Hiroya Tomita

Pantoate Kinase and Phosphopantothenate Synthetase, Two Novel Enzymes Necessary for CoA Biosynthesis in the Archaea

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 β-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. Vmax 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 β-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 β-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.

Biochemical and genetical analyses of the three mcm genes from the hyperthermophilic archaeon, Thermococcus kodakarensis

In eukaryotes, the replicative DNA helicase ‘core’ is the minichromosome maintenance (Mcm) complex (MCM), forming a heterohexameric complex consisting of six subunits (Mcm2‐7). Recent studies showed that the CMG (Cdc45–MCM–GINS) complex is the actual helicase body in the replication fork progression complex. In Archaea, Thermococcus kodakarensis harbors three genes encoding the Mcm homologs on its genome, contrary to most archaea, which have only one homolog. It is thus, of high interest, whether and how these three Mcms share their functions in DNA metabolism in this hyperthermophile. Here, we report the biochemical properties of two of these proteins, TkoMcm1 and TkoMcm3. In addition, their physical and functional interactions with GINS, possibly an essential factor for the initiation and elongation process of DNA replication, are presented through in vitro ATPase and helicase assays, and an in vivo immunoprecipitation assay. Gene disruption and product quantification analyses suggested that TkoMcm3 is essential for cell growth and plays a key role as the main DNA helicase in DNA replication, whereas TkoMcm1 also shares some function in the cells.

Characterization of Two Members among the Five ADP-Forming Acyl Coenzyme A (Acyl-CoA) Synthetases Reveals the Presence of a 2-(Imidazol-4-yl)Acetyl-CoA Synthetase in Thermococcus kodakarensis

The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative α subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining two acyl-CoA synthetase proteins from T. kodakarensis. The TK0944 and TK2127 genes encoding the two α subunits were each coexpressed with the β subunit-encoding TK0943 gene. In both cases, soluble proteins with an α2β2 structure were obtained and their activities toward various acids in the ADP-forming reaction were examined. The purified TK0944/TK0943 protein (ACS IIITk) accommodated a broad range of acids that corresponded to those generated in the oxidative metabolism of Ala, Val, Leu, Ile, Met, Phe, and Cys. In contrast, the TK2127/TK0943 protein exhibited relevant levels of activity only toward 2-(imidazol-4-yl)acetate, a metabolite of His degradation, and was thus designated 2-(imidazol-4-yl)acetyl-CoA synthetase (ICSTk), a novel enzyme. Kinetic analyses were performed on both proteins with their respective substrates. In T. kodakarensis, we found that the addition of histidine to the medium led to increases in intracellular ADP-forming 2-(imidazol-4-yl)acetyl-CoA synthetase activity, and 2-(imidazol-4-yl)acetate was detected in the culture medium, suggesting that ICSTk participates in histidine catabolism. The results presented here, together with those of previous studies, have clarified the substrate specificities of all five known NDP-forming acyl-CoA synthetase proteins in the Thermococcales.

Identification and characterization of an archaeal ketopantoate reductase and its involvement in regulation of coenzyme A biosynthesis

Coenzyme A (CoA) biosynthesis in bacteria and eukaryotes is regulated primarily by feedback inhibition towards pantothenate kinase (PanK). As most archaea utilize a modified route for CoA biosynthesis and do not harbour PanK, the mechanisms governing regulation of CoA biosynthesis are unknown. Here we performed genetic and biochemical studies on the ketopantoate reductase (KPR) from the hyperthermophilic archaeon Thermococcus kodakarensis. KPR catalyses the second step in CoA biosynthesis, the reduction of 2‐oxopantoate to pantoate. Gene disruption of TK1968, whose product was 20–29% identical to previously characterized KPRs from bacteria/eukaryotes, resulted in a strain with growth defects that were complemented by addition of pantoate. The TK1968 protein (Tk‐KPR) displayed reductase activity specific for 2‐oxopantoate and preferred NADH as the electron donor, distinct to the bacterial/eukaryotic NADPH‐dependent enzymes. Tk‐KPR activity decreased dramatically in the presence of CoA and KPR activity in cell‐free extracts was also inhibited by CoA. Kinetic studies indicated that CoA inhibits KPR by competing with NADH. Inhibition of ketopantoate hydroxymethyltransferase, the first enzyme of the pathway, by CoA was not observed. Our results suggest that CoA biosynthesis in T. kodakarensis is regulated by feedback inhibition of KPR, providing a feasible regulation mechanism of CoA biosynthesis in archaea.

CoA biosynthesis in archaea

CoA is a ubiquitous molecule in all three domains of life and is involved in various metabolic pathways. The enzymes and reactions involved in CoA biosynthesis in eukaryotes and bacteria have been identified. By contrast, the proteins/genes involved in CoA biosynthesis in archaea have not been fully clarified, and much has to be learned before we obtain a general understanding of how this molecule is synthesized. In the present paper, we review the current status of the research on CoA biosynthesis in the archaea, and discuss important questions that should be addressed in the near future.

Identification and characterization of a bacterial cytochrome P450 monooxygenase catalyzing the 3-nitration of tyrosine in rufomycin biosynthesis

Rufomycin is a circular heptapeptide with anti-mycobacterial activity and is produced by Streptomyces atratus ATCC 14046. Its structure contains three non-proteinogenic amino acids, N-dimethylallyltryptophan, trans-2-crotylglycine, and 3-nitrotyrosine (3NTyr). Although the rufomycin structure was already reported in the 1960s, its biosynthesis, including 3NTyr generation, remains unclear. To elucidate the rufomycin biosynthetic pathway, we assembled a draft genome sequence of S. atratus and identified the rufomycin biosynthetic gene cluster (ruf cluster), consisting of 20 ORFs (rufA–rufT). We found a putative heptamodular nonribosomal peptide synthetase encoded by rufT, a putative tryptophan N-dimethylallyltransferase encoded by rufP, and a putative trimodular type I polyketide synthase encoded by rufEF. Moreover, the ruf cluster contains an apparent operon harboring putative cytochrome P450 (rufO) and nitric oxide synthase (rufN) genes. A similar operon, txtDE, is responsible for the formation of 4-nitrotryptophan in thaxtomin biosynthesis; the cytochrome P450 TxtE catalyzes the 4-nitration of Trp. Therefore, we hypothesized that RufO should catalyze the Tyr 3-nitration. Disruption of rufO abolished rufomycin production by S. atratus, which was restored when 3NTyr was added to the culture medium of the disruptant. Recombinant RufO protein exhibited Tyr 3-nitration activity both in vitro and in vivo. Spectroscopic analysis further revealed that RufO recognizes Tyr as the substrate with a dissociation constant of ∼0.1 μm. These results indicate that RufO is an unprecedented cytochrome P450 that catalyzes Tyr nitration. Taken together with the results of an in silico analysis of the ruf cluster, we propose a rufomycin biosynthetic pathway in S. atratus.

Crystal structure of archaeal ketopantoate reductase complexed with coenzyme a and 2-oxopantoate provides structural insights into feedback regulation

Coenzyme A (CoA) plays essential roles in a variety of metabolic pathways in all three domains of life. The biosynthesis pathway of CoA is strictly regulated by feedback inhibition. In bacteria and eukaryotes, pantothenate kinase is the target of feedback inhibition by CoA. Recent biochemical studies have identified ketopantoate reductase (KPR), which catalyzes the NAD(P)H‐dependent reduction of 2‐oxopantoate to pantoate, as a target of the feedback inhibition by CoA in archaea. However, the mechanism for recognition of CoA by KPR is still unknown. Here we report the crystal structure of KPR from Thermococcus kodakarensis in complex with CoA and 2‐oxopantoate. CoA occupies the binding site of NAD(P)H, explaining the competitive inhibition by CoA. Our structure reveals a disulfide bond between CoA and Cys84 that indicates an irreversible inhibition upon binding of CoA. The structure also suggests the cooperative binding of CoA and 2‐oxopantoate that triggers a conformational closure and seems to facilitate the disulfide bond formation. Our findings provide novel insights into the mechanism that regulates biosynthesis of CoA in archaea. Proteins 2016; 84:374–382.

An ornithine ω-aminotransferase required for growth in the absence of exogenous proline in the archaeon Thermococcus kodakarensis

Aminotransferases are pyridoxal 5′-phosphate–dependent enzymes that catalyze reversible transamination reactions between amino acids and α-keto acids, and are important for the cellular metabolism of nitrogen. Many bacterial and eukaryotic ω-aminotransferases that use l-ornithine (Orn), l-lysine (Lys), or γ-aminobutyrate (GABA) have been identified and characterized, but the corresponding enzymes from archaea are unknown. Here, we examined the activity and function of TK2101, a gene annotated as a GABA aminotransferase, from the hyperthermophilic archaeon Thermococcus kodakarensis. We overexpressed the TK2101 gene in T. kodakarensis and purified and characterized the recombinant protein and found that it displays only low levels of GABA aminotransferase activity. Instead, we observed a relatively high ω-aminotransferase activity with l-Orn and l-Lys as amino donors. The most preferred amino acceptor was 2-oxoglutarate. To examine the physiological role of TK2101, we created a TK2101 gene–disruption strain (ΔTK2101), which was auxotrophic for proline. Growth comparison with the parent strain KU216 and the biochemical characteristics of the protein strongly suggested that TK2101 encodes an Orn aminotransferase involved in the biosynthesis of l-Pro. Phylogenetic comparisons of the TK2101 sequence with related sequences retrieved from the databases revealed the presence of several distinct protein groups, some of which having no experimentally studied member. We conclude that TK2101 is part of a novel group of Orn aminotransferases that are widely distributed at least in the genus Thermococcus, but perhaps also throughout the Archaea.

Regulation of coenzyme A biosynthesis in the hyperthermophilic bacterium Thermotoga maritima

UNLABELLED Regulation of coenzyme A (CoA) biosynthesis in bacteria and eukaryotes occurs through feedback inhibition targeting type I and type II pantothenate kinase (PanK), respectively. In contrast, the activity of type III PanK is not affected by CoA. As the hyperthermophilic bacterium Thermotoga maritima harbors only a single type III PanK (Tm-PanK), here we examined the mechanisms that regulate CoA biosynthesis in this organism. We first examined the enzyme responsible for the ketopantoate reductase (KPR) reaction, which is the target of feedback inhibition in archaea. A classical KPR homolog was not present on the T. maritima genome, but we found a homolog (TM0550) of the ketol-acid reductoisomerase (KARI) from Corynebacterium glutamicum, which exhibits KPR activity. The purified TM0550 protein displayed both KPR and KARI activities and was designated Tm-KPR/KARI. When T. maritima cell extract was subjected to anion-exchange chromatography, the fractions containing high levels of KPR activity also displayed positive signals in a Western blot analysis using polyclonal anti-TM0550 protein antisera, strongly suggesting that Tm-KPR/KARI was the major source of KPR activity in the organism. The KPR activity of Tm-KPR/KARI was not inhibited in the presence of CoA. We thus examined the properties of Tm-PanK and the pantothenate synthetase (Tm-PS) of this organism. Tm-PS was not affected by CoA. Surprisingly however, Tm-PanK was inhibited by CoA, with almost complete inhibition in the presence of 400 μM CoA. Our results suggest that CoA biosynthesis in T. maritima is regulated by feedback inhibition targeting PanK, although Tm-PanK is a type III enzyme. IMPORTANCE Bacteria and eukaryotes regulate the biosynthesis of coenzyme A (CoA) by feedback inhibition targeting type I or type II pantothenate kinase (PanK). The hyperthermophilic bacterium Thermotoga maritima harbors a single type III PanK (Tm-PanK), previously considered to be unaffected by CoA. By examining the properties of three enzymes involved in CoA biosynthesis in this organism, we found that Tm-PanK, although a type III enzyme, is inhibited by CoA. The results provide a feasible explanation of how CoA biosynthesis is regulated in T. maritima, which may also apply for other bacteria that harbor only type III PanK enzymes.

Crystal structure of ketopantoate reductase from Thermococcus kodakarensis complexed with NADP(.).

Coenzyme A (CoA) plays pivotal roles in a variety of metabolic pathways in all organisms. The biosynthetic pathway of CoA is strictly regulated by feedback inhibition. In the hyperthermophilic archaeon Thermococcus kodakarensis, ketopantoate reductase (KPR), which catalyzes the NAD(P)H-dependent reduction of 2-oxopantoate, is a target of feedback inhibition by CoA. The crystal structure of KPR from T. kodakarensis (Tk-KPR) complexed with CoA and 2-oxopantoate has previously been reported. The structure provided an explanation for the competitive inhibition mechanism. Here, further biochemical analyses of Tk-KPR and the crystal structure of Tk-KPR in complex with NADP(+) are reported. A mutational analysis implies that the residues in the binding pocket cooperatively contribute to the recognition of CoA. The structure reveals the same dimer architecture as the Tk-KPR-CoA-2-oxopantoate complex. Moreover, the positions of the residues involved in the dimer interaction are not changed by the binding of CoA and 2-oxopantoate, suggesting individual conformational changes of Tk-KPR monomers.