Discovery of a novel transcriptional regulator of sugar catabolism in archaea.

The haloarchaeon Haloferax volcanii degrades D-glucose via the semiphosphorylative Entner-Doudoroff pathway and D-fructose via a modified Embden-Meyerhof pathway. Here, we report the identification of GfcR, a novel type of transcriptional regulator that functions as an activator of both D-glucose and D-fructose catabolism. We find that in the presence of D-glucose, GfcR activates gluconate dehydratase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase and also acts as activator of the phosphotransferase system and of fructose-1,6-bisphosphate aldolase, which are involved in uptake and degradation of D-fructose. In addition, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase are activated by GfcR in the presence of D-fructose and also during growth on D-galactose and glycerol. Electrophoretic mobility shift assays indicate that GfcR binds directly to promoters of regulated genes. Specific intermediates of the degradation pathways of the three hexoses and of glycerol were identified as inducer molecules of GfcR. GfcR is composed of a phosphoribosyltransferase (PRT) domain with an N-terminal helix-turn-helix motif and thus shows homology to PurR of Gram-positive bacteria that is involved in the transcriptional regulation of nucleotide biosynthesis. We propose that GfcR of H. volcanii evolved from a PRT-like enzyme to attain a function as a transcriptional regulator of central sugar catabolic pathways in archaea.

Glucose Metabolism and Acetate Switch in Archaea: the Enzymes in Haloferax volcanii.

The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. Following our previous studies on key enzymes of this pathway, we now focus on the characterization of enzymes involved in 3-phosphoglycerate conversion to pyruvate, in anaplerosis, and in acetyl coenzyme A (acetyl-CoA) formation from pyruvate. These enzymes include phosphoglycerate mutase, enolase, pyruvate kinase, phosphoenolpyruvate carboxylase, and pyruvate-ferredoxin oxidoreductase. The essential function of these enzymes were shown by transcript analyses and growth experiments with respective deletion mutants. Furthermore, we show that H. volcanii-during aerobic growth on glucose-excreted significant amounts of acetate, which was consumed in the stationary phase (acetate switch). The enzyme catalyzing the conversion of acetyl-CoA to acetate as part of the acetate overflow mechanism, an ADP-forming acetyl-CoA synthetase (ACD), was characterized. The functional involvement of ACD in acetate formation and of AMP-forming acetyl-CoA synthetases (ACSs) in activation of excreted acetate was proven by using respective deletion mutants. Together, the data provide a comprehensive analysis of enzymes of the spED pathway and of anaplerosis and report the first genetic evidence of the functional involvement of enzymes of the acetate switch in archaea.IMPORTANCE In this work, we provide a comprehensive analysis of glucose degradation via the semiphosphorylative Entner-Doudoroff pathway in the haloarchaeal model organism Haloferax volcanii The study includes transcriptional analyses, growth experiments with deletion mutants. and characterization of all enzymes involved in the conversion of 3-phosphoglycerate to acetyl coenzyme A (acetyl-CoA) and in anaplerosis. Phylogenetic analyses of several enzymes indicate various lateral gene transfer events from bacteria to haloarchaea. Furthermore, we analyzed the key players involved in the acetate switch, i.e., in the formation (overflow) and subsequent consumption of acetate during aerobic growth on glucose. Together, the data provide novel aspects of glucose degradation, anaplerosis, and acetate switch in H. volcanii and thus expand our understanding of the unusual sugar metabolism in archaea.

d-Ribose Catabolism in Archaea: Discovery of a Novel Oxidative Pathway in Haloarcula Species.

The Haloarcula species H. marismortui and H. hispanica were found to grow on d-ribose, d-xylose, and l-arabinose. Here, we report the discovery of a novel promiscuous oxidative pathway of pentose degradation based on genome analysis, identification and characterization of enzymes, transcriptional analysis, and growth experiments with knockout mutants. Together, the data indicate that in Haloarcula spp., d-ribose, d-xylose, and l-arabinose were degraded to α-ketoglutarate involving the following enzymes: (i) a promiscuous pentose dehydrogenase that catalyzed the oxidation of d-ribose, d-xylose, and l-arabinose; (ii) a promiscuous pentonolactonase that was involved in the hydrolysis of ribonolactone, xylonolactone, and arabinolactone; (iii) a highly specific dehydratase, ribonate dehydratase, which catalyzed the dehydration of ribonate, and a second enzyme, a promiscuous xylonate/gluconate dehydratase, which was involved in the conversion of xylonate, arabinonate, and gluconate. Phylogenetic analysis indicated that the highly specific ribonate dehydratase constitutes a novel sugar acid dehydratase family within the enolase superfamily; and (iv) finally, 2-keto-3-deoxypentanonate dehydratase and α-ketoglutarate semialdehyde dehydrogenase catalyzed the conversion of 2-keto-3-deoxypentanonate to α-ketoglutarate via α-ketoglutarate semialdehyde. We conclude that the expanded substrate specificities of the pentose dehydrogenase and pentonolactonase toward d-ribose and ribonolactone, respectively, and the presence of a highly specific ribonate dehydratase are prerequisites of the oxidative degradation of d-ribose in Haloarcula spp. This is the first characterization of an oxidative degradation pathway of d-ribose to α-ketoglutarate in archaea.IMPORTANCE The utilization and degradation of d-ribose in archaea, the third domain of life, have not been analyzed so far. We show that Haloarcula species utilize d-ribose, which is degraded to α-ketoglutarate via a novel oxidative pathway. Evidence is presented that the oxidative degradation of d-ribose involves novel promiscuous enzymes, pentose dehydrogenase and pentonolactonase, and a novel sugar acid dehydratase highly specific for ribonate. This is the first report of an oxidative degradation pathway of d-ribose in archaea, which differs from the canonical nonoxidative pathway of d-ribose degradation reported for most bacteria. The data contribute to our understanding of the unusual sugar degradation pathways and enzymes in archaea.

l-Rhamnose catabolism in archaea.

The halophilic archaeon Haloferax volcanii utilizes l-rhamnose as a sole carbon and energy source. It is shown that l-rhamnose is taken up by an ABC transporter and is oxidatively degraded to pyruvate and l-lactate via the diketo-hydrolase pathway. The genes involved in l-rhamnose uptake and degradation form a l-rhamnose catabolism (rhc) gene cluster. The rhc cluster also contains a gene, rhcR, that encodes the transcriptional regulator RhcR which was characterized as an activator of all rhc genes. 2-keto-3-deoxy-l-rhamnonate, a metabolic intermediate of l-rhamnose degradation, was identified as inducer molecule of RhcR. The essential function of rhc genes for uptake and degradation of l-rhamnose was proven by the respective knockout mutants. Enzymes of the diketo-hydrolase pathway, including l-rhamnose dehydrogenase, l-rhamnonolactonase, l-rhamnonate dehydratase, 2-keto-3-deoxy-l-rhamnonate dehydrogenase and 2,4-diketo-3-deoxy-l-rhamnonate hydrolase, were characterized. Further, genes of the diketo-hydrolase pathway were also identified in the hyperthermophilic crenarchaeota Vulcanisaeta distributa and Sulfolobus solfataricus and selected enzymes were characterized, indicating the presence of the diketo-hydrolase pathway in these archaea. Together, this is the first comprehensive description of l-rhamnose catabolism in the domain of archaea.

Pentose degradation in archaea: Halorhabdus species degrade D-xylose, L-arabinose and D-ribose via bacterial-type pathways.

The degradation of the pentoses D-xylose, L-arabinose and D-ribose in the domain of archaea, in Haloferax volcanii and in Haloarcula and Sulfolobus species, has been shown to proceed via oxidative pathways to generate α-ketoglutarate. Here, we report that the haloarchaeal Halorhabdus species utilize the bacterial-type non-oxidative degradation pathways for pentoses generating xylulose-5-phosphate. The genes of these pathways are each clustered and were constitutively expressed. Selected enzymes involved in D-xylose degradation, xylose isomerase and xylulokinase, and those involved in L-arabinose degradation, arabinose isomerase and ribulokinase, were characterized. Further, D-ribose degradation in Halorhabdus species involves ribokinase, ribose-5-phosphate isomerase and D-ribulose-5-phosphate-3-epimerase. Ribokinase of Halorhabdus tiamatea and ribose-5-phosphate isomerase of Halorhabdus utahensis were characterized. This is the first report of pentose degradation via the bacterial-type pathways in archaea, in Halorhabdus species that likely acquired these pathways from bacteria. The utilization of bacterial-type pathways of pentose degradation rather than the archaeal oxidative pathways generating α-ketoglutarate might be explained by an incomplete gluconeogenesis in Halorhabdus species preventing the utilization of α-ketoglutarate in the anabolism.

Insights into the Substrate Specificity of Archaeal Entner-Doudoroff Aldolases: The Structures of Picrophilus torridus 2-Keto-3-deoxygluconate Aldolase and Sulfolobus solfataricus 2-Keto-3-deoxy-6-phosphogluconate Aldolase in Complex with 2-Keto-3-deoxy-6-phosphogluconate.

The thermoacidophilic archaea Picrophilus torridus and Sulfolobus solfataricus catabolize glucose via a nonphosphorylative Entner-Doudoroff pathway and a branched Entner-Doudoroff pathway, respectively. Key enzymes for these Entner-Doudoroff pathways are the aldolases, 2-keto-3-deoxygluconate aldolase (KDG-aldolase) and 2-keto-3-deoxy-6-phosphogluconate aldolase [KD(P)G-aldolase]. KDG-aldolase from P. torridus (Pt-KDG-aldolase) is highly specific for the nonphosphorylated substrate, 2-keto-3-deoxygluconate (KDG), whereas KD(P)G-aldolase from S. solfataricus [Ss-KD(P)G-aldolase] is an enzyme that catalyzes the cleavage of both KDG and 2-keto-3-deoxy-6-phosphogluconate (KDPG), with a preference for KDPG. The structural basis for the high specificity of Pt-KDG-aldolase for KDG as compared to the more promiscuous Ss-KD(P)G-aldolase has not been analyzed before. In this work, we report the elucidation of the structure of Ss-KD(P)G-aldolase in complex with KDPG at 2.35 Å and that of KDG-aldolase from P. torridus at 2.50 Å resolution. By superimposition of the active sites of the two enzymes, and subsequent site-directed mutagenesis studies, a network of four amino acids, namely, Arg106, Tyr132, Arg237, and Ser241, was identified in Ss-KD(P)G-aldolase that interact with the negatively charged phosphate group of KDPG, thereby increasing the affinity of the enzyme for KDPG. This KDPG-binding network is absent in Pt-KDG-aldolase, which explains the low catalytic efficiency of KDPG cleavage.

Key Enzymes of the Semiphosphorylative Entner-Doudoroff Pathway in the Haloarchaeon Haloferax volcanii: Characterization of Glucose Dehydrogenase, Gluconate Dehydratase, and 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase.

UNLABELLED: The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semiphosphorylative Entner-Doudoroff (spED) pathway. So far, the key enzymes of this pathway, glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (KDPGA), have not been characterized, and their functional involvement in glucose degradation has not been demonstrated. Here we report that the genes HVO_1083 and HVO_0950 encode GDH and KDPGA, respectively. The recombinant enzymes show high specificity for glucose and KDPG and did not convert the corresponding C4 epimers galactose and 2-keto-3-deoxy-6-phosphogalactonate at significant rates. Growth studies of knockout mutants indicate the functional involvement of both GDH and KDPGA in glucose degradation. GAD was purified from H. volcanii, and the encoding gene, gad, was identified as HVO_1488. GAD catalyzed the specific dehydration of gluconate and did not utilize galactonate at significant rates. A knockout mutant of GAD lost the ability to grow on glucose, indicating the essential involvement of GAD in glucose degradation. However, following a prolonged incubation period, growth of the Δgad mutant on glucose was recovered. Evidence is presented that under these conditions, GAD was functionally replaced by xylonate dehydratase (XAD), which uses both xylonate and gluconate as substrates. Together, the characterization of key enzymes and analyses of the respective knockout mutants present conclusive evidence for the in vivo operation of the spED pathway for glucose degradation in H. volcanii IMPORTANCE: The work presented here describes the identification and characterization of the key enzymes glucose dehydrogenase, gluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase and their encoding genes of the proposed semiphosphorylative Entner-Doudoroff pathway in the haloarchaeon Haloferax volcanii The functional involvement of the three enzymes was proven by analyses of the corresponding knockout mutants. These results provide evidence for the in vivo operation of the semiphosphorylative Entner-Doudoroff pathway in haloarchaea and thus expand our understanding of the unusual sugar degradation pathways in the domain Archaea.

XacR - a novel transcriptional regulator of D-xylose and L-arabinose catabolism in the haloarchaeon Haloferax volcanii.

The haloarchaeon Haloferax volcanii degrades D-xylose and L-arabinose via oxidative pathways to α-ketoglutarate. The genes involved in these pathways are clustered and were transcriptionally upregulated by both D-xylose and L-arabinose suggesting a common regulator. Adjacent to the gene cluster, a putative IclR-like transcriptional regulator, HVO_B0040, was identified. It is shown that HVO_B0040, designated xacR, encodes an activator of both D-xylose and L-arabinose catabolism: in ΔxacR cells, transcripts of genes involved in pentose catabolism could not be detected; transcript formation could be recovered by complementation, indicating XacR dependent transcriptional activation. Upstream activation promoter regions and nucleotide sequences that were essential for XacR-mediated activation of pentose-specific genes were identified by in vivo deletion and scanning mutagenesis. Besides its activator function XacR acted as repressor of its own synthesis: xacR deletion resulted in an increase of xacR promoter activity. A palindromic sequence was identified at the operator site of xacR promoter, and mutation of this sequence also resulted in an increase and thus derepression of xacR promoter activity. It is concluded that the palindromic sequence represents the binding site of XacR as repressor. This is the first report of a transcriptional regulator of pentose catabolism in the domain of archaea.

3-Phosphoglycerate is an allosteric activator of pyruvate kinase from the hyperthermophilic archaeon Pyrobaculum aerophilum.

Pyruvate kinase (PK) is a highly regulated enzyme that catalyzes the final step of glycolysis. PK from the hyperthermophilic archaeon Pyrobaculum aerophilum (PaPK) is distinguished from most PK enzymes of eukarya and bacteria by not responding to any known allosteric effectors and apparently exhibiting only cooperative regulation. We determined the crystal structure of PaPK to 2.2 Å resolution and, in a manner consistent with the lack of a response to conventional effectors, observed that the canonical allosteric site is occluded by a tyrosine. Unexpectedly, though, a bound sulfate was observed at a position equivalent to the 6'-phosphate of sugar effectors, suggesting an allosteric site, but for an unknown effector and sharing only the phosphate position. A search of three-carbon intermediates of glycolysis revealed 3-phosphoglycerate (3PG) as a potent allosteric activator of PaPK. The response was abolished by mutation of residues that contact the sulfate and of an arginine proposed to interact with the 3PG carboxylate group. Regulation of PK by 3PG is consistent with the ancestral glycolysis of hyperthermophilic archaea in which this intermediate is produced by an irreversible enzyme, glyceraldehyde 3-phosphate ferredoxin oxidoreductase. Coordinated regulation within the lower half of glycolysis contrasts sharply with conventional glycolysis in which 3PG is produced reversibly and PK is regulated by fructose 1,6-bisphosphate, the product of phosphofructokinase, an irreversible enzyme in the upper half of the pathway. Regulation of PaPK by a carboxylate molecule rather than a sugar phosphate may reflect a step in the evolution of glycolysis that predates the dominance of sugars in metabolism.

L-Arabinose degradation pathway in the haloarchaeon Haloferax volcanii involves a novel type of L-arabinose dehydrogenase.

The pathway of L-arabinose degradation was studied in the haloarchaeon Haloferax volcanii. It is shown that L-arabinose is oxidatively degraded to α-ketoglutarate. During growth on L-arabinose, L-arabinose dehydrogenase (L-AraDH) was induced. The enzyme was purified as a 130 kDa homotetrameric protein catalyzing the oxidation of L-arabinose with both NADP(+) and NAD(+). The gene encoding L-AraDH was identified as HVO_B0032 and recombinant L-AraDH showed similar properties as the native enzyme. The L-AraDH deletion mutant did not grow on L-arabinose, but grew unaffected on glucose and D-xylose, indicating a specific involvement in L-arabinose degradation. Phylogenetic analyses attribute the first archaeal L-AraDH to the extended short-chain dehydrogenase/reductase (SDRe) family, where it is part of a novel cluster and thus differs from known archaeal and bacterial pentose dehydrogenases. Further, cell extracts of H. volcanii catalyzed the NADP(+)-dependent conversion of L-arabinoate to α-ketoglutarate. The genes involved in that conversion were identified by analyses of transcripts and deletion mutants as HVO_B0038A, HVO_B0027 and HVO_B0039 recently reported to be involved in D-xylonate conversion to α-ketoglutarate in H. volcanii (Johnsen et al. 2009).

Fructose degradation in the haloarchaeon Haloferax volcanii involves a bacterial type phosphoenolpyruvate-dependent phosphotransferase system, fructose-1-phosphate kinase, and class II fructose-1,6-bisphosphate aldolase.

The halophilic archaeon Haloferax volcanii utilizes fructose as a sole carbon and energy source. Genes and enzymes involved in fructose uptake and degradation were identified by transcriptional analyses, deletion mutant experiments, and enzyme characterization. During growth on fructose, the gene cluster HVO_1495 to HVO_1499, encoding homologs of the five bacterial phosphotransferase system (PTS) components enzyme IIB (EIIB), enzyme I (EI), histidine protein (HPr), EIIA, and EIIC, was highly upregulated as a cotranscript. The in-frame deletion of HVO_1499, designated ptfC (ptf stands for phosphotransferase system for fructose) and encoding the putative fructose-specific membrane component EIIC, resulted in a loss of growth on fructose, which could be recovered by complementation in trans. Transcripts of HVO_1500 (pfkB) and HVO_1494 (fba), encoding putative fructose-1-phosphate kinase (1-PFK) and fructose-1,6-bisphosphate aldolase (FBA), respectively, as well as 1-PFK and FBA activities were specifically upregulated in fructose-grown cells. pfkB and fba knockout mutants did not grow on fructose, whereas growth on glucose was not inhibited, indicating the functional involvement of both enzymes in fructose catabolism. Recombinant 1-PFK and FBA obtained after homologous overexpression were characterized as having kinetic properties indicative of functional 1-PFK and a class II type FBA. From these data, we conclude that fructose uptake in H. volcanii involves a fructose-specific PTS generating fructose-1-phosphate, which is further converted via fructose-1,6-bisphosphate to triose phosphates by 1-PFK and FBA. This is the first report of the functional involvement of a bacterial-like PTS and of class II FBA in the sugar metabolism of archaea.

Metabolism of pentose sugars in the hyperthermophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius.

We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous pathway. On further investigation, the catalytic promiscuity of the first enzyme in this pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, D-xylose and L-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this pathway for the catabolism of D-xylose and L-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.

D-xylose degradation pathway in the halophilic archaeon Haloferax volcanii.

The pathway of D-xylose degradation in archaea is unknown. In a previous study we identified in Haloarcula marismortui the first enzyme of xylose degradation, an inducible xylose dehydrogenase (Johnsen, U., and Schönheit, P. (2004) J. Bacteriol. 186, 6198-6207). Here we report a comprehensive study of the complete D-xylose degradation pathway in the halophilic archaeon Haloferax volcanii. The analyses include the following: (i) identification of the degradation pathway in vivo following (13)C-labeling patterns of proteinogenic amino acids after growth on [(13)C]xylose; (ii) identification of xylose-induced genes by DNA microarray experiments; (iii) characterization of enzymes; and (iv) construction of in-frame deletion mutants and their functional analyses in growth experiments. Together, the data indicate that D-xylose is oxidized exclusively to the tricarboxylic acid cycle intermediate alpha-ketoglutarate, involving D-xylose dehydrogenase (HVO_B0028), a novel xylonate dehydratase (HVO_B0038A), 2-keto-3-deoxyxylonate dehydratase (HVO_B0027), and alpha-ketoglutarate semialdehyde dehydrogenase (HVO_B0039). The functional involvement of these enzymes in xylose degradation was proven by growth studies of the corresponding in-frame deletion mutants, which all lost the ability to grow on d-xylose, but growth on glucose was not significantly affected. This is the first report of an archaeal D-xylose degradation pathway that differs from the classical D-xylose pathway in most bacteria involving the formation of xylulose 5-phosphate as an intermediate. However, the pathway shows similarities to proposed oxidative pentose degradation pathways to alpha-ketoglutarate in few bacteria, e.g. Azospirillum brasilense and Caulobacter crescentus, and in the archaeon Sulfolobus solfataricus.

Acetate Metabolism in Archaea: Characterization of an Acetate Transporter and of Enzymes Involved in Acetate Activation and Gluconeogenesis in Haloferax volcanii.

The haloarchaeon Haloferax volcanii grows on acetate as sole carbon and energy source. The genes and proteins involved in uptake and activation of acetate and in gluconeogenesis were identified and analyzed by characterization of enzymes and by growth experiments with the respective deletion mutants. (i) An acetate transporter of the sodium: solute-symporter family (SSF) was characterized by kinetic analyses of acetate uptake into H. volcanii cells. The functional involvement of the transporter was proven with a Δssf mutant. (ii) Four paralogous AMP-forming acetyl-CoA synthetases that belong to different phylogenetic clades were shown to be functionally involved in acetate activation. (iii) The essential involvement of the glyoxylate cycle as an anaplerotic sequence was concluded from growth experiments with an isocitrate lyase knock-out mutant excluding the operation of the methylaspartate cycle reported for Haloarcula species. (iv) Enzymes involved in phosphoenolpyruvate synthesis from acetate, namely two malic enzymes and a phosphoenolpyruvate synthetase, were identified and characterized. Phylogenetic analyses of haloarchaeal malic enzymes indicate a separate evolutionary line distinct from other archaeal homologs. The exclusive function of phosphoenolpyruvate synthetase in gluconeogenesis was proven by the respective knock-out mutant. Together, this is a comprehensive study of acetate metabolism in archaea.

D-galactose catabolism in archaea: operation of the DeLey-Doudoroff pathway in Haloferax volcanii.

The haloarchaeon Haloferax volcanii was found to grow on D-galactose as carbon and energy source. Here we report a comprehensive analysis of D-galactose catabolism in H. volcanii. Genome analyses indicated a cluster of genes encoding putative enzymes of the DeLey-Doudoroff pathway for D-galactose degradation including galactose dehydrogenase, galactonate dehydratase, 2-keto-3-deoxygalactonate kinase and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase. The recombinant galactose dehydrogenase and galactonate dehydratase showed high specificity for D-galactose and galactonate, respectively, whereas KDPGal aldolase was promiscuous in utilizing KDPGal and also the C4 epimer 2-keto-3-deoxy-6-phosphogluconate as substrates. Growth studies with knock-out mutants indicated the functional involvement of galactose dehydrogenase, galactonate dehydratase and KDPGal aldolase in D-galactose degradation. Further, the transcriptional regulator GacR was identified, which was characterized as an activator of genes of the DeLey-Doudoroff pathway. Finally, genes were identified encoding components of an ABC transporter and a knock-out mutant of the substrate binding protein indicated the functional involvement of this transporter in D-galactose uptake. This is the first report of D-galactose degradation via the DeLey-Doudoroff pathway in the domain of archaea.

Uptake of D-xylose and L-arabinose in Haloferax volcanii involves an ABC transporter of the CUT1 subfamily.

Haloferax volcanii degrades D-xylose and L-arabinose via an oxidative pathway to α-ketoglutarate as an intermediate. The enzymes of this pathway are encoded by the xac gene cluster (xylose and arabinose catabolism) which also contains genes (xacGHIJK) that encode all components of a putative ABC transporter. The xacGHIJK genes encode one substrate binding protein, two transmembrane domains and two nucleotide binding domains. It is shown here, that xacGHIJK is upregulated by both D-xylose and L-arabinose mediated by the transcriptional regulator XacR, the general regulator of xac genes. Knock-out mutants of xacG and of xacGHIJK resulted in a reduced growth rate on both pentoses; wild type growth could be recovered by complementation in trans. Together, the data indicate that uptake of xylose and arabinose in H. volcanii is mediated by this ABC transporter. Pentose specific ABC transporters, homologous to that of H. volcanii, were identified in other haloarchaea suggesting a similar function in pentose uptake in these archaea. Sequence analyses attribute the haloarchaeal pentose ABC transporter to the CUT1 (carbohydrate uptake transporter 1) subfamily.

New views on an old enzyme: allosteric regulation and evolution of archaeal pyruvate kinases.

Pyruvate kinases (PKs) synthesize ATP as the final step of glycolysis in the three domains of life. PKs from most bacteria and eukarya are allosteric enzymes that are activated by sugar phosphates; for example, the feed-forward regulator fructose-1,6-bisphosphate, or AMP as a sensor of energy charge. Archaea utilize unusual glycolytic pathways, but the allosteric properties of PKs from these species are largely unknown. Here, we present an analysis of 24 PKs from most archaeal clades with respect to allosteric properties, together with phylogenetic analyses constructed using a novel mode of rooting protein trees. We find that PKs from many Thermoproteales, an order of crenarchaeota, are allosterically activated by 3-phosphoglycerate (3PG). We also identify five conserved amino acids that form the binding pocket for 3PG. 3PG is generated via an irreversible reaction in the modified glycolytic pathway of these archaea and therefore functions as a feed-forward regulator. We also show that PKs from hyperthermophilic Methanococcales, an order of euryarchaeota, are activated by AMP. Phylogenetic analyses indicate that 3PG-activated PKs form an evolutionary lineage that is distinct from that of sugar-phosphate activated PKs, and that sugar phosphate-activated PKs originated as AMP-regulated PKs in hyperthermophilic Methanococcales. Since the phospho group of sugar phosphates and 3PG overlap in the allosteric site, our data indicate that the allostery in PKs first started from a progenitor phosphate-binding site that evolved in two spatially distinct directions: one direction generated the canonical site that responds to sugar phosphates and the other gave rise to the 3PG site present in Thermoproteales. Overall, our data suggest an intimate connection between the allosteric properties and evolution of PKs.

Characterization of a pentonolactonase involved in D-xylose and L-arabinose catabolism in the haloarchaeon Haloferax volcanii.

Haloferax volcanii degrades the pentoses D-xylose and L-arabinose via an oxidative pathway to α-ketoglutarate as an intermediate. The initial dehydrogenases of the pathway, D-xylose dehydrogenase (XDH) and L-arabinose dehydrogenase (L-AraDH) catalyze the NADP+ dependent D-xylose and L-arabinose oxidation. It is shown here that the pentoses are oxidized to the corresponding lactones, D-xylono-γ-lactone and L-arabino-γ-lactone, rather than to the respective sugar acids. A putative lactonase gene, xacC, located in genomic vicinity of XDH and L-AraDH, was found to be transcriptionally upregulated by both D-xylose and L-arabinose mediated by the pentose-specific regulator XacR. The recombinant lactonase catalyzed the hydrolysis of D-xylono-γ-lactone and L-arabino-γ-lactone. This is the first report of a functional lactonase involved in sugar catabolism in the domain of archaea.

Succinyl-CoA:Mesaconate CoA-Transferase and Mesaconyl-CoA Hydratase, Enzymes of the Methylaspartate Cycle in Haloarcula hispanica.

Growth on acetate or other acetyl-CoA-generating substrates as a sole source of carbon requires an anaplerotic pathway for the conversion of acetyl-CoA into cellular building blocks. Haloarchaea (class Halobacteria) possess two different anaplerotic pathways, the classical glyoxylate cycle and the novel methylaspartate cycle. The methylaspartate cycle was discovered in Haloarcula spp. and operates in ∼40% of sequenced haloarchaea. In this cycle, condensation of one molecule of acetyl-CoA with oxaloacetate gives rise to citrate, which is further converted to 2-oxoglutarate and then to glutamate. The following glutamate rearrangement and deamination lead to mesaconate (methylfumarate) that needs to be activated to mesaconyl-C1-CoA and hydrated to ß-methylmalyl-CoA. The cleavage of ß-methylmalyl-CoA results in the formation of propionyl-CoA and glyoxylate. The carboxylation of propionyl-CoA and the condensation of glyoxylate with another acetyl-CoA molecule give rise to two C4-dicarboxylic acids, thus regenerating the initial acetyl-CoA acceptor and forming malate, its final product. Here we studied two enzymes of the methylaspartate cycle from Haloarcula hispanica, succinyl-CoA:mesaconate CoA-transferase (mesaconate CoA-transferase, Hah_1336) and mesaconyl-CoA hydratase (Hah_1340). Their genes were heterologously expressed in Haloferax volcanii, and the corresponding enzymes were purified and characterized. Mesaconate CoA-transferase was specific for its physiological substrates, mesaconate and succinyl-CoA, and produced only mesaconyl-C1-CoA and no mesaconyl-C4-CoA. Mesaconyl-CoA hydratase had a 3.5-fold bias for the physiological substrate, mesaconyl-C1-CoA, compared to mesaconyl-C4-CoA, and virtually no activity with other tested enoyl-CoA/3-hydroxyacyl-CoA compounds. Our results further prove the functioning of the methylaspartate cycle in haloarchaea and suggest that mesaconate CoA-transferase and mesaconyl-CoA hydratase can be regarded as characteristic enzymes of this cycle.