Biochemical Characterization of Pantoate Kinase, a Novel Enzyme Necessary for Coenzyme A Biosynthesis in the Archaea Hiroya Tomita,a Yuusuke Yokooji,a Takuya Ishibashi,a Tadayuki Imanaka,b,c and Haruyuki Atomia,c Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University Katsura, Nishikyo-ku, Kyoto, Japana; Department of Biotechnology, College of Life Sciences, Ritsumeikan University Noji-Higashi, Kusatsu, Shiga, Japanb; and JST, CREST, Sanbancho, Chiyoda-ku, Tokyo, Japanc Although bacteria and eukaryotes share a pathway for coenzyme A (CoA) biosynthesis, we previously clarified that most archaea utilize a distinct pathway for the conversion of pantoate to 4=-phosphopantothenate. Whereas bacteria/eukaryotes use pantothe- nate synthetase and pantothenate kinase (PanK), the hyperthermophilic archaeon Thermococcus kodakarensis utilizes two novel enzymes: pantoate kinase (PoK) and phosphopantothenate synthetase (PPS). Here, we report a detailed biochemical examina- tion of PoK from T. kodakarensis. Kinetic analyses revealed that the PoK reaction displayed Michaelis-Menten kinetics toward ATP, whereas substrate inhibition was observed with pantoate. PoK activity was not affected by the addition of CoA/acetyl-CoA. Interestingly, PoK displayed broad nucleotide specificity and utilized ATP, GTP, UTP, and CTP with comparable kcat/Km values. Sequence alignment of 27 PoK homologs revealed seven conserved residues with reactive side chains, and variant proteins were constructed for each residue. Activity was not detected when mutations were introduced to Ser104, Glu134, and Asp143, suggest- ing that these residues play vital roles in PoK catalysis. Kinetic analysis of the other variant proteins, with mutations S28A, H131A, R155A, and T186A, indicated that all four residues are involved in pantoate recognition and that Arg155 and Thr186 play important roles in PoK catalysis. Gel filtration analyses of the variant proteins indicated that Thr186 is also involved in dimer assembly. A sequence comparison between PoK and other members of the GHMP kinase family suggests that Ser104 and -Glu134 are involved in binding with phosphate and Mg2؉, respectively, while Asp143 is the base responsible for proton abstrac tion from the pantoate hydroxy group. oenzyme A (CoA) is an important coenzyme that is found in identified two novel enzymes, pantoate kinase (PoK) and phos- Call three domains of life. CoA forms high-energy thioester phopantothenate synthetase (PPS), that can convert pantoate to bonds with various carboxylic acids and plays a central role in 4=-phosphopantothenate, replacing the missing PS/PanK path- carbon and energy metabolism. Its derivatives, such as acetyl- way (Fig. 1)(33). In the PoK/PPS pathway, the order of the con- CoA, succinyl-CoA, malonyl-CoA, and hydroxymethylglu- densation reaction with ␤-alanine and the phosphorylation reac- taryl-CoA, are key metabolites in a wide range of pathways tion are reversed compared to the classical PS and PanK reactions. which include sugar breakdown and ␤-oxidation, as well as the Disruption of the individual genes in the hyperthermophilic ar- biosynthesis of fatty acids and isoprenoid compounds. chaeon Thermococcus kodakarensis led to strains that could only In bacteria and eukaryotes, the mechanism of CoA biosynthe- grow with the addition of exogenous CoA or 4=-phosphopanto- sis has been examined in detail, and it is now known that they thenate to the medium, confirming their involvement in CoA bio- share common reactions (14, 27). CoA is synthesized from pan- synthesis in this archaeon. Genes homologous to the two genes tothenate via five enzymatic reactions: pantothenate kinase (TK2141, encoding PoK, and TK1686, encoding PPS) are present (PanK), 4=-phosphopantothenoylcysteine synthetase (PPCS), 4=- in almost all archaeal genomes with the exception of those of the phosphopantothenoylcysteine decarboxylase (PPCDC), 4=-phos- Thermoplasmatales and Nanoarchaeum equitans, strongly suggest- phopantetheine adenylyltransferase (PPAT), and dephospho- ing that the majority of the archaea utilize this pathway for CoA CoA kinase (DPCK). In addition, microorganisms and plants can biosynthesis (33). Concerning the members of the Thermoplas- synthesize pantothenate from L-valine and ␤-alanine through matales, there are genes that display structural similarity with the four enzymatic reactions: aminotransferase (AT), ketopantoate bacterial/eukaryotic PanK in the genomes of Ferroplasma acidar- hydroxymethyltransferase (KPHMT), ketopantoate reductase manus and Picrophilus torridus. The PanK from P. torridus has (KPR), and pantothenate synthetase (PS). been examined and shown to exhibit PanK activity (28). The gene Although much knowledge has accumulated on CoA biosyn- that encodes PS in these organisms has yet to be identified. thesis in bacteria and eukaryotes, the corresponding pathway in Based on its primary structure, PoK is structurally related to archaea has not been well examined. A number of genes that dis- members of the GHMP kinase superfamily (33). This family com- play similarity to bacterial/eukaryotic genes are present on the archaeal genomes, including those encoding PPCS, PPCDC, and PPAT. Among these, the PPCS and PPCDC from Methanocaldo- Received 1 December 2011 Accepted 27 July 2012 coccus jannaschii (13) and the PPAT from Pyrococcus abyssi (1, 17) Published ahead of print 3 August 2012 have been biochemically characterized. A striking observation was Address correspondence to Haruyuki Atomi, [email protected]. that almost all of the archaeal genomes did not harbor genes cor- Supplemental material for this article may be found at http://jb.asm.org/. responding to PS and PanK. Genes predicted to be involved in Copyright © 2012, American Society for Microbiology. All Rights Reserved. these reactions have been studied in Methanosarcina mazei (7, 23). doi:10.1128/JB.06624-11 By taking a comparative genomics approach, we have previously Reproduced from J. Bacteriol. 194: 5434-5443 (2012). 120 inant route for CoA biosynthesis in the archaea, in this study, we have performed the first detailed biochemical analysis of PoK. Here, we focused on the reaction kinetics, substrate specificity, and moreover, identification of the amino acid residues involved in the catalysis of this novel enzyme, which can be considered a new archaeal member of the GHMP kinase superfamily. MATERIALS AND METHODS Strains, media, and culture conditions. Cultivation of T. kodakarensis KOD1 (2, 16) and its derivative strains was performed under anaerobic conditions at 85°C in a nutrient-rich medium (ASW-YT) or a synthetic medium (ASW-AA). ASW-YT medium consists of 0.8ϫ artificial seawa- ter (ASW), 5.0 g literϪ1 yeast extract, 5.0 g literϪ1 tryptone, and 0.8 mg literϪ1 resazurine. Prior to inoculation, 5.0 g literϪ1 sodium pyruvate (ASW-YT-Pyr medium) or 2.0 g literϪ1 elemental sulfur (ASW-YT-S0 medium) and Na2S were added to the medium until it became colorless. ASW-AA medium consisted of 0.8ϫ ASW, a mixture of 20 amino acids, modified Wolfe’s trace minerals, a vitamin mixture, and 2.0 g literϪ1 elemental sulfur (19, 25). In the case of plate culture used to isolate trans- formants, elemental sulfur and Na2S9H2O were replaced with 2 ml of a polysulfide solution (10 g Na2S9H2O and 3 g sulfur flowers in 15 ml H2O) per liter and 10 g literϪ1 Gelrite was added to solidify the medium. Specific modifications of the medium to isolate transformants are described in the respective sections below. Escherichia coli DH5␣ used for plasmid con- struction was cultivated at 37°C in Luria-Bertani (LB) medium containing ampicillin (100 mg literϪ1). Unless mentioned otherwise, all chemicals were purchased from Wako Pure Chemicals (Osaka, Japan) or Nacalai FIG 1 Two pathways from pantoate to 4=-phosphopantothenate. Bacteria and Tesque (Kyoto, Japan). eukaryotes use the PS/PanK pathway (left), whereas most archaea utilize the Production and purification of the wild-type pantoate kinase. The PoK/PPS pathway (right). TK2141 gene encoding pantoate kinase (PoK), with a His6 tag on its N terminus, was overexpressed in T. kodakarensis. The TK2141 overexpres- sion strain (ETK2141) (33) was cultivated in ASW-YT-Pyr for 12 h at prises a vast number of proteins that include the galactokinases, 85°C. Cells were harvested, resuspended in 20 mM potassium phosphate homoserine kinases, mevalonate kinases, and phosphomeval- buffer (pH 7.4) containing 0.5 M KCl and 40 mM imidazole, and dis- ϫ onate kinases (3). Many members of the GHMP family are in- rupted by sonication. After centrifugation (20,000 g, 15 min), the su- volved in intermediary metabolism and utilize ATP to phosphor- pernatant was applied to His GraviTrap (GE Healthcare Biosciences, Pis- ylate their specific substrates. These kinases harbor a common cataway, NJ), and the His-tagged proteins were eluted with 20 mM potassium phosphate (pH 7.4), 0.5 M KCl, and 0.5 M imidazole. After the loop sequence rich in glycine/serine residues (4), which, in the buffer was exchanged for 50 mM Tris-HCl (pH 8.0) using a PD-10 col- case of human mevalonate kinase (MvK), has been biochemically umn (GE Healthcare Biosciences), the sample was applied to anion-ex- 2ϩ shown to be involved in binding with Mg and ATP. The specific change chromatography (HiTrap Q HP; GE Healthcare Biosciences), and residue involved in this binding (Ser146) is highly conserved proteins were eluted with a linear gradient of NaCl (0 to 1.0 M) in 50 mM among the members of the GHMP family (4). Glu193 from this Tris-HCl (pH 8.0) at a flow rate of 2.5 ml minϪ1. The protein concentra- enzyme has also been shown to be involved in ATP binding (18). tion was determined with the Bio-Rad protein assay system (Bio-Rad, Furthermore, mutagenesis studies on Asp204 have indicated that Hercules, CA), using bovine serum albumin as a standard. this residue functions as the catalytic base to enhance the nucleo- Examinations of pantoate kinase activity. PoK activity was measured philicity of the hydroxy group to attack the ATP ␥-phosphate by quantifying the ADP generated from the PoK reaction with the pyru- (18). Both of these residues are conserved in a wide range of vate kinase/lactate dehydrogenase (PK/LDH) reaction.