E. coli Histidine Triad Nucleotide Binding Protein 1 (ecHinT) Is a Catalytic Regulator of D-Alanine Dehydrogenase (DadA) Activity In Vivo
Abstract
Histidine triad nucleotide binding proteins (Hints) are highly conserved members of the histidine triad (HIT) protein superfamily. Hints comprise the most ancient branch of this superfamily and can be found in Archaea, Bacteria, and Eukaryota. Prokaryotic genomes, including a wide diversity of both Gram-negative and Gram-positive bacteria, typically have one Hint gene encoded by hinT (ycfF in E. coli). Despite their ubiquity, the foundational reason for the wide-spread conservation of Hints across all kingdoms of life remains a mystery. In this study, we used a combination of phenotypic screening and complementation analyses with wild-type and hinT knock-out Escherichia coli strains to show that catalytically active ecHinT is required in E. coli for growth on D-alanine as a sole carbon source. We demonstrate that the expression of catalytically active ecHinT is essential for the activity of the enzyme D-alanine dehydrogenase (DadA) (equivalent to D-amino acid oxidase in eukaryotes), a necessary component of the D-alanine catabolic pathway. Site-directed mutagenesis studies revealed that catalytically active C-terminal mutants of ecHinT are unable to activate DadA activity. In addition, we have designed and synthesized the first cell-permeable inhibitor of ecHinT and demonstrated that the wild-type E. coli treated with the inhibitor exhibited the same phenotype observed for the hinT knock-out strain. These results reveal that the catalytic activity and structure of ecHinT is essential for DadA function and therefore alanine metabolism in E. coli. Moreover, they provide the first biochemical evidence linking the catalytic activity of this ubiquitous protein to the biological function of Hints in Escherichia coli.
Additional Information
© 2011 Bardaweel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: March 11, 2011; Accepted: May 11, 2011; Published: July 6, 2011. We thank Dr. Masayuki Sugawara for his help in conducting the Biolog screening experiments and for his helpful discussions. Funding was provided by the University of Minnesota. No external funding sources for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions. Conceived and designed the experiments: SB MS CRW. Performed the experiments: SB BG TFC. Analyzed the data: SB BG TFC MS CRW. Wrote the paper: SB MS CRW. The authors have declared that no competing interests exist.Attached Files
Published - pone.0020897.pdf
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Supplemental Material - pone.0020897.s003.doc
Supplemental Material - pone.0020897.s004.doc
Supplemental Material - pone.0020897.s005.doc
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Additional details
- PMCID
- PMC3130732
- Eprint ID
- 114033
- Resolver ID
- CaltechAUTHORS:20220323-565437000
- University of Minnesota
- Created
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2022-03-25Created from EPrint's datestamp field
- Updated
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2022-03-25Created from EPrint's last_modified field