Skip to main content

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

Wichelecki DJ, Vendiola JAF, Jones AM, Al-Obaidi N, Almo SC, Gerlt JA (2014) Biochemistry, 53, 5692-5699. PMCID: PMC4159206

This study investigates an important question: do physiological roles exist for low-efficiency enzymes? Three homologous, low-efficiency members of the Enolase Superfamily were found to be in the genome neighborhood of in vitro carbohydrate metabolism pathways, but none were found to be physiologically relevant. It is hypothesized that we are observing these low-efficiency enzymes in evolutionary flux: between retirement and repurposing. Enzymes without compelling physiological roles prove to be a challenge in the endeavor of accurate functional annotation. Notably - this investigation also includes the first successful attempt at large-scale ligand screening of transcriptional regulators to gain insight into the substrate of the regulated enzyme. Furthermore, this investigation led to the discovery of a novel L-gulonate catabolic pathway in Chromohalobacter salexigens DSM 3043.


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) Biochemistry 53, 2722-2731]. That study revealed that members of the ManD subgroup vary in substrate specificity and catalytic efficiency: high-efficiency (kcat/KM = 10(3)-10(4) M(-1) s(-1)) for dehydration of d-mannonate, low-efficiency (kcat/KM = 10-10(2) 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.

Link to PubMed »


Abstract Image

Figure 1: Genome neighborhood for CsManD in C. salexigens DSM3043. Carbohydrate metabolism genes are colored green. TRAP transporters are colored red. The GntR transcriptional regulator is colored orange.

Figure 2: Catabolic pathways for d-glucuronate in eubacteria. The pathway hypothesized for degradation of l-gulonate in C. salexigens has red arrows: CsGulDH (alcohol dehydrogenase), CsFR (fructuronate reductase), CsManD (d-mannonate dehydratase), and UxuA (d-mannonate dehydratase). Starting compounds are labeled in blue (d-glucuronate and l-gulonate).

Figure 3: qRT-PCR data for genes in the CsManD operon for cells grown on l-gulonate (top) or d-mannonate (bottom) vs growth on d-glucose. Cells were grown as described in Materials and Methods to an optical density of 0.4–0.5 at 600 nm (early log phase). Upregulation is observed for all genes on both l-gulonate and d-mannonate.

Figure 4: Growth curves for wild-type C. salexigens DSM3043 and various knockout strains. Growth was recorded in M9 minimal medium with 1.7 M NaCl and either l-gulonate (A) or d-mannonate (B) as the sole carbon source. The cultures were grown in triplicate.

Figure 5: Genome neighborhood of the gene cluster in E. coli CFT073. RspA (low-efficiency d-mannonate dehydratase, Uniprot entry Q8FHC7), RspB (l-gulonate dehydrogenase, Uniprot entry Q8FHC8), and RspD (fructuronate reductase, Uniprot entry Q8FHD0) are colored green. The hypothetical metabolite transporter (RspC, Uniprot entry Q8FHC9) is colored red. Hypothetical genes of unknown function are colored orange.

Figure 6: Genome neighborhoods of the previously described l-idonate catabolic pathway and the low-efficiency ManD-containing l-idonate catabolic pathway present in S. enterica subsp. enterica serovar Enteritidis str. P125109.

Figure 7: In vitro catabolic pathway for the consumption of l-idonate present in S. enterica subsp. enterica serovar Enteritidis str. P125109.

Reprinted with permission from Wichelecki et al. Biochemistry 53, 5692-5699. Copyright 2014 American Chemical Society.