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

Abstract

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 C 4 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 ⌬gad 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. IMPORTANCEThe 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. Glucose degradation in thermoacidophilic and halophilic archaea has been proposed to proceed via modified versions of the classical Entner-Doudoroff (ED) pathway of bacteria (1, 2) (Fig. 1). For the thermoacidophilic euryarchaeon Picrophilus torridus, a nonphosphorylative ED (npED) pathway was reported (3). Accordingly, glucose is converted to 2-keto-3-deoxygluconate (KDG) via glucose dehydrogenase (GDH) and gluconate dehydratase (GAD). KDG is then cleaved by a KDG aldolase to pyruvate and glyceraldehyde (GA), which is converted to a second molecule of pyruvate via glyceraldehyde dehydrogenase, 2-phosphoglycerate forming glycerate kinase, enolase, and pyruvate kinase. The pathway was proposed to be promiscuous, being involved in the degradation of both D-glucose and its C 4 epimer ...

Two distinct glyceraldehyde‐3‐phosphate dehydrogenases in glycolysis and gluconeogenesis in the archaeon Haloferax volcanii

“…Growth analyses of H. volcanii strain H26 and knock‐out mutants (Table S2) were performed aerobically at 42 °C in synthetic medium containing either 25 m m d ‐glucose or d ‐fructose or sodium‐pyruvate or 40 m m sodium‐acetate or 1% casaminoacids supplemented with 50 μ m uracil . For complementation experiments, the knock‐out mutants were transformed with pTA963 carrying the respective genes under the control of a tryptophanase promoter (see below, overexpression section).…”

“…The haloarchaea Haloferax volcanii and Haloarcula marismortui have been shown to degrade glucose and fructose via the semiphosphorylative ED pathway and modified EM pathway respectively. Both pathways involve GAP as an intermediate . In Haloarcula vallismortis , a phosphorylating NAD + dependent GAPDH has been characterised; however, its function in sugar metabolism has not been defined .…”

l‐Rhamnose catabolism in archaea

“…(Q1NEI8), Sulfobacillus thermosulfidooxidans (A0A1W1WQH4), Azotobacter vinelandii (C1DMY1); l ‐fuconate dehydratases: Xanthomonas campestris (Q8P3K2), Xenopus laevis (Q6INX4), Homo sapiens (Q7L5Y1); mandelate racemases (MR): Pseudomonas putida (P11444), Bradyrhizobium diazoefficiens (Q89D20), Paraburkholderia xenovorans (Q13JD6). For accession numbers of gluconate/xylonate dehydratase and enolase sequences see in Sutter et al ().…”

“…Haloferax volcanii H26 and deletion mutants grew aerobically at 42°C in 100 ml Erlenmeyer flasks containing 20 ml of synthetic medium (Sutter et al , ) supplemented with 20 mM l ‐rhamnose, d ‐glucose or 1% casamino acids. Due to the chromosomal deletion of the orotate phosphoribosyltransferase gene ( pyrE2 ) in H26, medium was supplemented with 50 µg ml –1 uracil.…”

Sucrose Metabolism in Haloarchaea: Reassessment Using Genomics, Proteomics, and Metagenomics

“…In Haloarcula vallismortis, Haloarcula marismortui, and Haloferax mediterranei glucose and fructose are mostly degraded by different pathways: glucose is degraded by the semiphosphorylative Entner-Doudoroff (spED) pathway, and fructose is degraded by a modified Embden-Meyerhof-Parnas (EMP) pathway (6)(7)(8)(9)(10)(11)(12). These haloarchaeal glycolytic pathways are variants of the classical Entner-Doudoroff (ED) and EMP pathways, respectively (10,(12)(13)(14)(15). In the haloarchaeal spED pathway, oxidation precedes phosphorylation: glucose is oxidized to gluconate (rather than being phosphorylated), and the phosphorylation step is deferred to later in the pathway using 3-deoxy-2-oxo-D-gluconate (KDG) as the substrate.…”