Novel Xylose Dehydrogenase in the Halophilic Archaeon Haloarcula marismortui

Abstract

During growth of the halophilic archaeon Haloarcula marismortui on D-xylose, a specific D-xylose dehydrogenase was induced. The enzyme was purified to homogeneity. It constitutes a homotetramer of about 175 kDa and catalyzed the oxidation of xylose with both NADP ؉ and NAD ؉ as cosubstrates with 10-fold higher affinity for NADP ؉ . In addition to D-xylose, D-ribose was oxidized at similar kinetic constants, whereas D-glucose was used with about 70-fold lower catalytic efficiency (k cat /K m ). With the N-terminal amino acid sequence of the subunit, an open reading frame (ORF)-coding for a 39.9-kDA protein-was identified in the partially sequenced genome of H. marismortui. The function of the ORF as the gene designated xdh and coding for xylose dehydrogenase was proven by its functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione, and substrates. Xylose dehydrogenase showed the highest sequence similarity to glucose-fructose oxidoreductase from Zymomonas mobilis and other putative bacterial and archaeal oxidoreductases. Activities of xylose isomerase and xylulose kinase, the initial reactions of xylose catabolism of most bacteria, could not be detected in xylose-grown cells of H. marismortui, and the genes that encode them, xylA and xylB, were not found in the genome of H. marismortui. Thus, we propose that this first characterized archaeal xylose dehydrogenase catalyzes the initial step in xylose degradation by H. marismortui.The utilization of sugars, in particular of hexoses and hexose polymers and-to a lesser extent-of pentoses, has been reported for various species in the domain Archaea. So far, only the catabolic pathways of hexoses and glucose polymers (e.g., maltose and starch) have been studied in detail in particular in hyperthermophilic, thermoacidophilic, and extremely halophilic archaea. Comparative analyses of glucose degradation pathways in these organisms revealed that the classical Embden-Meyerhof-(EM) or Entner-Doudoroff-(ED) pathway found in bacteria is not operative in archaea; they use instead modified versions of these pathways as follows (for reviews, see references 31 and 41). In hyperthermophilic eury-and crenarchaeota, glucose degradation proceeds predominantly via modified EM pathways, which differ from the classical EM pathway by the presence of several unusual glucokinases (ADP or ATP dependent) and 6-phosphofructokinases (ADP, ATP, or PP i dependent), novel enzymes of glucose-6-phosphate isomerization and of glyceraldehyde-3-phosphate oxidation, and pyruvate kinases with reduced regulatory potential (15,18,41).In thermoacidophilic archaea, Sulfolobus and Thermoplasma spp., glucose is degraded via a nonphosphorylated version of the ED pathway (22,31,41) by which glucose is oxidized to glycerate via the nonphosphorylated intermediates gluconate and 2-keto-3-deoxygluconate (KDG) involving glucose dehydrogenase, gluconate dehydrata...

Identification of the Missing Links in Prokaryotic Pentose Oxidation Pathways

“…In the halophile Haloarcula marismortui, a gene cluster was found on chromosome I that seems to contain all of the necessary components for D-Xyl oxidation, including a gene that has been identified as a D-Xyl dehydrogenase (19) (Fig. 5A).…”

“…solfataricus is a model archaeon for studying metabolism and information processing systems, such as transcription, translation, and DNA replication (37,38). Several halophilic and thermophilic Archaea have been reported to assimilate pentose sugars, but neither the catabolic pathways for these 5-carbon sugars nor the majority of its enzymes are known (17,19). To close this knowledge gap, we have studied the growth of S. solfataricus on the pentose D-Ara using a multidisciplinary genomics approach, and compared the results to growth on the hexose D-Glu.…”

“…Although the catabolism of hexoses is well studied (reviewed in Ref. 18), the pathways for pentose degradation have neither been established in Sulfolobus solfataricus, nor in any other member of the Archaea (19).…”

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

“…Also, glycolaldehyde is shuttled into the CAC after oxidation to glycoxylate via glycolate and further conversion to malate via malate synthase. In Archaea, pentose utilization has been reported for some aerobic halophiles and for Sulfolobus species (14,283,287,291,343,345). For other Archaea, including hyperthermophiles from the orders Thermococcales, Archaeoglobales, Thermoproteales, Desulfurococcales, and Pyrodictyales, no growth on pentoses has been reported, although many of these organisms are able to grow with hexoses or hexose polymers as carbon and energy sources (14).…”

Genetic Analysis of a Novel Pathway for d -Xylose Metabolism in Caulobacter crescentus

“…A few bacterial species have been shown to express XDH activity (3,6,28,30), but only one dehydrogenase with high specificity for D-xylose has been identified genetically (14), in the halophilic archeon Haloarcula marismortui. A pairwise BLAST comparison identified no significant similarity between the H. marismortui XDH and the C. crescentus XylB polypeptide sequences.…”