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

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.

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

“…This is considered a reason why TRAPs are especially widespread in marine microorganisms (7). Molecules known to be transported by TRAP transporters range from small organic acids including C4-dicarboxylates, larger sugar acids like N-acetylneuraminic acid (Neu5Ac), to amino acids (4,8). Most TRAP transporter substrates contain a carboxylic acid group, which is specifically recognized by the P domain of the transporter (9).…”

Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars

“…An integrated SSN/GNN analysis of DUF1537 proteins identified three predominant conserved genome neighborhoods and permitted the prediction that DUF1537 proteins catalyze ATP-dependent phosphorylation. We constrained the possible kinase substrates upon recognizing that some of the neighborhoods also encoded SBPs that bound four-carbon monoacid sugars (18). Directed by the prediction, we confirmed functions for DUF1537 proteins from two of the three predominant genome neighborhoods: the PdxA2-DUF1537 and the aldolase-DUF1537 neighborhoods.…”

“…We previously identified four SBPs that bind four-carbon acid sugars, including D-erythronate and L-erythronate (18). When the SSN for the TRAP SBP family (IPR018389) is filtered at an alignment score threshold of 80 (∼45% sequence identity) (SI Appendix, Fig.…”

“…As described in this article, we used in vitro and in vivo functional assignment of members of the DUF1537 family to further develop our large-scale genomic enzymology-based strategy for functional assignment of novel enzymes in novel microbial catabolic pathways (18). The strategy takes advantage of the frequently observed genomic colocation of microbial genes that encode the transport systems and enzymes for catabolism of an extracellular solute.…”

“…The first step in our strategy is the experimental screening of the ligand specificity for a transport system solute binding protein (SBP) (18). This step identifies the starting metabolite for the pathway and helps locate the genes that encode the enzyme components of the pathway.…”