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Identification of the in vivo function of the high-efficiency D-mannonate dehydratase in Caulobacter crescentus NA1000 from the enolase superfamily

Wichelecki DJ, Graff DC, Al-Obaidi N, Almo SC, Gerlt JA (2014) Biochemistry 53, 4087-4089 PMCID: PMC4082379

In line with the EFI practice of verifying in vitro activities with in vivo functions, Enolase Bridging Project researchers follow up on the discovery of a novel enolase activity with studies that confirm a physiological role for the enzyme in microbial glucuronate metabolism. This study also describes the first implementation of Thermofluor screening against transcriptional regulators in an attempt to understand regulation of a pathway.


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) Biochemistry 53, 2722–2731]. Despite extensive in vitro characterization, the in vivo physiological role of a ManD has yet to be established. In this study, we report the in vivo functional characterization of a high-efficiency ManD from Caulobacter crescentus NA1000 (UniProt entry B8GZZ7) by in vivo discovery of its essential role in d-glucuronate metabolism. This in vivo functional annotation may be extended to 50 additional proteins.

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Abstract Image

Figure 1: Degradation pathway of d-glucuronate in Escherichia coli. The dehydration of d-mannonate to 2-keto-3-deoxy-d-gluconate is performed by UxuA.

Figure 2: Genome neighborhoods of B8GZZ7, a high-efficiency ManD, (top) and canonical d-glucuronate catabolism genes (bottom). The genes directly involved in d-glucuronate metabolism are colored green.

Figure 3: Upregulation of the genes encoding B8GZZ7 and the d-glucuronate catabolism genes shown in Figure 2.

Reprinted with permission from Wichelecki et al. Biochemistry 53, 4087-4089. Copyright 2014 American Chemical Society.