Investigating the Physiological Roles of Low-Efficiency d-Mannonate and d-Gluconate Dehydratases in the Enolase Superfamily: Pathways for the Catabolism of l-Gulonate and l-Idonate

Abstract

The sequence/function space in the d-mannonate dehydratase subgroup (ManD) of the enolase superfamily was investigated to determine how enzymatic function diverges as sequence identity decreases [Wichelecki, D. J., et al. (2014) Biochemistry53, 2722–2731]. That study revealed that members of the ManD subgroup vary in substrate specificity and catalytic efficiency: high-efficiency (kcat/KM = 103–104 M–1 s–1) for dehydration of d-mannonate, low-efficiency (kcat/KM = 10–102 M–1 s–1) for dehydration of d-mannonate and/or d-gluconate, and no activity. Characterization of high-efficiency members revealed that these are ManDs in the d-glucuronate catabolic pathway {analogues of UxuA [Wichelecki, D. J., et al. (2014) Biochemistry 53, 4087–4089]}. However, the genomes of organisms that encode low-efficiency members of the ManDs subgroup encode UxuAs; therefore, these must have divergent physiological functions. In this study, we investigated the physiological functions of three low-efficiency members of the ManD subgroup and identified a novel physiologically relevant pathway for l-gulonate catabolism in Chromohalobacter salexigens DSM3043 as well as cryptic pathways for l-gulonate catabolism in Escherichia coli CFT073 and l-idonate catabolism in Salmonella enterica subsp. enterica serovar Enteritidis str. P125109. However, we could not identify physiological roles for the low-efficiency members of the ManD subgroup, allowing the suggestion that these pathways may be either evolutionary relics or the starting points for new metabolic potential.

Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes

“…Gul5DH activity was first observed in E. coli in 1980; however, the gene/protein was never identified. 49 Recently, we purified and annotated an authentic Gul5DH from C. salexigens , CsGul5DH; 30 however, CsGul5DH is from a nonorthologous Cog1063 cluster (SeqID 33% to HiGul5DH), although it is part of an analogous pathway for the degradation of l -gulonate.…”

“…The ΔDctP and ΔVanX knockouts in C. salexigens DSM3043 were made using overlap extension PCR as previously described. 30 Primers are given in Table S1 . For disruption of genes in the putative ethanolamine utilization operon in C. salexigens , the genomic region ∼1000 bp upstream and downstream of Csal_0678 and Csal_0679 was amplified from C. salexigens genomic DNA using Pfu Ultra high-fidelity DNA polymerase (Thermo) with primers Csal0678_Fwd and Csal0678_Rev or primers Csal0679_Fwd and Csal0679_Rev ( Table S1 ).…”

“…Gene disruption plasmids pCsal0678KOGm or pCsal0679KOGm were electroporated into E. coli WM6029 (obtained from W. Metcalf at the University of Illinois at Urbana–Champaign), and plasmids were introduced into C. salexigens by biparental mating following established protocols. 30 Cointegrates (single-crossover insertions) were selected by resistance to kanamycin and gentamicin. After selection on 10% sucrose medium, double-crossover events were identified by resistance to gentamicin and sensitivity to kanamycin and confirmed by genomic PCRs.…”

Substrate and metabolic promiscuities of d‐altronate dehydratase family proteins involved in non‐phosphorylative d‐arabinose, sugar acid, l‐galactose and l‐fucose pathways from bacteria

“…C and J). The epimerization of sugar acids at the configuration of the C5 position by the sequential activity of two dehydrogenases also occurs in l‐ gulonate and l‐ idonate metabolism from bacteria, in which d‐ KDGlu and/or d‐ KDGal subsequently enter the semi‐phosphorylative route (Wichelecki et al , ) (Fig. ).…”

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

“…Within their conserved barrel structure, mannonate dehydratases of this family share conserved ligand-binding sites for Mg 2+ , which are essential for the stabilisation of the enediolate intermediate [12]. Among mannonate dehydratases of the enolase superfamily, some representatives with diverse functions have been found, which are involved in the catabolism of sugar acids other than glucuronate or galacturonate [13]. Several crystal structures have been solved for mannonate dehydratases from the enolase superfamily, including enzymes from Chromohalobacter salexigens and Novosphingobium aromaticivorans [12,14].…”