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