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Salmonella typhimurium can synthesize thiamine in the absence of de novo purine biosynthesis by the alternative pyrimidine biosynthetic (APB) pathway (5, 7). Several mutants unable to utilize this pathway have been isolated, and apbA was the first locus characterized that was essential for thiamine synthesis in the absence of purine biosynthesis (1, 2, 6, 8, 15). apbA mutants were found to be prototrophic in a Pur⁺ background, as expected for a locus involved in an alternative pathway. Two conditions were identified under which mutations in apbA cause thiamine auxotrophy: first, if adenine is present in the medium and second, if the purF gene (whose product catalyzes the first step in de novo purine biosynthesis) is deleted. In both of these cases, the growth defect of the strain can be corrected by the addition of exogenous thiamine or pantothenate. The ability of pantothenate and thiamine to satisfy the same growth defect had been previously described but was not well understood (4, 8, 12). The apbA gene maps to 10 min and encodes a protein predicted to contain a flavin adenine dinucleotide-NAD binding site, a motif common to oxidoreductases in various organisms (6).

We recently showed that ApbA catalyzes the reduction of ketopantoic acid to pantoic acid, a reaction in the biosynthetic pathway for pantothenate (16). Two gene products had been implicated in the ketopantoic acid reductase activity in the cell. The ilvC gene product was shown to catalyze the reduction of ketopantoic acid in crude extracts under some conditions, although the primary reaction catalyzed by this enzyme is the reduction of -acetolactate or -aceto--hydroxybutyrate in the synthesis of branched-chain amino acids (16). Mutations which removed the residual ketopantoic acid reductase activity in ilvC mutants of Escherichia coli were isolated, and the lesion was mapped to 87 min. These mutations defined the panE locus and caused pantothenate auxotrophy only in strains defective for acetohydroxy acid isomeroreductase (IlvC) (10). Similar mutations were isolated in Salmonella typhimurium, and crude extracts of the panE ilvC double mutant had no ketopantoic acid reductase activity, consistent with the nutritional requirement of the strain (13, 16). From this work, it was suggested that both IlvC and PanE have ketopantoic acid reductase activity and that together they account for 100% of the cellular activity.

Our identification of a ketopantoic acid reductase activity associated with purified ApbA caused us to re-evaluate the past work on panE. We report here that panE mutations previously reported to map to 87 min are allelic with apbA and map at 10 min on both the S. typhimurium and E. coli chromosomes. Results presented here clarify the position of the final pantothenate biosynthetic gene and raise interesting questions as to the connection between pantothenate and synthesis of thiamine by the APB pathway.

The identification of a ketopantoic acid reductase activity of ApbA (9) meant that either PanE and IlvC do not provide 100% of the cellular activity or, alternatively, that apbA and panE are allelic. To distinguish between these possibilities, we obtained one of the strains containing an original panE mutation (DU6611 ilvC8 panE61) from the Salmonella Genetic Stock Center. As expected, this strain required both branched-chain amino acids (ilvC) and pantothenate (panE). The results described below demonstrated that apbA and panE are allelic.

panE61 was transductionally linked to apbA. Phage P22 was grown on an apbA⁺ strain containing Tn10d(Tc) (17) linked to apbA [DM86; zba-8007::Tn10d(Tc)] (6). This lysate was used to transduce strain DU6611 (panE61 ilvC8) to tetracycline resistance (Tc^r), and the transductants were scored for growth in the absence of pantothenate (panE⁺). Of the 50 Tc^r transductants, 32 required only branched-chain amino acids, indicating that the zba-8007::Tn10d(Tc) insertion was linked to a region sufficient to restore growth without pantothenate to the panE61-containing strain. From these results, we concluded that the panE mutation in strain DU6611 maps at 10 min, not at 87 min, as had been previously reported.

panE61 causes an APB phenotype. One of the clones from the above-described transduction that had retained its pantothenate requirement [DM4593; ilvC8 zba-8007::Tn10d(Tc) panE61] was saved, and a P22 lysate was grown on it. This lysate was used to transduce wild-type strain LT2 to Tc^r and the transductants were screened for the ability to grow on minimal glucose medium in the presence of adenine. Sixty-four percent of the Tc^r transductants were unable to grow on this medium unless either exogenous thiamine or pantothenate was provided; i.e., they had the phenotype previously described for apbA mutants (6). In addition, the panE61 mutation prevented thiamine synthesis in a purF mutant background, the diagnostic phenotype for a mutant defective in the APB pathway (data not shown).

panE and apbA are allelic. To confirm that apbA and panE are allelic, the apbA gene was sequenced from an isogenic set of PanE^+/ mutant strains. Primers were designed both upstream and downstream of apbA based on the previously determined sequence (GenBank accession no. U09529). These primers were used to amplify the apbA gene from the chromosomes of strains DM4593 (zba-8007:Tn10d panE61 ilvC8) and DM4594 (zba-8007:Tn10d ilvC8) by PCR as has been described (19). With each strain, the fragments resulting from three independent amplifications were sequenced by the University of Wisconsin-Madison Biotechnology Center. This sequence analysis identified a single base change in the coding sequence of apbA in DM4593, the panE-containing strain, compared to DM4594. This change resulted in a predicted alanine-to-threonine change at amino acid 119, a residue conserved in the three identified ApbA homologs (E. coli, S. typhimurium, and Archaeoglobus fulgidus). To reflect these findings, ApbA and PanE are used interchangeably throughout the remainder of this report.

Ketopantoic acid reductase activity is reduced in apbA mutant backgrounds. To confirm previous reports that IlvC and PanE account for all of the ketopantoic acid reductase activity in the cell, this activity was measured in crude extracts of various mutant strains. Table 1 shows the ketopantoic acid reductase levels in crude extract of wild-type and mutant strains. Appropriate strains were grown overnight in rich medium at 37°C with shaking, and cell extracts (ca. 5 mg of protein per ml) were generated by sonication. The assay for ketopantoic acid reductase was performed as previously described (9, 11). Briefly, the assay mixture contained 2.5 µmol of ketopantoic acid, 0.5 µmol of NADPH, and 200 µl of crude extract as a source of enzyme in a total volume of 2.0 ml of 100 mM potassium phosphate buffer, pH 7.5. Mixtures were incubated at 42°C for 2 min prior to initiation of the reaction by addition of ketopantoic acid. NADPH oxidation was monitored at 340 nm to determine the rate of the reaction. As can be seen in Table 1, an apbA mutant strain contained ~40% of the ketopantoic acid reductase activity of a wild-type strain. This remaining activity was eliminated by an ilvC mutation, as indicated by the lack of reductase activity in strain DM3498 (ilvC apbA). These assays showed that, together, the ApbA (PanE) and IlvC proteins account for all of the detectable ketopantoic acid reductase activity in the cell. Taken together, the above results clearly demonstrate that apbA and panE are allelic and encode the pantothenate biosynthetic enzyme ketopantoic acid reductase.

Is PanE involved in thiamine biosynthesis? Previous results showed that thiamine and pantothenate were equally proficient at correcting the nutritional requirement of apbA strains in either a Pur⁺ or a purF genetic background (6, 8a). With the identification of apbA as the pantothenate biosynthetic gene panE, our focus shifted from understanding how pantothenate satisfies the growth requirement of a putative thiamine biosynthetic mutant to addressing how a lesion in pantothenate biosynthesis could result in a defect in the APB pathway for thiamine synthesis.

Pantoic acid is not a precursor to thiamine via the APB pathway. In the first model suggested above, pantoic acid would be a precursor to thiamine via the APB pathway, demanding that the pantoic acid synthesized by PanE donate carbon to both pantothenate and thiamine. To initiate dissecting the role of PanE in thiamine biosynthesis, we synthesized uniformly labeled ¹⁴C-pantoic acid to address the question of whether pantoic acid is a direct precursor to thiamine via the APB pathway. First, [U-¹⁴C]ketoisovalerate was synthesized from [U-¹⁴C]valine by the method of Meister (14). [U-¹⁴C]pantoic acid was then synthesized from the [U-¹⁴C]ketoisovalerate by the method of King et al. (11). Identification and radiochemical purity were confirmed by multiple descending paper chromatography systems as described by Benson et al. (3). A 0.009-µmol sample of [U-¹⁴C]pantoic acid was added to a culture of purF apbA ilvC cells growing in 10 ml of minimal medium containing adenine and branched-chain amino acids. After overnight growth at 37°C with shaking, the thiamine was extracted from the cells and quantified by using a previously published procedure (18). No radioactivity was found to be associated with the thiamine purified from purF apbA ilvC cells grown in this manner, although in a control experiment, radiolabeled pantothenate was recovered. Based upon the pantoic acid specific activity (0.59 mCi/µmol) and the quantity of thiamine isolated, we expected to detect at least 12,000 counts associated with the thiamine-containing fraction if pantoic acid donated at least one carbon atom to thiamine. From these experiments, we concluded that pantoate is not a precursor to thiamine via the APB pathway.