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Title: Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes

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. Here in this paper, 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 anmore » integrated strategy for realizing the full value of amassing genome sequence data.« less
Authors:
 [1] ;  [1] ;  [2] ;  [3] ;  [1] ;  [4] ;  [4] ;  [3] ;  [5] ;  [3] ;  [6] ;  [1] ;  [1] ;  [1] ;  [5] ;  [7] ;  [8] ;  [2] ;  [9] ;  [1]
  1. Albert Einstein College of Medicine, Bronx, NY (United States). Dept. of Biochemistry
  2. Univ. of California, San Francisco, CA (United States). Dept. of Pharmaceutical Chemistry
  3. Univ. of Illinois, Urbana-Champaign, IL (United States). Inst. for Genomic Biology
  4. Univ. of Illinois, Urbana-Champaign, IL (United States). Inst. for Genomic Biology, Dept. of Biochemistry
  5. Griffith Univ., Brisbane, QLD (Australia). Eskitis Inst. for Drug Discovery
  6. Sanford-Burnham Medical Research Inst., La Jolla, CA (United States); Russian Academy of Sciences (RAS), Moscow (Russian Federation). A.A.Kharkevich Inst. for Information Transmission Problems
  7. Sanford-Burnham Medical Research Inst., La Jolla, CA (United States)
  8. Univ. of Illinois, Urbana-Champaign, IL (United States). Dept. of Biochemistry
  9. Univ. of Illinois, Urbana-Champaign, IL (United States). Inst. for Genomic Biology, Dept. of Biochemistry and Dept. of Chemistry
Publication Date:
OSTI Identifier:
1178823
Grant/Contract Number:
U54GM093342; U54GM094662; AC02-06CH11357
Type:
Accepted Manuscript
Journal Name:
Biochemistry
Additional Journal Information:
Journal Volume: 54; Journal Issue: 3; Journal ID: ISSN 0006-2960
Publisher:
American Chemical Society (ACS)
Research Org:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org:
National Institutes of Health (NIH); USDOE Office of Science (SC)
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY