Journal of Bacteriology, November 2003, p . 6732-6735, Vol . 185, No . 22

 
As an essential enzyme in the synthesis and salvage of guanine nucleotides, guanylate kinase has been the subject of numerous investigations . In addition, the biomedical role of human Gmk in the activation of antiviral agents has attracted significant attention (2, 13, 14) . As a result, a large amount of information has amassed about the biochemistry, genetics, and regulation of these enzymes for a variety of organisms (2, 5, 6, 8, 12, 13, 15, 17, 20, 23) and a high-resolution structure for the Saccharomyces cerevisiae enzyme (GUK1) is available (21) . Most guanylate kinases have significant sequence similarity, and the conserved motifs allow the prediction of substrate binding sites . Near the N terminus of the proteins is a canonical nucleoside triphosphate binding motif, GXXXXGK, reflecting the phosphate binding loop (P-loop) that is present in many ATP/GTP binding proteins . This site has been assigned to ATP binding, and the GMP binding site was shown to contain additional conserved residues just C terminal to the P-loop (21) .

Since the activity of Gmk is essential for growth (e.g., null mutations are lethal), few studies have probed the consequences of altered guanylate kinase activity for cellular metabolism . Mutations in the GUK1 gene of S . cerevisiae were identified for their ability to bypass the purine biosynthetic gene repression normally caused by exogenous adenine (14) . In this study, multiple metabolic phenotypes were described for strains that had a detectable, but >10-fold-decreased, level of GUK activity . The authors attributed the phenotypes of these mutants to the accumulation of the substrate GMP that resulted from the decrease in GUK activity (14) . The molecular nature of the causative mutations was not explored in the yeast study, and potential kinetic differences in the mutant enzymes were not addressed .

We report the identification of a mutation in the Salmonella gene that encodes guanylate kinase (gmk) . This mutation causes a cold-sensitive growth defect that can be corrected by the addition of exogenous adenine . The mutant protein displayed a significantly increased apparent K_m for ATP, yet this difference could not fully account for the growth behavior with changing temperature . On the basis of the results reported herein and the known regulatory properties of enzymes involved in the biosynthesis and salvage of purines, a model to explain the role of temperature and adenine is proposed .

Initial observations. In the course of screening mutants generated by localized hydroxylamine mutagenesis (11) around the pyrE locus, a cold-sensitive mutant was identified . Phenotypic characterization showed that this mutant strain (DM922) was able to grow at 37°C on minimal medium but required exogenous adenine for growth at 30°C .

A requirement for adenine at low temperature is caused by a mutation in gmk. A plasmid clone able to complement the growth defect of strain DM922 was isolated from a plasmid library of Sau3A partially digested chromosomal DNA from S . enterica . The complementing plasmid contained a 3.5-kb insert . Subsequent cloning of a 1.2-kb fragment into the mid-copy vector pSU19 (16) provided a plasmid (pMK1) that was able to complement the growth defect in DM922 . Sequence analysis determined that this fragment contained the entire gmk locus and no other complete open reading frames (data not shown) . In Salmonella and Escherichia coli, the gmk locus is the promoter proximal gene in the spoT operon and encodes the essential protein guanylate kinase (8) . Since gmk is <10 kb from the pyrE locus, growth allowed by plasmid pMK1 was considered to be due to functional complementation rather than to multicopy suppression by an unlinked gene .

The gmk gene was amplified by PCR from the wild-type strain LT2 and mutant strain DM922 . Comparison of the nucleotide sequences revealed that the gmk gene from DM922 contained a single base substitution (GA) consistent with the hydroxylamine mutagenesis . This base substitution at nucleotide 56 of the coding sequence results in a serine-to-asparagine (S19N) change in the protein at a location immediately adjacent to the ATP binding site of the protein (13) .

A pair of isogenic strains differing at the gmk locus were constructed and subjected to liquid growth analyses at different temperatures . The data from representative experiments are shown in Table 1 . The gmk101 strain had a significant growth defect at 30°C in minimal medium . Growth was restored by the addition of adenine (>0.1 mM) or by elevated temperature . The addition of other purines, including adenosine, guanosine, and hypoxanthine, had no effect on the growth of the gmk101 mutant at 30°C (data not shown) .

 
The S19N variant of Gmk has an increased K_m for ATP. The biochemical characterization of the mutant and wild-type enzymes was performed . The wild-type and mutant gmk genes were cloned into pET-20b (Novagen, Madison, Wis.) to generate a 6-His tag fusion to the carboxy terminus of the protein . Gmk and GmkS19N proteins were overexpressed in E . coli BL21(DE3) . The resulting 24-kDa proteins were purified using standard nickel column affinity column chromatography and determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be >95% pure . Guanylate kinase activity was measured in an assay coupled to lactate dehydrogenase activity, as has been previously described (8, 17) . Initial velocity measurements were made using 5.1 nmol of either mutant or wild-type enzyme per reaction . The results of representative experiments are shown in Table 2 and Fig . 2 . Data were analyzed and fitted to the Michaelis-Menten equation using the enzyme kinetics template of a Prism program (GraphPad, San Diego, Calif.) . Pseudo first-order kinetics were used to determine the apparent K_m and V_max values presented . The kinetic constants found for the wild-type enzyme were consistent with those reported for the E . coli enzyme (17) . As shown in Table 2, the apparent GMP K_m values of the wild-type and mutant enzymes (10^-4 M) were not significantly different . Kinetic analyses to determine the K_m for ATP were carried out at 30 and 37°C . At both temperatures, the two proteins had comparable V_max values . In contrast, there was a significant difference between the mutant and wild-type enzymes in their apparent K_m values for ATP . The data showed that a more than 20-fold difference in K_m values for ATP existed between the two enzymes . This finding was consistent with a model in which the larger asparagine residue of the mutant protein obstructed the ATP binding site and the serine residue of the wild-type protein did not . The experimental data used to determine the apparent K_m value for ATP (plotted as initial velocity versus ATP concentration) are presented in Fig . 2 .

 
The temperature profile of Gmk activity does not explain the in vivo temperature-dependent phenotype. A temperature profile of the mutant and wild-type enzyme activity was performed to identify a parameter that would correlate with the temperature-dependent phenotype in vivo . Initial velocity was measured from 25 to 52°C . Over this temperature range the change in initial velocity was linear, with slopes of 0.0016 and 0.0037 for the mutant and wild-type protein, respectively (data not shown) . These slopes indicted that the disparity in initial velocity between the wild-type and mutant protein increased with temperature . In contrast, the phenotypic difference between the two strains decreased with temperature, as indicated by the prototrophic growth of the mutant strain at 37 but not at 30°C . This result indicated the temperature-dependent growth of the mutant was not a direct consequence of the altered kinetics of GmkS19N .

Accumulation of GMP contributes to two phenotypes associated with gmk101. Two simple explanations for the temperature-dependent phenotype were considered and eliminated . First, similar ATP pools (0.5 mM) were detected with an Enliten ATP assay system (Promega, Madison, Wis.) at both temperatures, eliminating the possibility that increased ATP levels at 30°C could overcome the difference in K_m value of GmkS19N . Although the presence of exogenous adenine has been reported to increase ATP pool size by 40%, other purine sources result in a similar elevation of pool size and yet fail to allow growth of the gmk101 mutant (1) . Second, regulation of gmk expression was considered . Polyclonal antibodies were obtained from M . Cashel (National Institutes of Health) and used in immunoblot analyses to qualitatively assess accumulation of Gmk in mutant and wild-type strains at the two relevant temperatures . No significant difference in Gmk accumulation levels was noted .

Considering the data presented above, a general model to explain the purine requirement at 30°C and the specificity for adenine caused by the gmk101 mutation was suggested . Reduced activity of Gmk results in the accumulation of GMP in the cell (14) . Since GMP is an allosteric inhibitor of adenylsuccinate synthetase (PurA) (7, 22), a partial starvation for AMP occurred in the gmk101 mutant strain . The starvation was exacerbated at 30°C, resulting in the adenine requirement; we suggest two possible means for this . First, the enzymatic activity of PurA may be decreased at 30°C . There is a precedent for a temperature dependence of biosynthetic enzymes (e.g., that of PurH and MetA) (4, 19) . To the best of our knowledge, a temperature profile for PurA has not been reported . Second, a small increase in ppGpp levels would prevent AMP formation, since ppGpp is the most potent inhibitor of PurA, occupying a site distinct from that of GMP (7, 22) . The metabolism of ppGpp is complex; thus, there are multiple factors that could generate the postulated increase . For instance, in one report a mutant defective in ndk (nucleoside diphosphate kinase) (Fig . 1) was found have a cold-sensitive growth defect (9, 18) . At the low temperature the ppGpp levels in this mutant were elevated with respect to those seen at the permissive temperature . While the mechanism of this effect was not clear, the similarity to the result described herein suggests that the proposed scenario is feasible .

The specificity for adenine for satisfying the mutant growth requirement can be explained by a regulatory role for GMP . GMP allosterically inhibits guanine phosphoribosyl transferase (Gpt), the primary route for the conversion of hypoxanthine and guanine to their respective nucleotides (3, 10) (Fig . 1) . While a single purine source can be converted to all others in a wild-type strain, a block in Gpt would slow, or prevent, the generation of adenine derivatives from the others (22), since ultimately this conversion must go through IMP via adenylsuccinate synthetase (PurA) . Somewhat surprisingly, the preferred route for conversion of adenosine to adenine is via a route through IMP that also depends on Gpt and PurA . The direct phosphorolysis of adenosine to adenine is of minor quantitative importance (emphasized by the fact that an adenine-requiring mutant [e.g., purA or purB] grows poorly on adenosine) (22) . Thus, specificity for adenine is consistent with the known routes of salvage in cases in which PurA is allosterically inhibited .

Interestingly, when the de novo purine pathway is blocked via mutation (purG) in a gmk101 mutant, the specificity for adenine is lost (data not shown) . This result is consistent with the above model and suggests that the distribution of flux from IMP can be different when purines are utilized solely via the salvage pathways .

Conclusions. The work described here identified a lesion in an essential gene and probed the metabolic consequences of partially disrupting the relevant metabolic activity . Two clear phenotypes were noted, and a model to explain them is presented . Many of the purine biosynthetic and salvage enzymes are allosterically regulated positively and negatively by various purine nucleotides and ppGpp . As shown here, significant metabolic disruptions can result from the sum of these multiple effects .

The nucleotide sequence for the gmk gene was deposited in GenBank under accession number AF140283 .

This work was supported by competitive grant GM47296 from the NIH . Funds were also provided from a 21st Century Scientist Scholars Award from the J . S . McDonnell Foundation .

Present address: Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109-1065 .

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