(Received for publication, November 9, 1994; and in revised form, January 23, 1995)
From the
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
.
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), ()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, ()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.
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.
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) .
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).
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.
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).
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.
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. ()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
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U01881[GenBank].