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 ␤-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 ␤-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 ␤-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.Coenzyme A (CoA) 2 and its derivative 4Ј-phosphopantetheine are essential cofactors in numerous metabolic pathways, including the tricarboxylic acid cycle, the ␤-oxidation pathway, and fatty acid and polyketide biosynthesis pathways. Acyl-CoA derivatives are key intermediates in energy metabolism due to their high energy thioester bonds and have been identified in all three domains of life.The mechanism of CoA biosynthesis in bacteria and eukaryotes has been well examined and involves common enzymatic conversions (1-3). CoA is synthesized from pantothenate via five enzymatic reactions; pantothenate kinase (PanK), 4Ј-phosphopantothenoylcysteine synthetase (PPCS), 4Ј-phosphopantothenoylcysteine decarboxylase (PPCDC), 4Ј-phosphopantetheine adenylyltransferase (PPAT), and dephospho-CoA kinase (DPCK). Although many animals rely on exogenous pantothenate to initiate CoA biosynthesis, microorganisms and plants can synthesize pantothenate from 2-oxoisovalerate and ␤-alanine. This is a three-step pathway catalyzed by ketopantoate hydroxymethyltransferase (KPHMT), ketopantoate reductase, and pantothenate synthetase (PS).In contrast to the wealth of knowledge on CoA biosynthesis in bacteria ...

Biochemical Characterization of Pantoate Kinase, a Novel Enzyme Necessary for Coenzyme A Biosynthesis in the Archaea

“…The amounts of PK/LDH in the second reaction mixture were 7.4 U ml Ϫ1 /9.3 U ml Ϫ1 for ADP, 22.2 U ml Ϫ1 /27.9 U ml Ϫ1 for GDP and UDP, and 37.0 U ml Ϫ1 /46.5 U ml Ϫ1 for CDP. When pyrophosphate or triphosphate was examined, the PoK reaction mixture with 4 mM pyrophosphate or triphosphate (Sigma-Aldrich) and 11.4 g ml Ϫ1 PoK was coupled to the PPS reaction (33), and the formation of 4=-phosphopantothenate was examined with high-performance liquid chromatography. The PoK reaction was carried out at 75°C for 60 min.…”

“…Pantoate kinase (PoK, encoded by TK2141) was expressed as an insoluble protein in E. coli (33), and therefore, it was expressed in its native host, T. kodakarensis ETK2141. As a result, recombinant PoK harboring a His 6 tag sequence at the N terminus was purified to apparent homogeneity by nickel chelate affinity chromatography and anion-exchange chromatography.…”

“…By taking a comparative genomics approach, we have previously identified two novel enzymes, pantoate kinase (PoK) and phosphopantothenate synthetase (PPS), that can convert pantoate to 4=-phosphopantothenate, replacing the missing PS/PanK pathway ( Fig. 1) (33). In the PoK/PPS pathway, the order of the condensation reaction with ␤-alanine and the phosphorylation reaction are reversed compared to the classical PS and PanK reactions.…”

“…Disruption of the individual genes in the hyperthermophilic archaeon Thermococcus kodakarensis led to strains that could only grow with the addition of exogenous CoA or 4=-phosphopantothenate to the medium, confirming their involvement in CoA biosynthesis in this archaeon. Genes homologous to the two genes (TK2141, encoding PoK, and TK1686, encoding PPS) are present in almost all archaeal genomes with the exception of those of the Thermoplasmatales and Nanoarchaeum equitans, strongly suggesting that the majority of the archaea utilize this pathway for CoA biosynthesis (33). Concerning the members of the Thermoplasmatales, there are genes that display structural similarity with the bacterial/eukaryotic PanK in the genomes of Ferroplasma acidarmanus and Picrophilus torridus.…”

“…Based on its primary structure, PoK is structurally related to members of the GHMP kinase superfamily (33). This family com-prises a vast number of proteins that include the galactokinases, homoserine kinases, mevalonate kinases, and phosphomevalonate kinases (3).…”