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Title: PLP and GABA trigger GabR-mediated transcription regulation in Bacillus subtilis via external aldimine formation

Abstract

Here, the Bacillus subtilis protein regulator of the gabTD operon and its own gene (GabR) is a transcriptional activator that regulates transcription of gamma-aminobutyric acid aminotransferase (GABA-AT; GabT) upon interactions with pyridoxal-5'-phosphate (PLP) and GABA, and thereby promotes the biosynthesis of glutamate from GABA. We show here that the external aldimine formed between PLP and GABA is apparently responsible for triggering the GabR-mediated transcription activation. Details of the "active site" in the structure of the GabR effector-binding/oligomerization (Eb/O) domain suggest that binding a monocarboxylic.-amino acid such as GABA should be preferred over dicarboxylic acid ligands. A reactive GABA analog, (S)-4-amino-5-fluoropentanoic acid (AFPA), was used as a molecular probe to examine the reactivity of PLP in both GabR and a homologous aspartate aminotransferase (Asp-AT) from Escherichia coli as a control. A comparison between the structures of the Eb/O-PLP-AFPA complex and Asp-AT-PLP-AFPA complex revealed that GabR is incapable of facilitating further steps of the transamination reaction after the formation of the external aldimine. Results of in vitro and in vivo assays using full-length GabR support the conclusion that AFPA is an agonistic ligand capable of triggering GabR-mediated transcription activation via formation of an external aldimine with PLP.

Authors:
 [1];  [2];  [3];  [4];  [5];  [6];  [7];  [4];  [8];  [9];  [7]
  1. Loyola Univ. Chicago, Chicago, IL (United States); Weill Cornell Medical College, New York, NY (United States)
  2. Argonne National Lab. (ANL), Lemont, IL (United States)
  3. Tufts Univ. School of Medicine, Boston, MA (United States)
  4. Northwestern Univ., Evanston, IL (United States)
  5. Northwestern Univ., Evanston, IL (United States); Univ. of Mississippi School of Pharmacy, University, MS (United States)
  6. Brandeis Univ., Waltham, MA (United States); Columbia Univ., New York, NY (United States)
  7. Loyola Univ. Chicago, Chicago, IL (United States)
  8. Brandeis Univ., Waltham, MA (United States); Weill Cornell Medical College, New York, NY (United States)
  9. Brandeis Univ., Waltham, MA (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
National Institutes of Health (NIH); USDOE Office of Science (SC), Basic Energy Sciences (BES)
OSTI Identifier:
1373733
Grant/Contract Number:  
AC02-06CH11357
Resource Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 114; Journal Issue: 15; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; MocR; GabR; PLP; external aldimine; transcription activation

Citation Formats

Wu, Rui, Sanishvili, Ruslan, Belitsky, Boris R., Juncosa, Jose I., Le, Hoang V., Lehrer, Helaina J. S., Farley, Michael, Silverman, Richard B., Petsko, Gregory A., Ringe, Dagmar, and Liu, Dali. PLP and GABA trigger GabR-mediated transcription regulation in Bacillus subtilis via external aldimine formation. United States: N. p., 2017. Web. doi:10.1073/pnas.1703019114.
Wu, Rui, Sanishvili, Ruslan, Belitsky, Boris R., Juncosa, Jose I., Le, Hoang V., Lehrer, Helaina J. S., Farley, Michael, Silverman, Richard B., Petsko, Gregory A., Ringe, Dagmar, & Liu, Dali. PLP and GABA trigger GabR-mediated transcription regulation in Bacillus subtilis via external aldimine formation. United States. doi:10.1073/pnas.1703019114.
Wu, Rui, Sanishvili, Ruslan, Belitsky, Boris R., Juncosa, Jose I., Le, Hoang V., Lehrer, Helaina J. S., Farley, Michael, Silverman, Richard B., Petsko, Gregory A., Ringe, Dagmar, and Liu, Dali. Mon . "PLP and GABA trigger GabR-mediated transcription regulation in Bacillus subtilis via external aldimine formation". United States. doi:10.1073/pnas.1703019114. https://www.osti.gov/servlets/purl/1373733.
@article{osti_1373733,
title = {PLP and GABA trigger GabR-mediated transcription regulation in Bacillus subtilis via external aldimine formation},
author = {Wu, Rui and Sanishvili, Ruslan and Belitsky, Boris R. and Juncosa, Jose I. and Le, Hoang V. and Lehrer, Helaina J. S. and Farley, Michael and Silverman, Richard B. and Petsko, Gregory A. and Ringe, Dagmar and Liu, Dali},
abstractNote = {Here, the Bacillus subtilis protein regulator of the gabTD operon and its own gene (GabR) is a transcriptional activator that regulates transcription of gamma-aminobutyric acid aminotransferase (GABA-AT; GabT) upon interactions with pyridoxal-5'-phosphate (PLP) and GABA, and thereby promotes the biosynthesis of glutamate from GABA. We show here that the external aldimine formed between PLP and GABA is apparently responsible for triggering the GabR-mediated transcription activation. Details of the "active site" in the structure of the GabR effector-binding/oligomerization (Eb/O) domain suggest that binding a monocarboxylic.-amino acid such as GABA should be preferred over dicarboxylic acid ligands. A reactive GABA analog, (S)-4-amino-5-fluoropentanoic acid (AFPA), was used as a molecular probe to examine the reactivity of PLP in both GabR and a homologous aspartate aminotransferase (Asp-AT) from Escherichia coli as a control. A comparison between the structures of the Eb/O-PLP-AFPA complex and Asp-AT-PLP-AFPA complex revealed that GabR is incapable of facilitating further steps of the transamination reaction after the formation of the external aldimine. Results of in vitro and in vivo assays using full-length GabR support the conclusion that AFPA is an agonistic ligand capable of triggering GabR-mediated transcription activation via formation of an external aldimine with PLP.},
doi = {10.1073/pnas.1703019114},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 15,
volume = 114,
place = {United States},
year = {2017},
month = {3}
}

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