Carbon dioxide fixation in 'Archaeoglobus lithotrophicus': are there multiple autotrophic pathways?

Several representatives of the euryarchaeal class Archaeoglobi are able to grow facultative autotrophically using the reductive acetyl-CoA pathway, with 'Archaeoglobus lithotrophicus' being an obligate autotroph. However, genome sequencing revealed that some species harbor genes for key enzymes of other autotrophic pathways, i.e. 4-hydroxybutyryl-CoA dehydratase of the dicarboxylate/hydroxybutyrate cycle and the hydroxypropionate/hydroxybutyrate cycle and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the Calvin-Benson cycle. This raised the question of whether only one or multiple autotrophic pathways are operating in these species. We searched for the presence of enzyme activities specific for the dicarboxylate/hydroxybutyrate or the hydroxypropionate/hydroxybutyrate cycles in 'A. lithotrophicus', but such enzymes could not be detected. Low Rubisco activity was detected that could not account for the carbon dioxide (CO(2)) fixation rate; in addition, phosphoribulokinase activity was not found. The generation of ribulose 1,5-bisphosphate from 5-phospho-D-ribose 1-pyrophosphate was observed, but not from AMP; these sources for ribulose 1,5-bisphosphate have been proposed before. Our data indicate that the reductive acetyl-CoA pathway is the only functioning CO(2) fixation pathway in 'A. lithotrophicus'.     

Complete genome sequence of the thermoacidophilic crenarchaeon Thermoproteus uzoniensis 768-20

Thermoproteus uzoniensis 768-20 is a thermoacidophilic anaerobic crenarchaeon isolated from a solfataric field in Kamchatka, Russia. The complete genome sequence reveals genes for protein and carbohydrate-active enzymes, beta-oxidation of fatty acids, the Embden-Meyerhof and Entner-Doudoroff pathways for glucose metabolism, the tricarboxylic acid cycle, the dicarboxylate/4-hydroxybutyrate cycle, hydrogenase, and sulfur reductase.     

Identification of missing genes and enzymes for autotrophic carbon fixation in crenarchaeota

Two autotrophic carbon fixation cycles have been identified in Crenarchaeota. The dicarboxylate/4-hydroxybutyrate cycle functions in anaerobic or microaerobic autotrophic members of the Thermoproteales and Desulfurococcales. The 3-hydroxypropionate/4-hydroxybutyrate cycle occurs in aerobic autotrophic Sulfolobales; a similar cycle may operate in autotrophic aerobic marine Crenarchaeota. Both cycles form succinyl-coenzyme A (CoA) from acetyl-CoA and two molecules of inorganic carbon, but they use different means. Both cycles have in common the (re)generation of acetyl-CoA from succinyl-CoA via identical intermediates. Here, we identified several missing enzymes/genes involved in the seven-step conversion of succinyl-CoA to two molecules of acetyl-CoA in Thermoproteus neutrophilus (Thermoproteales), Ignicoccus hospitalis (Desulfurococcales), and Metallosphaera sedula (Sulfolobales). The identified enzymes/genes include succinyl-CoA reductase, succinic semialdehyde reductase, 4-hydroxybutyrate-CoA ligase, bifunctional crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase, and beta-ketothiolase. 4-Hydroxybutyryl-CoA dehydratase, which catalyzes a mechanistically intriguing elimination of water, is well conserved and rightly can be considered the key enzyme of these two cycles. In contrast, several of the other enzymes evolved from quite different sources, making functional predictions based solely on genome interpretation difficult, if not questionable.     

Computation of metabolic fluxes and efficiencies for biological carbon dioxide fixation

With rising energy prices and concern over the environmental impact of fossil fuel consumption, the push to develop biomass derived fuels has increased significantly. Although most global carbon fixation occurs via the Calvin Benson Bassham cycle, there are currently five other known pathways for carbon fixation; the goal of this study was to determine the thermodynamic efficiencies of all six carbon fixation pathways for the production of biomass using flux balance analysis. The three chemotrophic pathways, the reductive acetyl-CoA pathway, the 3-hydroxypropionate/4-hydroxybutyrate cycle and the dicarboxylate/4-hydroxybutyrate cycle, were found to be more efficient than photoautotrophic carbon fixation pathways. However, as hydrogen is not freely available, the energetic cost of hydrogen production from sunlight was calculated and included in the overall energy demand, which results in a 5 fold increase in the energy demand of chemoautotrophic carbon fixation. Therefore, when the cost of hydrogen production is included, photoautotrophic pathways are more efficient. However, the energetic cost for the production of 12 metabolic precursors was found to vary widely across the different carbon fixation pathways; therefore, different pathways may be more efficient at producing products from a single precursor than others. The results of this study have significant impact on the selection or design of autotrophic organisms for biofuel or biochemical production. Overall biomass production from solar energy is most efficient in organisms using the reductive TCA cycle, however, products derived from one metabolic precursor may be more efficiently produced using other carbon fixation pathways.     

Sugar utilization and its control in hyperthermophiles

Many hyperthermophilic microorganisms show heterotrophic growth on a variety of carbohydrates. There has been considerable fundamental and applied interest in the utilization of glucose and its α- and β-polymers by hyperthermophiles. While glycolysis by Bacteria at high temperatures shows conventional characteristics, it has been found that glucose catabolism by hyperthermophilic Archaea differs from the canonical glycolytic pathways, involves novel enzymes, and shows a unique control. This review addresses these aspects with specific attention to Pyrococcus furiosus, which is one of the best studied hyperthermophilic Archaea, has the capacity to grow on a variety of sugars including the marine β-(1,3)-linked glucose polymer laminarin, and has been found to contain three novel glycolytic enzymes, two ADP-dependent kinases, and a ferredoxin-dependent glyceraldehyde-3-phosphate oxidoreductase. Received: January 22, 1998 / Accepted: February 16, 1998     

The role of log-normal dynamics in the evolution of biochemical pathways

The study of the scale-free topology in non-biological and biological networks and the dynamics that can explain this fascinating property of complex systems have captured the attention of the scientific community in the last years. Here, we analyze the biochemical pathways of three organisms (Methanococcus jannaschii, Escherichia coli, Saccharomyces cerevisiae) which are representatives of the main kingdoms Archaea, Bacteria and Eukaryotes during the course of the biological evolution. We can consider two complementary representations of the biochemical pathways: the enzymes network and the chemical compounds network. In this article, we propose a stochastic model that explains that the scale-free topology with exponent in the vicinity of gamma approximately 3/2 found across these three organisms is governed by the log-normal dynamics in the evolution of the enzymes network. Precisely, the fluctuations of the connectivity degree of enzymes in the biochemical pathways between evolutionary distant organisms follow the same conserved dynamical principle, which in the end is the origin of the stationary scale-free distribution observed among species, from Archaea to Eukaryotes. In particular, the log-normal dynamics guarantees the conservation of the scale-free distribution in evolving networks. Furthermore, the log-normal dynamics also gives a possible explanation for the restricted range of observed exponents gamma in the scale-free networks (i.e., gamma > or = 3/2). Finally, our model is also applied to the chemical compounds network of biochemical pathways and the Internet network.     

Autotrophic Carbon Dioxide Assimilation in Thermoproteales Revisited

For Crenarchaea, two new autotrophic carbon fixation cycles were recently described. Sulfolobales use the 3-hydroxypropionate/4-hydroxybutyrate cycle, with acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the carboxylating enzyme. Ignicoccus hospitalis (Desulfurococcales) uses the dicarboxylate/4-hydroxybutyrate cycle, with pyruvate synthase and phosphoenolpyruvate carboxylase being responsible for CO₂ fixation. In the two cycles, acetyl-CoA and two inorganic carbons are transformed to succinyl-CoA by different routes, whereas the regeneration of acetyl-CoA from succinyl-CoA proceeds via the same route. Thermoproteales would be an exception to this unifying concept, since for Thermoproteus neutrophilus, the reductive citric acid cycle was proposed as a carbon fixation mechanism. Here, evidence is presented for the operation of the dicarboxylate/4-hydroxybutyrate cycle in this archaeon. All required enzyme activities were detected in large amounts. The key enzymes of the cycle were strongly upregulated under autotrophic growth conditions, indicating their involvement in autotrophic CO₂ fixation. The corresponding genes were identified in the genome. ¹⁴C-labeled 4-hydroxybutyrate was incorporated into the central building blocks in accordance with the key position of this compound in the cycle. Moreover, the results of previous ¹³C-labeling studies, which could be reconciled with a reductive citric acid cycle only when some assumptions were made, were perfectly in line with the new proposal. We conclude that the dicarboxylate/4-hydroxybutyrate cycle is operating in CO₂ fixation in the strict anaerobic Thermoproteales as well as in Desulfurococcales.Two new autotrophic carbon fixation cycles have recently been discovered in the Crenarchaea, one of the two subgroups of the Archaea. The 3-hydroxypropionate/4-hydroxybutyrate cycle functions in the aerobic autotrophic Sulfolobales (7) and the dicarboxylate/4-hydroxybutyrate cycle (Fig. ​(Fig.1)1) in the anaerobic autotrophic Ignicoccus hospitalis, belonging to the Desulfurococcales (27). These pathways have in common the synthesis of succinyl-coenzyme A (CoA) from acetyl-CoA and two inorganic carbons, although this is accomplished in quite different ways and using different carboxylases. In the 3-hydroxypropionate/4-hydroxybutyrate cycle, acetyl-CoA/propionyl-CoA carboxylase fixes two molecules of bicarbonate, and in the dicarboxylate/4-hydroxybutyrate cycle, pyruvate synthase and phosphoenolpyruvate (PEP) carboxylase are the two carboxylating enzymes. Yet, the regenerations of acetyl-CoA, the primary CO₂ acceptor, from succinyl-CoA are similar in the two pathways.Open in a separate windowFIG. 1.Dicarboxylate/4-hydroxybutyrate cycle for autotrophic CO₂ fixation, as proposed for T. neutrophilus. Enzymes: 1, pyruvate synthase (reduced MV); 2, pyruvate-water dikinase; 3, PEP carboxylase; 4, malate dehydrogenase (NADH); 5, fumarate hydratase; 6, fumarate reductase (reduced MV); 7, succinyl-CoA synthetase (ADP forming); 8, succinyl-CoA reductase (NADPH); 9, succinic semialdehyde reductase (NADPH); 10, 4-hydroxybutyrate-CoA ligase (AMP forming); 11, 4-hydroxybutyryl-CoA dehydratase; 12, crotonyl-CoA hydratase; 13, (S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺); 14, acetoacetyl-CoA β-ketothiolase. Fd_red, reduced ferredoxin.Acetyl-CoA regeneration is as follows. The CO₂ fixation product succinyl-CoA is reduced to 4-hydroxybutyrate, which is activated to 4-hydroxybutyryl-CoA and then dehydrated to crotonyl-CoA by 4-hydroxybutyryl-CoA dehydratase. This radical [4Fe-4S] and flavin adenine dinucleotide-containing dehydratase (11, 37) is considered a key enzyme of the 4-hydroxybutyrate part of each pathway. Its product, crotonyl-CoA, is further converted to acetoacetyl-CoA and then to two acetyl-CoA molecules, closing the cycle and generating an additional molecule of acetyl-CoA for biosynthesis. Therefore, two different autotrophic pathways in different crenarchaeal orders share many common enzymes and intermediates.In this context, the order Thermoproteales would constitute an exception within the Crenarchaea, since the reductive citric acid cycle was proposed for Thermoproteus neutrophilus (6, 48-50, 55) and Pyrobaculum islandicum (26). T. neutrophilus is a strictly anaerobic hyperthermophilic archaeon growing autotrophically by reducing sulfur with hydrogen at 85°C and neutral pH (19). It can also assimilate organic compounds, such as acetate or succinate, but only in the presence of CO₂ and H₂, i.e., in a mixotrophic way (48).In the reductive citric acid cycle, succinyl-CoA is further transformed with 2 CO₂ to citrate, followed by citrate cleavage to oxaloacetate and acetyl-CoA. This requires two characteristic enzymes, 2-oxoglutarate synthase (2-oxoglutarate-ferredoxin oxidoreductase) and ATP citrate lyase. The proposal of the functioning of the reductive citric acid cycle in T. neutrophilus was based on the results of a ¹³C retrobiosynthetic analysis of the central carbon metabolism, using ¹³C-labeled succinate and acetate as an additional carbon source, following its incorporation into cellular building blocks. The ¹³C enrichment data of, e.g., glutamate, which is directly derived from 2-oxoglutarate, were consistent with the operation of a reductive citric acid cycle only when further assumptions were made (55). The activities of the enzymes of this cycle were demonstrated with extracts of autotrophically grown cells. However, the measured 2-oxoglutarate synthase and ATP-citrate lyase activity levels were very low and could not support the reported growth rate under autotrophic conditions (6, 48).The recent sequencing of the genome of Pyrobaculum aerophilum, belonging to the Thermoproteales (20), revealed a surprising feature, the presence of a 4-hydroxybutyryl-CoA dehydratase gene without the presence of an ATP-citrate lyase gene. Similar gene patterns are found in the genomes of T. neutrophilus as well as Pyrobaculum calidifontis and P. islandicum, sequenced by the DOE Joint Genome Institute (http://www.jgi.doe.gov/). This indicates a possible functioning of the dicarboxylate/4-hydroxybutyrate cycle in Thermoproteales and brings into question the involvement of the reductive citric acid cycle in autotrophic CO₂ fixation. This study has reinvestigated the pathway of autotrophic CO₂ fixation in Thermoproteus neutrophilus. We provide different lines of evidence for the operation of the dicarboxylate/4-hydroxybutyrate cycle.     

R-stereoselective amidase from Rhodococcus erythropolis No. 7 acting on 4-chloro-3-hydroxybutyramide

Ethyl (S)-4-chloro-3-hydroxybutyrate is an intermediate for the synthesis of Atorvastatin, a chiral drug used for hypercholesterolemia. A Rhodococcus erythropolis strain (No. 7) able to convert 4-chloro-3-hydroxybutyronitrile into 4-chloro-3-hydroxybutyric acid has recently been isolated from soil. This activity has been regarded as having been caused by the successive actions of the nitrile hydratase and amidase. In this instance, the corresponding amidase gene was cloned from the R. erythropolis strain and expressed in Escherichia coli cells. A soluble active form of amidase enzyme was obtained at 18 degrees . The Ni column-purified recombinant amidase was found to have a specific activity of 3.89 U/mg toward the substrate isobutyramide. The amidase was found to exhibit a higher degree of activity when used with midchain substrates than with short-chain ones. Put differently, amongst the various amides tested, isobutyramide and butyramide were found to be hydrolyzed the most rapidly. In addition to amidase activity, the enzyme was found to exhibit acyltransferase activity when hydroxyl amine was present. This dual activity has also been observed in other enzymes belonging to the same amidase group (E.C. 3.5.1.4). Moreover, the purified enzyme was proven to be able to enantioselectively hydrolyze 4-chloro-3-hydroxybutyramide into the corresponding acid. The e.e. value was measured to be 52% when the conversion yield was 57%. Although this e.e. value is low for direct commercial use, molecular evolution could eventually result in this amidase being used as a biocatalyst for the production of ethyl (S)-4-chloro-3-hydroxybutyrate.     

Malonic Semialdehyde Reductase from the Archaeon Nitrosopumilus maritimus Is Involved in the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle

The recently described ammonia-oxidizing archaea of the phylum Thaumarchaeota are highly abundant in marine, geothermal, and terrestrial environments. All characterized representatives of this phylum are aerobic chemolithoautotrophic ammonia oxidizers assimilating inorganic carbon via a recently described thaumarchaeal version of the 3-hydroxypropionate/4-hydroxybutyrate cycle. Although some genes coding for the enzymes of this cycle have been identified in the genomes of Thaumarchaeota, many other genes of the cycle are not homologous to the characterized enzymes from other species and can therefore not be identified bioinformatically. Here we report the identification and characterization of malonic semialdehyde reductase Nmar_1110 in the cultured marine thaumarchaeon Nitrosopumilus maritimus. This enzyme, which catalyzes the reduction of malonic semialdehyde with NAD(P)H to 3-hydroxypropionate, belongs to the family of iron-containing alcohol dehydrogenases and is not homologous to malonic semialdehyde reductases from Chloroflexus aurantiacus and Metallosphaera sedula. It is highly specific to malonic semialdehyde (K_m, 0.11 mM; V_max, 86.9 μmol min⁻¹ mg⁻¹ of protein) and exhibits only low activity with succinic semialdehyde (K_m, 4.26 mM; V_max, 18.5 μmol min⁻¹ mg⁻¹ of protein). Homologues of N. maritimus malonic semialdehyde reductase can be found in the genomes of all Thaumarchaeota sequenced so far and form a well-defined cluster in the phylogenetic tree of iron-containing alcohol dehydrogenases. We conclude that malonic semialdehyde reductase can be regarded as a characteristic enzyme for the thaumarchaeal version of the 3-hydroxypropionate/4-hydroxybutyrate cycle.     

Glycoproteins in prokaryotes

Rather recently it has become clear that prokaryotes (Archaea and Bacteria) are able to glycosylate proteins. A literature survey revealed the different types of glycoproteins. They include mainly surface layer (S-layer) proteins, flagellins, and polysaccharide-degrading enzymes. Only in a few cases is structural information available. Many different structures have been observed that display much more variation than that observed in eukaryotes. A few studies have given evidence for the function of the prokaryotic glycoprotein glycans. Also from the biosynthetic point of view, information is rather scarce. Due to their different cell structure, prokaryotes have to use mechanisms different from those found in eukaryotes to glycosylate proteins. However, from the fragmented data available for the prokaryotic glycoproteins, similarities with the eukaryotic system can be noticed. Received: 24 February 1997 / Accepted: 13 May 1997     

Enzymology of one-carbon metabolism in methanogenic pathways

Methanoarchaea, the largest and most phylogenetically diverse group in the Archaea domain, have evolved energy-yielding pathways marked by one-carbon biochemistry featuring novel cofactors and enzymes. All of the pathways have in common the two-electron reduction of methyl-coenzyme M to methane catalyzed by methyl-coenzyme M reductase but deviate in the source of the methyl group transferred to coenzyme M. Most of the methane produced in nature derives from acetate in a pathway where the activated substrate is cleaved by CO dehydrogenase/acetyl-CoA synthase and the methyl group is transferred to coenzyme M via methyltetrahydromethanopterin or methyltetrahydrosarcinapterin. Electrons for reductive demethylation of the methyl-coenzyme M originate from oxidation of the carbonyl group of acetate to carbon dioxide by the synthase. In the other major pathway, formate or H2 is oxidized to provide electrons for reduction of carbon dioxide to the methyl level and reduction of methyl-coenzyme to methane. Methane is also produced from the methyl groups of methanol and methylamines. In these pathways specialized methyltransferases transfer the methyl groups to coenzyme M. Electrons for reduction of the methyl-coenzyme M are supplied by oxidation of the methyl groups to carbon dioxide by a reversal of the carbon dioxide reduction pathway. Recent progress on the enzymology of one-carbon reactions in these pathways has raised the level of understanding with regard to the physiology and molecular biology of methanogenesis. These advances have also provided a foundation for future studies on the structure/function of these novel enzymes and exploitation of the recently completed sequences for the genomes from the methanoarchaea Methanobacterium thermoautotrophicum and Methanococcus jannaschii.     

Pathways for phosphoinositide synthesis.

In eukaryotic cells, phosphatidylinositol can be phosphorylated on the inositol ring by a series of kinases to produce at least seven distinct phosphoinositides. These lipids have been implicated in a variety of cellular processes, including calcium regulation, actin rearrangement, vesicle trafficking, cell survival and mitogenesis. The phosphorylated lipids can act as precursors of second messengers or act directly to recruit specific signaling proteins to the membrane. A number of the kinases responsible for producing these lipids have been purified and their cDNA clones have been isolated. The most well characterized of these enzymes are the phosphoinositide 3-kinases. However, progress has also been made in the characterization of phosphatidylinositol 4-kinases and phosphatidylinositol-4-phosphate 5-kinases. In addition, new pathways involving phosphatidylinositol-5-phosphate 4-kinases, phosphatidylinositol-3-phosphate 5-kinases and phosphatidylinositol-3-phosphate 4-kinases have recently been described. The various enzymes and pathways involved in the synthesis of cellular phosphoinositides will be discussed.     

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. 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 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.Coenzyme A (CoA)² and its derivative 4′-phosphopantetheine are essential cofactors in numerous metabolic pathways, including the tricarboxylic acid cycle, the β-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), 4′-phosphopantothenoylcysteine synthetase (PPCS), 4′-phosphopantothenoylcysteine decarboxylase (PPCDC), 4′- 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 β-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 and eukaryotes, the corresponding pathway in the Archaea remains unclear (4). Sequence data indicate that the bacterial PPCS and PPCDC homologs and eukaryotic PPAT homologs are found on almost all of the archaeal genomes. The archaeal PPCS and PPCDC genes are fused in many cases, and the bifunctional protein from Methanocaldococcus jannaschii has been shown to exhibit both activities (5). The PPAT homolog from Pyrococcus abyssi has also been studied and confirmed to exhibit the expected PPAT activity (6). Bacterial KPHMT and ketopantoate reductase homologs can also be found, to a lesser extent, on the archaeal genomes. They are not found in the methanogens and Thermoplasmatales, and the fact that the structural similarity among archaeal enzymes is not higher than that toward enzymes from hyperthermophilic bacteria suggests that the archaeal KPHMT and ketopantoate reductase are a result of horizontal gene transfer from bacteria (4). In addition, there are candidate genes distantly related to bacterial/eukaryotic DPCK. However, PS homologs are not found in any of the archaeal genomes, and PanK homologs are found only in a few exceptional cases. Recently, Genschel and co-workers have taken a comparative genomics approach to predict the genes corresponding to the archaeal PS and PanK genes, and have also described the identification of a structurally novel PS from Methanosarcina mazei (4, 7).In this study, we describe the identification of the enzymes responsible for the conversion of pantoate to 4′-phosphopantothenate in Thermococcus kodakaraensis. The organism is a hyperthermophilic archaeon isolated from Kodakara Island, Japan (8, 9). The complete genome sequence is available (10), and gene disruption systems have been developed (11–13). To our surprise, the conversion of pantoate to 4′-phosphopantothenate in T. kodakaraensis is not brought about by the two classic enzyme reactions catalyzed by PS and PanK, but by two novel enzyme reactions; phosphorylation of pantoate (pantoate kinase) followed by the condensation of 4-phosphopantoate and β-alanine (4′-phosphopantothenate synthetase or 4-phosphopantoate:β-alanine ligase). Homologs of these two genes are distributed on almost all of the archaeal genomes, suggesting that the Archaea utilize different chemistry in the conversion from pantoate to 4′-phosphopantothenate.     

Epimerase (Msed_0639) and Mutase (Msed_0638 and Msed_2055) Convert (S)-Methylmalonyl-Coenzyme A (CoA) to Succinyl-CoA in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Cycle

Crenarchaeotal genomes encode the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle for carbon dioxide fixation. Of the 13 enzymes putatively comprising the cycle, several of them, including methylmalonyl-coenzyme A (CoA) epimerase (MCE) and methylmalonyl-CoA mutase (MCM), which convert (S)-methylmalonyl-CoA to succinyl-CoA, have not been confirmed and characterized biochemically. In the genome of Metallosphaera sedula (optimal temperature [T(opt)], 73°C), the gene encoding MCE (Msed_0639) is adjacent to that encoding the catalytic subunit of MCM-α (Msed_0638), while the gene for the coenzyme B(12)-binding subunit of MCM (MCM-β) is located remotely (Msed_2055). The expression of all three genes was significantly upregulated under autotrophic compared to heterotrophic growth conditions, implying a role in CO(2) fixation. Recombinant forms of MCE and MCM were produced in Escherichia coli; soluble, active MCM was produced only if MCM-α and MCM-β were coexpressed. MCE is a homodimer and MCM is a heterotetramer (α(2)β(2)) with specific activities of 218 and 2.2 μmol/min/mg, respectively, at 75°C. The heterotetrameric MCM differs from the homo- or heterodimeric orthologs in other organisms. MCE was activated by divalent cations (Ni(2+), Co(2+), and Mg(2+)), and the predicted metal binding/active sites were identified through sequence alignments with less-thermophilic MCEs. The conserved coenzyme B(12)-binding motif (DXHXXG-SXL-GG) was identified in M. sedula MCM-β. The two enzymes together catalyzed the two-step conversion of (S)-methylmalonyl-CoA to succinyl-CoA, consistent with their proposed role in the 3-HP/4-HB cycle. Based on the highly conserved occurrence of single copies of MCE and MCM in Sulfolobaceae genomes, the M. sedula enzymes are likely to be representatives of these enzymes in the 3-HP/4-HB cycle in crenarchaeal thermoacidophiles.     

Complete genome sequence of strain 1860, a crenarchaeon of the genus Pyrobaculum able to grow with various electron acceptors

Strain 1860, a novel member of the genus Pyrobaculum, is a hyperthermophilic organotrophic crenarchaeon growing anaerobically with various electron acceptors. The complete genome sequence reveals genes for several membrane-bound oxidoreductases, the Embden-Meyerhof and Entner-Doudoroff pathways for glucose metabolism, the tricarboxylic acid cycle, the glyoxylate cycle, and the dicarboxylate/4-hydroxybutyrate cycle.     

A family portrait of the RIO kinases

The RIO family of atypical serine protein kinases has been first characterized only recently. It consists of enzymes that contain a unique domain with a characteristic kinase sequence motif and usually some additional domains. At least two RIO proteins, Rio1 and Rio2, are present in organisms varying from Archaea to humans, with a third Rio3 subfamily present only in multicellular eukaryotes. Yeast Rio1 and Rio2 proteins have been implicated in the processing of 20 S pre-rRNA and are necessary for survival of the cells. Crystal structures of Archaeoglobus fulgidus Rio1 and Rio2 have shown that whereas the overall fold of these enzymes resembles typical protein kinases, some of the structural domains, particularly those involved in peptide substrate binding, are not present. The mode of binding of nucleotides also differs from that found in typical protein kinases. Although it has been shown that both Rio1 and Rio2 have the enzymatic activity of kinases and are capable of autophosphorylation, the biological substrates of RIO proteins and their full biological role still remain to be discovered.     

MetaCyc and AraCyc. Metabolic pathway databases for plant research

MetaCyc (http://metacyc.org) contains experimentally determined biochemical pathways to be used as a reference database for metabolism. In conjunction with the Pathway Tools software, MetaCyc can be used to computationally predict the metabolic pathway complement of an annotated genome. To increase the breadth of pathways and enzymes, more than 60 plant-specific pathways have been added or updated in MetaCyc recently. In contrast to MetaCyc, which contains metabolic data for a wide range of organisms, AraCyc is a species-specific database containing only enzymes and pathways found in the model plant Arabidopsis (Arabidopsis thaliana). AraCyc (http://arabidopsis.org/tools/aracyc/) was the first computationally predicted plant metabolism database derived from MetaCyc. Since its initial computational build, AraCyc has been under continued curation to enhance data quality and to increase breadth of pathway coverage. Twenty-eight pathways have been manually curated from the literature recently. Pathway predictions in AraCyc have also been recently updated with the latest functional annotations of Arabidopsis genes that use controlled vocabulary and literature evidence. AraCyc currently features 1,418 unique genes mapped onto 204 pathways with 1,156 literature citations. The Omics Viewer, a user data visualization and analysis tool, allows a list of genes, enzymes, or metabolites with experimental values to be painted on a diagram of the full pathway map of AraCyc. Other recent enhancements to both MetaCyc and AraCyc include implementation of an evidence ontology, which has been used to provide information on data quality, expansion of the secondary metabolism node of the pathway ontology to accommodate curation of secondary metabolic pathways, and enhancement of the cellular component ontology for storing and displaying enzyme and pathway locations within subcellular compartments.     

Perspectives in anti-infective drug design. The late steps in the biosynthesis of the universal terpenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate

A mevalonate-independent pathway for the biosynthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) that has been elucidated during the last decade is essential in plants, many eubacteria and apicomplexan parasites, but is absent in Archaea and animals. The enzymes of the pathway are potential targets for the development of novel antibiotic, antimalarial and herbicidal agents. This review is focused on the late steps of this pathway. The intermediate 2C-methyl-D-erythritol 2,4-cyclodiphosphate is converted into IPP and DMAPP via 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate by the consecutive action of the iron-sulfur proteins IspG and IspH. IPP and DMAPP can be interconverted by IPP isomerase which is essential in microorganisms using the mevalonate pathway, whereas its presence is optional in microorganisms using the non-mevalonate pathway. A hitherto unknown family of IPP isomerases using FMN as coenzyme has been discovered recently in Archaea and certain eubacteria.     

Archaeal DNA replication: spotlight on a rapidly moving field

The replication of DNA is a fundamental step in the cell cycle, which must be coordinated with cell division to ensure that the daughter cells inherit the same genomic material as the parental cell. The recently published complete genome sequences of some archaeal species together with preliminary biochemical studies suggest that the Archaea quite likely duplicate their chromosome by using replication machinery that seems to be a simplified version of the eukaryotic machinery, although their metabolic facets and their cellular morphology are prokaryotic-like. This review is focused on recent progress on the structural and functional analysis of proteins and enzymes involved in the initiation and elongation steps of DNA replication in Archaea. Differences between the genome replication apparatus of the Euryarchaea and the Crenarchaea (the two main phylogenetic divisions of the Archaea domain) are highlighted.