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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. Various halophilic archaea, including Haloarcula marismortui and Haloferax volcanii, have been reported to utilize fructose as carbon and energy sources (29,34,46). The pathway of fructose degradation has been studied so far mainly in the Haloarcula species H. vallismortis and H. marismortui (6,7,29). On the basis of enzyme analyses, a modified version of the Embden-Meyerhof (EM) pathway has been proposed, involving fructose phosphorylation via ketohexokinase to fructose-1-phosphate, which is further phosphorylated to fructose-1,6-bisphosphate (FBP) by fructose-1-phosphate kinase (1-PFK). FBP is subsequently cleaved by FBP aldolase (FBA) to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, which are degraded to pyruvate following classical enzymes of the EM pathway. In vivo evidence for the operation of an EM pathway in fructose degradation was demonstrated in H. marismortui by labeling experiments with [13 C]fructose using growing cultures (29). With the same labeling techniques, glucose degradation in H. marismortui was shown to be degraded in vivo via an Entner-Doudoroff (ED) type pathway (29), which is in accordance with the proposed semiphosphorylated ED pathway for glucose degradation in haloarchaea (43).Although several enzymes of the proposed modified EM pathway...

a b s t r a c tThe oxidative pentose phosphate pathway (OPPP), catalyzing the oxidation of glucose-6-phosphate to ribulose-5-phosphate is ubiquitous in eukarya and bacteria but has not yet been reported in archaea. In haloarchaea a putative 6-phosphogluconate dehydrogenase (6PGDH) is annotated, whereas a gene coding for glucose-6-phosphate dehydrogenase (Glc6PDH) could not be identified. Here we report the purification and characterization of a novel type of Glc6PDH in Haloferax volcanii that is not related to bacterial and eukaryal Glc6PDHs and the encoding gene is designated as azf (archaeal zwischenferment). Further, recombinant H. volcanii 6PGDH was characterized. Deletion mutant analyses indicate that both, Glc6PDH and 6PGDH, are functionally involved in pentose phosphate formation in vivo. This is the first report on the operation of the OPPP in the domain of archaea.

The halophilic archaeon Haloferax volcanii has been proposed to degrade glucose via the semi-phosphorylative Entner-Doudoroff pathway, involving 2-keto-3-deoxygluconate kinase (KDGK) as key enzyme. So far, neither the enzyme has been characterized nor the encoding gene has been identified. In the genome of H. volcanii, two genes, HVO_0549 (kdgK1) and HVO_A0328 (kdgK2), are annotated encoding putative KDGK-1 and KDGK-2. To identify the physiological role of both kinases, transcriptional regulation analyses of both genes and growth experiments of the respective deletion mutants were performed on different sugars. Further, recombinant KDGK-1 and KDGK-2 were characterized. Together, the data indicate that KDGK-1 represents the functional constitutively expressed KDG kinase in glucose degradation, whereas KDGK-2 is an inducible 2-keto-3-deoxygalactonate kinase likely involved in d-galactose catabolism.

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.