Pantoate kinase and phosphopantothenate synthetase, two novel enzymes necessary for CoA biosynthesis in the Archaea

Abstract

Bacteria/eukaryotes share a common pathway for coenzyme A (CoA) biosynthesis. Although archaeal genomes harbor homologs for most of these enzymes, homologs of bacterial/eukaryotic pantothenate synthetase (PS) and pantothenate kinase (PanK) are missing. PS catalyzes the ATP-dependent condensation of pantoate and beta-alanine to produce pantothenate, whereas PanK catalyzes the ATP-dependent phosphorylation of pantothenate to produce 4'-phosphopantothenate. When we examined the cell-free extracts of the hyperthermophilic archaeon Thermococcus kodakaraensis, PanK activity could not be detected. A search for putative kinase-encoding genes widely distributed in Archaea, but not present in bacteria/eukaryotes, led to four candidate genes. Among these genes, TK2141 encoded a protein with relatively low PanK activity. However, higher levels of activity were observed when pantothenate was replaced with pantoate. V(max) values were 7-fold higher toward pantoate, indicating that TK2141 encoded a novel enzyme, pantoate kinase (PoK). A search for genes with a distribution similar to TK2141 led to the identification of TK1686. The protein product catalyzed the ATP-dependent conversion of phosphopantoate and beta-alanine to produce 4'-phosphopantothenate and did not exhibit PS activity, indicating that TK1686 also encoded a novel enzyme, phosphopantothenate synthetase (PPS). Although the classic PS/PanK system performs condensation with beta-alanine prior to phosphorylation, the PoK/PPS system performs condensation after phosphorylation of pantoate. Gene disruption of TK2141 and TK1686 led to CoA auxotrophy, indicating that both genes are necessary for CoA biosynthesis in T. kodakaraensis. Homologs of both genes are widely distributed among the Archaea, suggesting that the PoK/PPS system represents the pathway for 4'-phosphopantothenate biosynthesis in the Archaea.

FIGURE 1.

SDS-PAGE analysis of the recombinant TK2141 (3 μg, left panel) and TK1686 (4 μg, right panel) proteins after purification. After electrophoresis, the gel was stained with Coomassie Brilliant Blue.

FIGURE 2.

The classic bacterial/eukaryotic pathway and the novel archaeal pathway for the formation of 4′-phosphopantothenate from pantoate.

FIGURE 3.

Chromatograms of the reaction products after the PoK and PPS reactions. A COSMOSIL 5C₁₈-PAQ column was used for separation. The peaks with approximate retention times of 4.7, 5.6, 6.5, and 9.4 min correspond to ATP, ADP, 4′-phosphopantothenate, and AMP, respectively. The peak corresponding to 4′-phosphopantothenate is enlarged in the insets. Line colors indicate reaction times of 0 (green), 5 (blue), 10 (red), and 15 (black) min. All reactions were performed at 75 °C. A, the reaction mixture included both PoK and PPS along with ATP, pantoate, and β-alanine (see “Experimental Procedures”) and was applied to HPLC after various periods of time. B, an initial reaction was performed with PoK, ATP, and pantoate (1 h). After stopping the first reaction and removing PoK, an aliquot was added to a second reaction mixture, including PPS, ATP, and β-alanine. The second reaction mixture was applied to HPLC after various periods of time.

FIGURE 4.

A, the plasmid and recombination strategies to construct the gene disruption strains of TK2141 (left) and TK1686 (right). T. kodakaraensis KUW1 (ΔpyrF and ΔtrpE) was used as the host strain. Cells that had undergone the initial single crossover insertion were enriched by growing cells in medium without uracil. Cells that had undergone the second, pop-out recombination were selected with resistance toward 5-fluoroorotic acid. B, PCR analyses of the TK2141 (left) and TK1686 (right) loci. The positions of the primers used are indicated with arrowheads in A.

FIGURE 5.

Growth characteristics of T. kodakaraensis KUW1, ΔTK2141 and ΔTK1686 in the presence (left) or absence (right) of 1 ml CoA. The cells were cultured in ASW-YT medium with 0.2% elemental sulfur at 85 °C. Symbols: circles, KUW1; squares, ΔTK2141; and triangles, ΔTK1686.