Experimental strategies for functional annotation and metabolism discovery

Targeted screening of solute binding proteins and unbiased panning of metabolomes

Matthew W. Vetting, Nawar Al-Obaidi, Suwen Zhao, Brian San Francisco, Jungwook Kim, Daniel J. Wichelecki, Jason T. Bouvier, Jose O. Solbiati, Hoan Vu, Xinshuai Zhang, Dmitry A. Rodionov, James D. Love, Brandan S. Hillerich, Ronald D. Seidel, Ronald J. Quinn, Andrei L. Osterman, John E Cronan, Matthew P. Jacobson, John Alan Gerlt, Steven C. Almo

Research output: Contribution to journalArticle

Abstract

The rate at which genome sequencing data is accruing demands enhanced methods for functional annotation and metabolism discovery. Solute binding proteins (SBPs) facilitate the transport of the first reactant in a metabolic pathway, thereby constraining the regions of chemical space and the chemistries that must be considered for pathway reconstruction. We describe high-throughput protein production and differential scanning fluorimetry platforms, which enabled the screening of 158 SBPs against a 189 component library specifically tailored for this class of proteins. Like all screening efforts, this approach is limited by the practical constraints imposed by construction of the library, i.e., we can study only those metabolites that are known to exist and which can be made in sufficient quantities for experimentation. To move beyond these inherent limitations, we illustrate the promise of crystallographic- and mass spectrometric-based approaches for the unbiased use of entire metabolomes as screening libraries. Together, our approaches identified 40 new SBP ligands, generated experiment-based annotations for 2084 SBPs in 71 isofunctional clusters, and defined numerous metabolic pathways, including novel catabolic pathways for the utilization of ethanolamine as sole nitrogen source and the use of d-Ala-d-Ala as sole carbon source. These efforts begin to define an integrated strategy for realizing the full value of amassing genome sequence data. (Graph Presented).

Original languageEnglish (US)
Pages (from-to)909-931
Number of pages23
JournalBiochemistry
Volume54
Issue number3
DOIs
StatePublished - Jan 27 2015

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Metabolome
Metabolism
Carrier Proteins
Screening
Libraries
Metabolic Networks and Pathways
Genes
Genome
Fluorometry
Ethanolamine
Protein Transport
Metabolites
Proteins
Nitrogen
Carbon
Throughput
Ligands
Scanning
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Experimental strategies for functional annotation and metabolism discovery : Targeted screening of solute binding proteins and unbiased panning of metabolomes. / Vetting, Matthew W.; Al-Obaidi, Nawar; Zhao, Suwen; San Francisco, Brian; Kim, Jungwook; Wichelecki, Daniel J.; Bouvier, Jason T.; Solbiati, Jose O.; Vu, Hoan; Zhang, Xinshuai; Rodionov, Dmitry A.; Love, James D.; Hillerich, Brandan S.; Seidel, Ronald D.; Quinn, Ronald J.; Osterman, Andrei L.; Cronan, John E; Jacobson, Matthew P.; Gerlt, John Alan; Almo, Steven C.

In: Biochemistry, Vol. 54, No. 3, 27.01.2015, p. 909-931.

Research output: Contribution to journalArticle

Vetting, MW, Al-Obaidi, N, Zhao, S, San Francisco, B, Kim, J, Wichelecki, DJ, Bouvier, JT, Solbiati, JO, Vu, H, Zhang, X, Rodionov, DA, Love, JD, Hillerich, BS, Seidel, RD, Quinn, RJ, Osterman, AL, Cronan, JE, Jacobson, MP, Gerlt, JA & Almo, SC 2015, 'Experimental strategies for functional annotation and metabolism discovery: Targeted screening of solute binding proteins and unbiased panning of metabolomes', Biochemistry, vol. 54, no. 3, pp. 909-931. https://doi.org/10.1021/bi501388y
Vetting, Matthew W. ; Al-Obaidi, Nawar ; Zhao, Suwen ; San Francisco, Brian ; Kim, Jungwook ; Wichelecki, Daniel J. ; Bouvier, Jason T. ; Solbiati, Jose O. ; Vu, Hoan ; Zhang, Xinshuai ; Rodionov, Dmitry A. ; Love, James D. ; Hillerich, Brandan S. ; Seidel, Ronald D. ; Quinn, Ronald J. ; Osterman, Andrei L. ; Cronan, John E ; Jacobson, Matthew P. ; Gerlt, John Alan ; Almo, Steven C. / Experimental strategies for functional annotation and metabolism discovery : Targeted screening of solute binding proteins and unbiased panning of metabolomes. In: Biochemistry. 2015 ; Vol. 54, No. 3. pp. 909-931.
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