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

Journal Article · · Biochemistry
DOI:https://doi.org/10.1021/bi501388y· OSTI ID:1178823
 [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
+ Show Author Affiliations

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 an integrated strategy for realizing the full value of amassing genome sequence data.

Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Organization:
National Institutes of Health (NIH); USDOE Office of Science (SC)
Grant/Contract Number:
U54GM093342; U54GM094662; AC02-06CH11357
OSTI ID:
1178823
Journal Information:
Biochemistry, Vol. 54, Issue 3; ISSN 0006-2960
Publisher:
American Chemical Society (ACS)Copyright Statement
Country of Publication:
United States
Language:
ENGLISH
Citation Metrics:
Cited by: 74 works
Citation information provided by
Web of Science

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Cited By (11)

Converting a Periplasmic Binding Protein into a Synthetic Biosensing Switch through Domain Insertion journal January 2019
Evolution of cyclohexadienyl dehydratase from an ancestral solute-binding protein journal April 2018
Structural and biochemical characterization of Rv0187, an O-methyltransferase from Mycobacterium tuberculosis journal May 2019
Radical-mediated C-S bond cleavage in C2 sulfonate degradation by anaerobic bacteria journal April 2019
Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars journal July 2016
The substrate-binding protein in bacterial ABC transporters: dissecting roles in the evolution of substrate specificity journal October 2015
Uneven distribution of cobamide biosynthesis and dependence in bacteria predicted by comparative genomics journal November 2018
Organic matter processing by microbial communities throughout the Atlantic water column as revealed by metaproteomics journal December 2017
Linking Bacillus cereus Genotypes and Carbohydrate Utilization Capacity journal June 2016
Tripartite ATP-Independent Periplasmic (TRAP) Transporters and Tripartite Tricarboxylate Transporters (TTT): From Uptake to Pathogenicity journal February 2018
Genomic Reconstruction of Carbohydrate Utilization Capacities in Microbial-Mat Derived Consortia journal July 2017

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