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

Abstract

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...

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

“…Hence, the reaction catalyzed by either ferredoxin-dependent glyceraldehyde-3-phosphate:ferredoxin oxidoreductase (GAPOR) or NAD(P) ϩ -dependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) is irreversible in Archaea. Only mesophilic extreme halophiles growing at the expense of sugars rely on the GAPDH/PGK couple for GAP oxidation in the modified EMP and ED pathways utilized for fructose and glucose degradation, respectively (70,123,152). Also, in most methanogens, neither GAPOR nor GAPN has been identified, which might reflect the inability of most methanogenic species to degrade sugars (36,153).…”

“…volcanii, also represents a PFK-B member (123). Enzyme characterization revealed an ϳ70-kDa homodimeric structure (38-kDa subunits), and sequence comparison showed considerable sequence identities with E. coli 1-PFK (FruK) (124).…”

“…Although these archaeal-type class I enzymes have been reported for most Archaea, including many extreme halophiles, and therefore might represent the typical archaeal FBPA (126,137), for Haloferax species as well as some other haloarchaea, class II enzymes have been reported and characterized in detail (123,132). These halophilic class II aldolases represent the only archaeal class II aldolases known so far.…”

“…In accordance with the E. coli enzyme, the halophilic class II aldolases are 100-kDa homodimers (50-kDa subunits), share the conserved residues involved in catalysis, and were described to prefer Fe 2ϩ or Mn 2ϩ instead of Zn 2ϩ as a bivalent metal ion. Phylogenetic analyses revealed that the haloarchaeal sequences form a distinct third subfamily within the class II FBPA family, and it has been speculated that haloarchaea acquired these enzymes from Bacteria in a horizontal gene transfer event (123,132). The archaeal class I aldolases have been proposed to represent early descendants of an ancient lineage separated very early from the lineages of classical class I and class II aldolases (126).…”

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

“…It seems that these organisms use the PTS mainly to transport and phosphorylate fructose (3,17) or to phosphorylate dihydroxyacetone (18). A previous deduction that archaea are devoid of any PTS is therefore no longer valid (6).…”