Identification of the <i>in Vivo</i> Function of the High-Efficiency d‑Mannonate Dehydratase in <i>Caulobacter crescentus</i> NA1000 from the Enolase Superfamily

The d-mannonate dehydratase (ManD) subgroup of the enolase superfamily contains members with varying catalytic activities (high-efficiency, low-efficiency, or no activity) that dehydrate d-mannonate and/or d-gluconate to 2-keto-3-deoxy-d-gluconate [Wichelecki, D. J., et al. (2014) <i>Biochemistry</i> <i>53</i>, 2722–2731]. Despite extensive <i>in vitro</i> characterization, the <i>in vivo</i> physiological role of a ManD has yet to be established. In this study, we report the <i>in vivo</i> functional characterization of a high-efficiency ManD from <i>Caulobacter crescentus</i> NA1000 (UniProt entry B8GZZ7) by <i>in vivo</i> discovery of its essential role in d-glucuronate metabolism. This <i>in vivo</i> functional annotation may be extended to ∼50 additional proteins

Enzymatic and Structural Characterization of rTSγ Provides Insights into the Function of rTSβ

In humans, the gene encoding a reverse thymidylate synthase (<i>rTS</i>) is transcribed in the reverse direction of the gene encoding thymidylate synthase (<i>TS</i>) that is involved in DNA biosynthesis. Three isoforms are found: α, β, and γ, with the transcript of the α-isoform overlapping with that of <i>TS</i>. rTSβ has been of interest since the discovery of its overexpression in methotrexate and 5-fluorouracil resistant cell lines. Despite more than 20 years of study, none of the rTS isoforms have been biochemically or structurally characterized. In this study, we identified rTSγ as an l-fuconate dehydratase and determined its high-resolution crystal structure. Our data provide an explanation for the observed difference in enzymatic activities between rTSβ and rTSγ, enabling more informed proposals for the possible function of rTSβ in chemotherapeutic resistance

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

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) <i>Biochemistry</i> <i>53</i>, 2722–2731]. That study revealed that members of the ManD subgroup vary in substrate specificity and catalytic efficiency: high-efficiency (<i>k</i>_cat/<i>K</i>_M = 10³–10⁴ M^–1 s^–1) for dehydration of d-mannonate, low-efficiency (<i>k</i>_cat/<i>K</i>_M = 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) <i>Biochemistry 53</i>, 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 <i>Chromohalobacter salexigens</i> DSM3043 as well as cryptic pathways for l-gulonate catabolism in <i>Escherichia coli</i> CFT073 and l-idonate catabolism in <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar <i>Enteritidis</i> 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

Prediction of enzymatic pathways by integrative pathway mapping.

The functions of most proteins are yet to be determined. The function of an enzyme is often defined by its interacting partners, including its substrate and product, and its role in larger metabolic networks. Here, we describe a computational method that predicts the functions of orphan enzymes by organizing them into a linear metabolic pathway. Given candidate enzyme and metabolite pathway members, this aim is achieved by finding those pathways that satisfy structural and network restraints implied by varied input information, including that from virtual screening, chemoinformatics, genomic context analysis, and ligand -binding experiments. We demonstrate this integrative pathway mapping method by predicting the L-gulonate catabolic pathway in Haemophilus influenzae Rd KW20. The prediction was subsequently validated experimentally by enzymology, crystallography, and metabolomics. Integrative pathway mapping by satisfaction of structural and network restraints is extensible to molecular networks in general and thus formally bridges the gap between structural biology and systems biology

Discovery of Function in the Enolase Superfamily: d‑Mannonate and d‑Gluconate Dehydratases in the d‑Mannonate Dehydratase Subgroup

The continued increase in the size of the protein sequence databases as a result of advances in genome sequencing technology is overwhelming the ability to perform experimental characterization of function. Consequently, functions are assigned to the vast majority of proteins via automated, homology-based methods, with the result that as many as 50% are incorrectly annotated or unannotated (Schnoes et al. PLoS Comput. Biol. 2009, 5 (12), e1000605). This manuscript describes a study of the d-mannonate dehydratase (ManD) subgroup of the enolase superfamily (ENS) to investigate how function diverges as sequence diverges. Previously, one member of the subgroup had been experimentally characterized as ManD [dehydration of d-mannonate to 2-keto-3-deoxy-d-mannonate (equivalently, 2-keto-3-deoxy-d-gluconate)]. In this study, 42 additional members were characterized to sample sequence–function space in the ManD subgroup. These were found to differ in both catalytic efficiency and substrate specificity: (1) high efficiency (<i>k</i>_cat/<i>K</i>_M = 10³ to 10⁴ M^–1 s^–1) for dehydration of d-mannonate, (2) low efficiency (<i>k</i>_cat/<i>K</i>_M = 10¹ to 10² M^–1 s^–1) for dehydration of d-mannonate and/or D-gluconate, and 3) no-activity with either d-mannonate or d-gluconate (or any other acid sugar tested). Thus, the ManD subgroup is not isofunctional and includes d-gluconate dehydratases (GlcDs) that are divergent from the GlcDs that have been characterized in the mandelate racemase subgroup of the ENS (Lamble et al. FEBS Lett. 2004, 576, 133–136) (Ahmed et al. Biochem. J. 2005, 390, 529–540). These observations signal caution for functional assignment based on sequence homology and lay the foundation for the studies of the physiological functions of the GlcDs and the promiscuous ManDs/GlcDs