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.

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.

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.

Corrigendum: Thermostable Branched-Chain Amino Acid Transaminases From the Archaea Geoglobus acetivorans and Archaeoglobus fulgidus: Biochemical and Structural Characterization.

[This corrects the article DOI: 10.3389/fbioe.2019.00007.].

Thermostable Branched-Chain Amino Acid Transaminases From the Archaea Geoglobus acetivorans and Archaeoglobus fulgidus: Biochemical and Structural Characterization.

Two new thermophilic branched chain amino acid transaminases have been identified within the genomes of different hyper-thermophilic archaea, Geoglobus acetivorans, and Archaeoglobus fulgidus. These enzymes belong to the class IV of transaminases as defined by their structural fold. The enzymes have been cloned and over-expressed in Escherichia coli and the recombinant enzymes have been characterized both biochemically and structurally. Both enzymes showed high thermostability with optimal temperature for activity at 80 and 85°C, respectively. They retain good activity after exposure to 50% of the organic solvents, ethanol, methanol, DMSO and acetonitrile. The enzymes show a low activity to (R)-methylbenzylamine but no activity to (S)-methylbenzylamine. Both enzymes have been crystallized and their structures solved in the internal aldimine form, to 1.9 Å resolution for the Geoglobus enzyme and 2.0 Å for the Archaeoglobus enzyme. Also the Geoglobus enzyme structure has been determined in complex with the amino acceptor α-ketoglutarate and the Archaeoglobus enzyme in complex with the inhibitor gabaculine. These two complexes have helped to determine the conformation of the enzymes during enzymatic turnover and have increased understanding of their substrate specificity. A comparison has been made with another (R) selective class IV transaminase from the fungus Nectria haematococca which was previously studied in complex with gabaculine. The subtle structural differences between these enzymes has provided insight regarding their different substrate specificities.

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.

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.

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).