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Characterisation of the First Archaeal Mannonate Dehydratase from Thermoplasma Acidophilum and Its Potential Role in the Catabolism of D-Mannose

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Characterisation of the First Archaeal Mannonate Dehydratase from Thermoplasma Acidophilum and Its Potential Role in the Catabolism of D-Mannose catalysts Article Characterisation of the First Archaeal Mannonate Dehydratase from Thermoplasma acidophilum and Its Potential Role in the Catabolism of D-Mannose Dominik Kopp 1, Robert Willows 1,2 and Anwar Sunna 1,2,* 1 Department of Molecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia; [email protected] (D.K.); [email protected] (R.W.) 2 Biomolecular Discovery and Design Research Centre, Macquarie University, Sydney, New South Wales 2109, Australia * Correspondence: [email protected]; Tel.: +61-2-9850-4220 Received: 13 February 2019; Accepted: 24 February 2019; Published: 3 March 2019 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. Keywords: mannonate dehydratase; mannose metabolism; Thermoplasma acidophilum; mannono-1,4-lactone; 2-keto-3-deoxygluconate; aldohexose dehydrogenase 1. Introduction Mannonate dehydratases (EC 4.2.1.8) catalyse the conversion of mannonate to 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 [1–4]. 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 also 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 (Figure1A). 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]. Catalysts 2019, 9, 234; doi:10.3390/catal9030234 www.mdpi.com/journal/catalysts Catalysts 2019, 9, x FOR PEER REVIEW 2 of 14 Catalysts 2019, 9, 234 2 of 14 Mannonate dehydratases are represented in different enzyme families, such as the xylose isomeraseMannonate-like superfamily dehydratases or arethe representedenolase superfamily. in different Mannonate enzyme dehydratases families, such encoded as the xylosein the isomerase-likebacterial hexuronate superfamily gene clusters or the usually enolase belong superfamily. to the xylose Mannonate isomerase dehydratases-like superfamily. encoded Crystal in thestructures bacterial have hexuronate been solved gene for clusters xylose isomerase usually belong-like mannonate to the xylose dehydratases isomerase-like from superfamily.Streptococcus Crystalsuis, E. coli structures K12 and haveEnterococcus been solved faecalis forin native xylose form isomerase-like and in complex mannonate with Mn2+ dehydratases ions [10,11]. His311 from Streptococcusand Tyr325 suisin the, E. binding coli K12 pock andetEnterococcus were identified faecalis asin crucial native for form the and activity in complex of the withmannonate Mn2+ ionsdehydratases [10,11]. His311 from andGram Tyr325-positive in the bacteria binding, such pocket as S. were suis. identifiedHowever, asin crucialGram-negative for the activity bacteria of (e.g. the mannonateE. coli K12) dehydratases, an additional from inserted Gram-positive sequence in bacteria, the binding such aspocketS. suis rende. However,red the in dehydratase Gram-negative less bacteriaactive [11]. (e.g., MembersE. coli K12), of the an additionalenolase superfamily inserted sequence show a in conserved the binding structure pocket renderedand reaction the dehydratasemechanism, lessbut activediffer [in11 ].their Members physiological of the enolase functions. superfamily Within their show conserved a conserved barrel structure structure, and reactionmannonate mechanism, dehydratases but differ of this in their family physiological share conserved functions. ligand Within-bind theiring conservedsites for Mg barrel2+, which structure, are mannonateessential for dehydratases the stabilisation of thisof the family enediolate share intermediate conserved ligand-binding [12]. Among mannonate sites for Mg dehydratases2+, which are of essentialthe enolase for superfamily, the stabilisation some of therepresentatives enediolate intermediate with diverse [ 12functions]. Among have mannonate been found dehydratases, which are ofinvolved the enolase in the superfamily, catabolism some of sugar representatives acids other with than diverse glucuronate functions or havegalacturonate been found, [13]. which Several are involvedcrystal structures in the catabolism have been of sugar solved acids for other mannonate than glucuronate dehydratases or galacturonate from the enolase [13]. Several superfamily crystal, structuresincluding haveenzymes been from solved Chromohalobacter for mannonate salexigens dehydratases and fromNovosphingobium the enolase superfamily,aromaticivorans including [12,14]. enzymesHowever, from to theChromohalobacter best of our knowledge, salexigens and no archaealNovosphingobium mannonate aromaticivorans dehydratase[12 has,14 ].been However, investigated to the bestso far. of our knowledge, no archaeal mannonate dehydratase has been investigated so far. Here,Here, wewe usedused genomicgenomic analysisanalysis toto identifyidentify thethe firstfirst functionalfunctional mannonatemannonate dehydratasedehydratase fromfrom thethe thermoacidophilicthermoacidophilic archaeon archaeonThermoplasma Thermoplasma acidophilum acidophilum(TaManD). (TaMan The archaealD). The mannonate archaeal dehydratasemannonate wasdehydratase recombinantly-expressed was recombinantly in E.- coliexpressed, purified in and E. functionally coli, purified characterised. and functionally TaManD characterised. showed high aminoTaManD acid showed sequence high identity amino toacid bacterial sequence mannonate identity to dehydratases bacterial mannonate from the dehydratases xylose isomerase-like from the superfamily.xylose isomerase In the-like genome superfamily. of T. acidophilum In the genome, the geneof T. acidophilum encoding for, the TaManD gene encoding is located for adjacent TaManD to is anlocated aldohexose adjacent dehydrogenase to an aldohexose (AldT), dehydrogenase which has been (AldT), shown which previously has been to catalyse shown thepreviously oxidation to ofcatalyse mannose the tooxidation mannonate of withmannose high to specificity mannonate [15, 16with]. However, high specificity the physiological [15,16]. However, significance the ofphysiological the oxidation sig andnificance its products of the wereoxidation not investigated and its products further. were We identifiednot investigated the products further. of We an AldT-mediatedidentified the products oxidation of of an D-mannose AldT-mediated using NMRoxidation spectroscopy of D-mannose and confirmed using NMR that spectroscopy TaManD is able and toconfirmed convert the that products TaManD to is KDG. able Thisto convert demonstrates the products that in to principle, KDG. This a mannosedemonstrates metabolism that in principle, based on AldTa mannose and ManD metabolism is possible based in T.on acidophilum AldT and ManD(Figure is1 B).possible in T. acidophilum (Figure 1B). A UxaC UxuB UxuA KdgK glucuronate fructuronate mannonate KDG KDPG galucturonate tagaturonate altronate UxaC UxaB UxaA B CCM AldT D-mannonate UxuA KDGA D-mannose KDG CCM D-mannono- 1,4-lactone FigureFigure 1.1.Hexuronate Hexuronate metabolism metabolism in inE. E. coli coliand and possible possible mannose mannose catabolism catabolism in T. in acidophilum T. acidophilum.(A) Role. (A) ofRoleuxuA ofmannnonate uxuA mannnonate dehdyratase dehdyratase in dissimilation in dissimilation of hexuronates of hexuronates in E. coli adapted in E. from coli Peekhausadapted from and ConwayPeekhaus [8 ].and (B) RoleConway of uxuA [8].mannonate (B) Role dehydrataseof uxuA mannonate in a possible dehydratase mannose metabolism in a possible in T. acidophilum mannose. UxaC:metabolism hexuronate in T. acidophilum isomerase, UxuB:. UxaC: mannonate hexuronate
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