Identification of the Missing Links in Prokaryotic Pentose Oxidation Pathways

Abstract

The pentose metabolism of Archaea is largely unknown. Here, we have employed an integrated genomics approach including DNA microarray and proteomics analyses to elucidate the catabolic pathway for D-arabinose in Sulfolobus solfataricus. During growth on this sugar, a small set of genes appeared to be differentially expressed compared with growth on D-glucose. These genes were heterologously overexpressed in Escherichia coli, and the recombinant proteins were purified and biochemically studied. This showed that D-arabinose is oxidized to 2-oxoglutarate by the consecutive action of a number of previously uncharacterized enzymes, including a D-arabinose dehydrogenase, a D-arabinonate dehydratase, a novel 2-keto-3-deoxy-D-arabinonate dehydratase, and a 2,5-dioxopentanoate dehydrogenase. Promoter analysis of these genes revealed a palindromic sequence upstream of the TATA box, which is likely to be involved in their concerted transcriptional control. Integration of the obtained biochemical data with genomic context analysis strongly suggests the occurrence of pentose oxidation pathways in both Archaea and Bacteria, and predicts the involvement of additional enzyme components. Moreover, it revealed striking genetic similarities between the catabolic pathways for pentoses, hexaric acids, and hydroxyproline degradation, which support the theory of metabolic pathway genesis by enzyme recruitment.Pentose sugars are a ubiquitous class of carbohydrates with diverse biological functions. Ribose and deoxyribose are major constituents of nucleic acids, whereas arabinose and xylose are building blocks of several plant cell wall polysaccharides. Many prokaryotes, as well as yeasts and fungi, are able to degrade these polysaccharides, and use the released five-carbon sugars as a sole carbon and energy source. At present, three main catabolic pathways have been described for pentoses. The first is present in Bacteria and uses isomerases, kinases, and epimerases to convert D-and L-arabinose (Ara) and D-xylose (Xyl) into D-xylulose 5-phosphate (Fig. 1A), which is further metabolized by the enzymes of the phosphoketolase or pentose phosphate pathway. The genes encoding the pentose-converting enzymes are often located in gene clusters in bacterial genomes, for example, the araBAD operon for L-Ara (1), the xylAB operon for D-Xyl (2), and the darK-fucPIK gene cluster for D-Ara (3). The second catabolic pathway for pentoses converts D-Xyl into D-xylulose 5-phosphate as well, but the conversions are catalyzed by reductases and dehydrogenases instead of isomerases and epimerases (Fig. 1B). This pathway is commonly found in yeasts, fungi, mammals, and plants, but also in some bacteria (4 -6). In a third pathway, pentoses such as L-Ara, D-Xyl, D-ribose, and D-Ara are metabolized non-phosphorylatively to either 2-oxoglutarate (2-OG) 4 or to pyruvate and glycolaldehyde (Fig. 1C). The conversion to 2-OG, which is a tricarboxylic acid cycle intermediate, proceeds via the subsequent action of a pentose dehydrogenase, a pentonolactonase, a pentoni...

Metabolism of Pentose Sugars in the Hyperthermophilic Archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius

“…However, the data do not give a good fit to the classical substrate inhibition equation, indicating that other factors may influence the dependence of enzyme velocity on substrate concentration. Interestingly, the purified D-arabinonate dehydratase identified in S. solfataricus is also subject to what appears to be substrate inhibition (8).…”

“…Brouns et al (8) reported that S. solfataricus gene SSO3117 encodes an aldehyde dehydrogenase that, when recombinantly expressed, possessed catalytic activity with 2,5-dioxopentanoate, glyceraldehyde, and glycolaldehyde. We have confirmed this observation, found that the enzyme is specific for NADP ϩ , and report the kinetic parameters for the substrates pentanedial (as an analog of 2,5-dioxopentanoate) and glycolaldehyde (Table 1).…”

“…The genes for two of these three enzymes have been cloned and expressed, and the recombinant enzymes have been characterized. Furthermore, through in vivo metabolite tracer studies, we have shown that D-xylose is partitioned between this newly discovered pathway involving aldol cleavage and subsequent glyoxylate formation, and the pathway was identified for the metabolism of D-arabinose involving direct conversion to 2-oxoglutarate (8).…”

“…2,5-Dioxopentanoate dehydrogenase (2-oxoglutarate semialdehyde dehydrogenase) assays were performed at 70°C in 1 ml of 100 mM HEPES, pH 7.5, as described in Brouns et al (8). The assay mixture contained 1 mM NADP ϩ and 10 mM pentanedial, and the reaction was started by the addition of enzyme.…”

l-Hydroxyproline andd-Proline Catabolism in Sinorhizobium meliloti

“…␣-KGSA dehydrogenase enzymes involved in the catabolism of L-arabinose, D-glucarate, D-galactarate, and hydroxy-L-proline have also been characterized (25,52,53). To investigate whether the reducedgrowth phenotype of the hypH mutant on trans-4-L-Hyp may be due to the presence of other ␣-KGSADH-like enzymes, we searched the S. meliloti genome for other annotated dehydrogenases and identified the most similar as AraE, which functions as an ␣-KGSADH in the catabolism of L-arabinose (18).…”