Met may have one of two interpretations. It could have been processed off of both subunits identically, or these results could mean that the dAK subunit is blocked when expressed in E. coli, and that only dGK could be sequenced. The latter seems more likely, since only about 25% of the total protein appeared to be sequenceable. The purified cloned dGK/dAK migrated at the same rate on SDS-polyacrylamide gels as the wild-type dGK/dAK, both at pH 9.5 (the pH in the running gel of the Laemmli system) and at pH 6.8 in the multiphasic zone electrophoresis 3328.IV buffer system(25) , consisting of BisTris/TES and BisTris/Cl buffers, employed by Moos et al.(26) . In the latter buffer system, the subunits were resolved into two bands, just as they were with wild-type enzyme (9) (results not shown).

Sequence Alignment of dAdo Kinase and dGuo Kinase

Sequences of the dAK and dGK genes cloned from Lactobacillus were analyzed by the computer software DNAStar, revealing a 65% identity overall in the two DNA sequences. Furthermore, alignment of amino acid sequences inferred from the dAK and dGK genes reveals 61% identity between the two subunits in the 215 amino acids which overlap (Fig. 3). Regions that are highly conserved in other groups of proteins are marked in boldface type. Three regions in each of the two peptide sequences seem to be highly conserved in other phosphotransferase enzymes: (i) glycine-rich sequences, characteristic of ATP-binding sites(13) , located very near the N terminus of each peptide; (ii) sequences containing the Asp-Arg-Ser motif, 78-80 amino acid residues from the N termini, and which by analogy with many viral thymidine kinases (27) might be related to the nucleoside binding sites; and (iii) arginine-rich regions, located about two-thirds of the way along the length of each peptide, thought to participate in the binding of ATP phosphate groups.


Figure 3: Homology of derived amino acid sequences of dAdo kinase (dAK) and dGuo kinase (dGK). 61% identity in the 215-amino acid overlap (DNAStar AANW). A colon (:) denotes amino acids that are positively related using the probability of acceptable mutation matrix, a blank denotes negatively related amino acids, and a period (.) indicates a neutral relationship. Consensus regions, sites i, ii, iii, and iv, described in the text, are indicated in bold.



Recent reports of sequences inferred from several mammalian deoxycytidine kinase clones prompted us to attempt multiple alignment with human(28) , mouse(29) , and rat (GenBank accession no. L33894) dCKs (which are nearly identical to one another). These results are shown in Fig. 4. Groups of residues in common are found over the entire sequence, especially for dGK, and fairly close homology with the glycine- and arginine-rich regions is seen, as with the viral TKs. Moreover, the DRS motifs of the Lactobacillus dGK and dAK both align with a considerably longer dCK sequence (beginning with a glutamate rather than an aspartate, yielding the consensus sequence D/E-R-S-I/V-Y-X-D). These similarities suggest that the Lactobacillus dAK and dGK, in particular, bear a closer relationship with mammalian deoxycytidine kinases than with various thymidine kinases for which the homology lies mainly in the ATP-binding motifs and the very limited DRS/H triad. Evidence for the functional significance of the DRS motif in Lactobacillus dGK/dAK is the subject of a companion study(30) .


Figure 4: Multiple alignment of dGK and dAK sequences with mammalian deoxycytidine kinases. mdCK, mouse; hdCK, human; rdCK, rat. The solid bullet marks residues common to all five sequences; the open bullet marks residues found in all three dCKs plus dGK. Sequence comparisons were facilitated by the Blast E-mail server at the National Center for Biotechnology Information at the National Library of Medicine (43) and the AllAll E-mail server of the Computational Biochemistry Research Group at the ETH in Zürich, Switzerland.



Finally, it is interesting to note that one other region, residues 153-161 of the dGK subunit (site iv), is homologous to the highly conserved region G-2 of p21 and other Ras-like proteins(31, 32) , as seen in Fig. 5. There is nearly a perfect match between this dGK motif and that of the Ras consensus, whereas only 4 of 9 dAK residues are identical with those of the Ras motif. Perhaps the most significant observation to be made now is the fact that the dGK sequence includes the threonine, which, in the p21 sequence, is critical for GTP hydrolysis. While the closely related structure, dGTP, is bound by dGK, there is no evidence that it is hydrolyzed there. Thus, it would appear that this sequence has been adapted to a somewhat different function in the Lactobacillus dGK.


Figure 5: Comparison of dGK and dAK residues 153-161 with the region G-2 consensus sequence of Ras-like proteins(31) . Residues in common are in boldface.




DISCUSSION

dGK/dAK plays an essential role in generating the deoxyribonucleotide precursors, dGTP and dATP, for DNA metabolism in L. acidophilus R-26. Recently, workers in this laboratory have purified the paired wild-type dGK/dAK to homogeneity by the successive application of dCTP-Sepharose and dATP-Sepharose affinity chromatography and have demonstrated that the enzyme is composed of two nonidentical subunits of similar molecular mass (26 KDa)(9, 22) . N-terminal amino acid sequences of each subunit of dGK/dAK, as well as of dCK/dAK, have also been determined. The technique of differential tryptic digestion of one subunit (with the other subunit under the protection of its respective triphosphate end product) has pinpointed the dGuo phosphorylation site to one subunit, whereas the dAdo phosphorylation site is on the other(9) .

Clone GTM-K48 contains two intact genes, separated by a 21-bp spacer and a single translation termination codon. This tandem arrangement, with a functional promoter upstream and a transcription terminator loop downstream, suggests that a polycistronic message for the two subunits is transcribed from a new operon of Lactobacillus. The amino acid sequence inferred from the upstream gene matches the known N-terminal sequences of dAK, and the downstream gene corresponds to those of either dGK or dCK (after processing). However, the gene products that are readily expressed in active form in the E. coli host are specific for dGuo and dAdo only, not for dCyd. Identities of the two genes as those of dGK/dAK are further supported by the allosteric interaction and product inhibition patterns characteristic of dGK/dAK(30) . The cloned enzyme appears to be identical to the wild-type Lactobacillus enzyme in virtually every respect. It was purified to homogeneity by dATP-Sepharose affinity chromatography exactly like the wild-type enzyme(22) . Also like wild-type enzyme, the pure cloned enzyme exhibits an apparent subunit mass of 26 kDa upon SDS-polyacrylamide gel electrophoresis (Laemmli system, pH 9.5), and it is resolved into nonidentical subunits of 25 and 26 kDa by the multiphasic zone electrophoresis 3328.IV buffer system at pH 6.8.

These results, taken together with the genetic and kinetic data from our companion study(30) , clearly demonstrate that the first of the tandem cloned genes encodes dAK and the second gene encodes dGK. This tandem arrangement of the genes on the Lactobacillus chromosome, the presence of a functional promoter sequence preceding the dAK gene, and the apparent transcription terminator loop following the dGK gene strongly suggest that a polycistronic message is encoded in the transcription step, and this is a subject for further investigation.

Further study is needed also to learn whether the apparent deletion of the N-terminal threonine and valine (codons 2 and 3) from wild-type dAK subunits, but with retention of the initiating methionine, represents some new type of post-transcriptional or -translational modification occurring in lactobacilli. Given the fact that the first 19 codons of dAK are identical with those of dGK, it is difficult to see how such a mechanism could be applied selectively to dAK.

Another surprising observation to be drawn from the inferred amino acid sequences, is that, unlike mammalian dCKs which require thiols for activity(33, 34) , neither subunit of the cloned dGK/dAK contains a cysteine (nor is thiol required for activity). The absence of cysteine from wild-type enzyme has been confirmed by amino acid analysis of carboxymethylated protein. (^3)The dAK subunit has but one tryptophan, while dGK has two.

Codon usages of the dGK/dAK genes are similar to other L. acidophilus genes in that they appear to have a preference for U and A in the third base of synonymous codons. This bias against codons ending in G or C also appears in Lactobacillus helveticus and Lactobacillus lactis, but is less pronounced in other Lactobacillus species(35) .

A search of GenBank has yielded both some predictable comparisons and some surprising new relationships. Multiple alignment of the dAK and dGK amino acid sequences with those of the thymidine kinases (not shown) has revealed two sequences common to most purine nucleotide-binding proteins (sites i and iii highlighted in Fig. 3), plus the very short DRS/H motif (site ii), which appears to be required for TK activity and maps close to residues which, when mutagenized, affect the K(m) for thymidine in herpesviral thymidine kinases(36, 37, 38) . However, except for these conserved motifs, the bacterial dGK/dAK bears little homology with the thymidine kinases from various sources. The highly conserved arginine-rich region with the consensus sequence, R-X-X-X-R-X-R, and found at amino acid residue positions 140-146 (site iii) of dGK and dAK, is a common element of all the deoxynucleoside kinases sequenced so far and is also a conserved site in adenylate kinases (27, 39) The corresponding third Arg residue (Arg-118) of chicken adenylate kinase has been shown to be essential for activity in chicken adenylate kinase by the site-directed mutagenesis and NMR studies of Yan et al.(40) . Replacement of Arg-138 by methionine, or even lysine, while causing no apparent structural perturbation compared with wild-type enzyme, resulted in inactivation of the enzyme. Kinetic and structural results suggested that Arg-138 stabilizes the transition state, and to a small extent, the ground state ternary complex, thus implicating this Arg as a candidate involved in transferring the phosphoryl group. Based on refined crystallographic data, Dreusicke et al.(41) also find that the arginines in this region of porcine adenylate kinase bind substrate phosphoryl groups. The arginines in this conserved site of Lactobacillus dGK/dAK may therefore have a similar function.

The considerably higher sequence homology with mammalian dCKs, contrasted with TKs, is interesting in the light of their functional and regulatory similarities. Mammalian dCKs phosphorylate dAdo and dGuo in addition to dCyd, albeit at a common catalytic site. Also, there are similarities in the apparent mode of end product inhibition of dGK/dAK and mammalian dCKs. Whereas thymidine kinase appears to behave like a classical allosterically inhibited enzyme having cooperative kinetics and an inhibitor site(8, 42) , there is evidence that human dCK, at least, is controlled by the nonallosteric multisubstrate inhibitor mechanism we have postulated for these Lactobacillus enzymes (12) . The longer consensus sequence accompanying the D/ERS motifs suggests functional importance, which by analogy with TKs may somehow contribute to deoxynucleoside binding. However, it seems unlikely to be a specificity determinant, since these larger sites are nearly identical in the two subunits of dGK/dAK.

The presence of a highly conserved sequence, -YDPTLEDYY- (Fig. 3, site iv) characteristic of Ras-like proteins, is certainly unexpected in the deoxynucleoside kinases. The threonine of this motif, found in the dGK but not dAK of our sequences, is critical in p21 for the hydrolysis of GTP, and that hydrolysis is accompanied by a large conformational change affecting the entire loop. In the realm of pure speculation, it is tempting to postulate that the large one-directional effects on conformation and activity induced in the dAK subunit upon binding dGTP or dGuo might be mediated through contact with just such a site.


FOOTNOTES

*
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.

(^1)
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).

(^2)
S. Ikeda, personal communication.

(^3)
S. Ikeda, personal communication.


REFERENCES

  1. Hoff-Jorgensen, E. (1952) Biochem. J. 50, 400-403 [Medline] [Order article via Infotrieve]
  2. Durham, J. P., and Ives, D. H. (1971) Biochim. Biophys. Acta 228, 9-25 [Medline] [Order article via Infotrieve]
  3. Wachsman, J. T., and Morgan, D. D. (1973) Appl. Microbiol. 25, 506-508 [Medline] [Order article via Infotrieve]
  4. Rima, B. K., and Takahashi, I. (1977) J. Bacteriol. 129, 574-579 [Medline] [Order article via Infotrieve]
  5. McElwain, M. C., Chandler, D. K. F., Barile, M. F., Young, T. F., Tryon, V. V., Davis, J. W., Jr., Petzel, J. P., Chang, C.-J., Williams, M. V., and Pollack, J. D. (1988) Int. J. Syst. Bacteriol. 38, 417-423
  6. Firshein, W., and Hasselbacher, P. (1970) Biochim. Biophys. Acta 204, 60-81 [Medline] [Order article via Infotrieve]
  7. Mollgaard, H. (1980) J. Biol. Chem. 255, 8216-8220 [Abstract/Free Full Text]
  8. Okazaki, R., and Kornberg, A. (1964) J. Biol. Chem. 239, 275-284 [Free Full Text]
  9. Ikeda, S., Ma, G. T., and Ives, D. H. (1994) Biochemistry 33, 5328-5334 [Medline] [Order article via Infotrieve]
  10. Deibel, M. R., Jr., Reznik, R. B., and Ives, D. H. (1977) J. Biol. Chem. 252, 8240-8244 [Medline] [Order article via Infotrieve]
  11. Chakravarty, R., Ikeda, S., and Ives, D. H. (1984) Biochemistry 23, 6235-6240 [Medline] [Order article via Infotrieve]
  12. Kim, M.-Y., and Ives, D. H. (1989) Biochemistry 28, 9043-9047 [Medline] [Order article via Infotrieve]
  13. Ikeda, S., Swenson, R. P., and Ives, D. H. (1988) Biochemistry 27, 8648-8652 [Medline] [Order article via Infotrieve]
  14. Deibel, M. R., Jr., and Ives, D. H. (1977) J. Biol. Chem. 252, 8235-8239 [Medline] [Order article via Infotrieve]
  15. Deibel, M. R., Jr., and Ives, D. H. (1978) Methods Enzymol. 51, 346-354 [Medline] [Order article via Infotrieve]
  16. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Science 239, 487-494 [Medline] [Order article via Infotrieve]
  17. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  18. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  19. Mihovilovic, M., and Lee, J. E. (1989) BioTechniques 7, 14-16 [Medline] [Order article via Infotrieve]
  20. Gyllensten, U. B., and Erlich, H. A. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7652-7656 [Abstract]
  21. McCabe, P. C. (1990) in PCR Protocols: A Guide to Methods and Application (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds) pp. 76-83, Academic Press, New York
  22. Ikeda, S., and Ives, D. H. (1994) Biochemistry 33, 13373-13381 [Medline] [Order article via Infotrieve]
  23. Graves, M. C., and Rabinowitz, J. C. (1986) J. Biol. Chem. 261, 11409-11415 [Abstract/Free Full Text]
  24. Platt, T. (1986) Annu. Rev. Biochem. 55, 339-372 [CrossRef][Medline] [Order article via Infotrieve]
  25. Jovin, T. M. (1973) Biochemstry 12, 891-898
  26. Moos, M., Jr., Nguyen, N. Y., and Liu, T.-Y. (1988) J. Biol. Chem. 263, 6005-6008 [Abstract/Free Full Text]
  27. Balasubramaniam, N. K., Veerisetty, V., and Gentry, G. A. (1990) J. Gen. Virol. 71, 2979-2987 [Abstract]
  28. Chottiner, E. G., Shewach, D. S., Datta, N. S., Ashcraft, E., Gribbin, D., Ginsburg, D., Fox, I. H., and Mitchell, B. S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1531-1535 [Abstract]
  29. Karlsson, A., Johansson, M., and Eriksson, S. (1994) J. Biol. Chem. 269, 24374-24378 [Abstract/Free Full Text]
  30. Hong, Y. S., Ma, G. T., and Ives, D. H. (1995) J. Biol. Chem. 270, 6602-6606 [Abstract/Free Full Text]
  31. Bourne, H. R., Sanders, D. A., and McCormick, F. (1991) Nature 349, 117-127 [CrossRef][Medline] [Order article via Infotrieve]
  32. Markby, D., Onrust, R., and Bourne, H. R. (1993) Science 262, 1895-1901 [Medline] [Order article via Infotrieve]
  33. Durham, J. P., and Ives, D. H. (1970) J. Biol. Chem. 245, 2276-2284 [Abstract/Free Full Text]
  34. Datta, N. S., Shewach, D. S., Hurley, M. C., Mitchell, B. S., and Fox, I. H. (1989) Biochemistry 28, 114-123 [Medline] [Order article via Infotrieve]
  35. Pouwels, P. H., and Leunissen, J. A. M. (1994) Nucleic Acids Res. 22, 929-936 [Abstract]
  36. Dube, D. K., Parker, J. D., French, D. C., Cahill, D. S., Dube, S., Horwitz, M. S. Z., Munir, K. M., and Loeb, L. A. (1991) Biochemistry 30, 11760-11767 [Medline] [Order article via Infotrieve]
  37. Gentry, G. A. (1992) Pharmacol. Ther. 54, 319-355 [CrossRef][Medline] [Order article via Infotrieve]
  38. Munir, K. M., French, D. C., Dube, D. K., and Loeb, L. A. (1992) J. Biol. Chem. 267, 6584-6589 [Abstract/Free Full Text]
  39. Schulz, G. E., Schiltz, E., Tomasselli, A. G., Frank, R., Brune, M., Wittinghofer, A., and Schirmer, R. H. (1986) Eur. J. Biochem. 161, 127-132 [Abstract]
  40. Yan, H., Shi, Z., and Tsai, M.-D. (1990) Biochemistry 29, 6385-6392 [Medline] [Order article via Infotrieve]
  41. Dreusicke, D., Karplus, P. A., and Schulz, G. E. (1988) J. Mol. Biol. 199, 359-371 [Medline] [Order article via Infotrieve]
  42. Cheng, Y.-C., and Prusoff, W. H. (1974) Biochemistry 13, 1179-1185 [Medline] [Order article via Infotrieve]
  43. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1994) J. Mol. Biol. 215, 403-410 [CrossRef]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.