The number of newly reported protein sequences inferred from genome sequencing continues to grow at a rate that severely outpaces the assignment of function through comparative genomics or direct biochemical analysis. This situation results in a large proportion of unannotated and misannotated proteins sequence, precluding the discovery of novel enzymes activities and metabolic pathways important to 1) understanding the contributions of the gut microbiome to human health, 2) the realization of new chemical processes for industry and 3) our understanding of critical environmental issues, including global nutrient cycles and the evolution of complex microbial communities. Accordingly, the development of strategies, tools and infrastructure for enhanced functional assignment represents major challenges to the post-genomic biological community. 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 only study those metabolites that are both 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 70 isofunctional clusters and defined numerous metabolic pathways, including novel pathways for the utilization of ethanolamine as sole nitrogen source and the use of D-Ala-D-Ala as sole carbon source. These experimental approaches are applicable to a wide range of protein families and begin to define an integrated strategy for realizing the full value of amassing genome sequence data.

Experimental strategies for functional annotation and metabolism discovery: Targeted screening of solute binding proteins and unbiased panning of metabolomes

249th National Meeting (2015)

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