Abstract
Mannonate dehydratases catalyse the dehydration reaction from mannonate to 2-keto-3-deoxygluconate as part of the hexuronic acid metabolism in bacteria. Bacterial mannonate dehydratases present in this gene cluster usually belong to the xylose isomerase-like superfamily, which have been the focus of structural, biochemical and physiological studies. Mannonate dehydratases from archaea have not been studied in detail. Here, we identified and characterised the first archaeal mannonate dehydratase (TaManD) from the thermoacidophilic archaeon Thermoplasma acidophilum. The recombinant TaManD enzyme was optimally active at 65 °C and showed high specificity towards D-mannonate and its lactone, D-mannono-1,4-lactone. The gene encoding for TaManD is located adjacent to a previously studied mannose-specific aldohexose dehydrogenase (AldT) in the genome of T. acidophilum. Using nuclear magnetic resonance (NMR) spectroscopy, we showed that the mannose-specific AldT produces the substrates for TaManD, demonstrating the possibility for an oxidative metabolism of mannose in T. acidophilum. Among previously studied mannonate dehydratases, TaManD showed closest homology to enzymes belonging to the xylose isomerase-like superfamily. Genetic analysis revealed that closely related mannonate dehydratases among archaea are not located in a hexuronate gene cluster like in bacteria, but next to putative aldohexose dehydrogenases, implying a different physiological role of mannonate dehydratases in those archaeal species.
Highlights
Introduction2-keto-3-deoxygluconate (KDG) and have been studied as part of the hexuronic acid gene cluster in several organisms, such as Escherichia coli, Bacillus stearothermophilus, Bacillus subtilis and Erwinia chrysanthemi [1,2,3,4]
Mannonate dehydratases (EC 4.2.1.8) catalyse the conversion of mannonate to2-keto-3-deoxygluconate (KDG) and have been studied as part of the hexuronic acid gene cluster in several organisms, such as Escherichia coli, Bacillus stearothermophilus, Bacillus subtilis and Erwinia chrysanthemi [1,2,3,4]
We identified the protein product of the Ta0753 gene as a functional mannonate dehydratase from T. acidophilum by heterologous expression in E. coli, and compared its properties and genomic context to other previously studied mannonate dehydratases
Summary
2-keto-3-deoxygluconate (KDG) and have been studied as part of the hexuronic acid gene cluster in several organisms, such as Escherichia coli, Bacillus stearothermophilus, Bacillus subtilis and Erwinia chrysanthemi [1,2,3,4]. The hexuronate gene cluster encodes enzymes involved in the metabolism of glucuronate and galacturonate [5]. Glucuronate is a common sugar acid present in glucuronoxylan, a constituent of plant cell walls which can serve as the only carbon source for growth of some bacteria [3,6,7]. Glucuronate is present in the mucus layer of mammals, providing a carbon source for anaerobic gut bacteria, such as E. coli [8,9]. As part of the hexuronate metabolism, mannonate dehydratase converts mannonate to KDG (Figure 1A). In E. coli, and some species of Erwinia, KDG is phosphorylated and cleaved into pyruvate and 3-phosphoglycerate, which can be further metabolised in the tricarboxylic acid cycle, or the Entner-Doudoroff pathway [4,5]
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