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.

Figure 1

TRAP SBP ligands and structures prior to this study. TRAP SBP SSN network at an e-value of 10^–120. In the network, each node is labeled with its cluster number, and each color represents a unique function (see Table S2 for ligand to color mapping). Only a small number of sequences have their functions annotated and/or have their structures determined. The known functions prior to our study are shown by the larger colored nodes. Sequences with PDB structures are shown as diamonds with red borders, labeled with the PDB ID. If there is more than one PDB structure for a sequence, then only one is listed.

Figure 2

Schematic of TRAP SBP ligands determined in this study. These ligands were determined either by DSF and/or were co-purified (CO) ligands observed by crystallography. Co-purified ligands that were confirmed by X-FTMS or ESI-FTMS are marked by (MS). Those ligands that are novel for the TRAP SBP family, as defined by this study, are indicated by an asterisk.

Figure 3

Annotated 10^–120 TRAP SBP SSN network with data from this study. Targets are colored by ligand(s). The ligands determined prior to this study are shown by the smaller colored nodes (also visualized in Figure 1), whereas those determined here are shown by larger colored nodes. Sequences with PDB structures are shown as diamonds with red borders, labeled with the PDB ID. See Table S2 for a mapping of ligand, cluster number, and color and Table S3 for the number of sequences that map to those clusters.

Figure 4

DSF of the TRAP SBP BH2673 from Bacillus halodurans. Denaturation of BH2673 as a function of temperature as observed by increase in fluorescence of the indicator dye SYPRO Orange, which binds nonspecifically to hydrophobic surfaces. At higher temperatures, the intrinsic fluorescence degrades due to the formation of protein aggregates and dye dissociation. Ligands and their calculated ΔT_m’s are shown. The light blue circles map where the stereochemistries of the C2–C5 hydroxyls of the weaker hits differ from that of the top hit, d-gluconate.

Figure 5

Functional implications from d-glucuronate/d-galacturonate TRAP SBPs. (A) Genome context of Bpro_3107, a TRAP SBP shown to bind d-galacturonate and d-glucuronate by DSF, and the encoded catabolic pathway predicted to convert these ligands to central metabolites. The newly annotated uronate dehydrogenase (Udh) and lactonase (UxuL) are shown in indigo and yellow, respectively. Gene annotations are listed as obtained from KEGG. The TctC proteins are the SBP component of the TTT family of transporters, whose colocalization within this operon/regulon suggests alternative entry points into the pathway, perhaps at galactarate or glucarate. (B) Interactions of the related TRAP SBP, Apre_1383 with d-glucuronate. Hydrogen bonds are shown as dashed lines, and hydrophobic contacts are represented by an arc with spokes radiating toward the ligand atoms that they contact. (C) Interactions of the TRAP SBP Bamb_6123 with d-galacturonate. Residues that interact with the ligand in an analogous fashion within panels B and C are shown in bold.

Figure 6

Functional implications from l-galactonate/l-gulonate TRAP SBPs. (A) Genome context of HI0052, a TRAP SBP shown to bind l-gulonate by DSF, and the encoded catabolic pathway that would convert l-gulonate to glycerol 3-phosphate and pyruvate. The newly annotated l-gulonate 5-dehydrogenase is shown in pink. Gene annotations are listed as obtained from KEGG. (B) Genome context of Asuc_0158, a TRAP SBP shown by DSF to bind l-galactonate, and the encoded catabolic pathway that would convert l-galactonate to glycerol 3-phosphate and pyruvate. The newly annotated l-galactonate 5-dehydrogenase is shown in pink. (C) Interactions of the related TRAP SBP, HICG_00826 with l-gulonate. Hydrogen bonds are shown as dashed lines, and hydrophobic contacts are represented by an arc with spokes radiating toward the ligand atoms that they contact. (D) Interactions of the TRAP SBP Asuc_0158 with l-galactonate. Residues that interact with the ligand in an analogous fashion within panels C and D are shown in bold.

Figure 7

Functional implications from d-Ala-d-Ala TRAP SBPs. (A) Genome environment of the three TRAP SBPs that had DSF hits on the dipeptide d-Ala-d-Ala. Genes putatively assigned for the transport and catabolic degradation of d-Ala-d-Ala are shown in color. (B) Interactions of Csal_0660 with d-Ala-d-Ala. Hydrogen bonds are shown as dashed lines, and hydrophobic contacts are represented by an arc with spokes radiating toward the ligand atoms that they contact. (C) ¹H NMR verification of CsVanX (Csal_0663) dipeptisase activity on d-Ala-d-Ala. The control spectrum is show on the bottom, and the reaction is shown on top (glycerol from the enzyme prep is present between 3.6 and 3.4 ppm). Insets show magnifications of the control and reaction peaks. (D) Fold change in transcript measured by qRT-PCR for Csal_0660 genome neighborhood related genes when C. salexigens is grown on d-Ala-d-Ala versus d-glucose as a carbon source. (E) Growth curves of wild-type C. salexigens versus a deletion mutant of the d-Ala-d-Ala TRAP SBP (ΔCsDctP) or deletion mutant of the d-Ala-d-Ala dipeptidase (ΔCsVanX).

Figure 8

TRAP SBP co-purified ligands. Omit maps for adventitiously bound ligands contoured at 3 RMSD and the associated TRAP SBPs genomic environment. The genome environment for Dde_0634 was substituted with that of the related TRAP transporter from Geobacter sulfurreducens (KN400_2073-75), for which more genes were colocated. Gene annotations are those found in KEGG. Co-purified ethanolamine is shown in Figure 9.

Figure 9

Functional implications from ethanolamine TRAP SBP. (A) 3 RMSD omit map for a co-purified ethanolamine ligand bound to the TRAP SBP Csal_0678 from Chromohalobacter salexigens. (B) Binding interactions of ethanolamine with Csal_0678. The amine is coordinated by the carbonyl of Trp215 and the side chains of Glu220 and Asp155, whereas the ethanolamine oxygen is coordinated by Tyr241 and Glu220. In Csal_0678, the highly conserved TRAP SBP arginine is replaced by phenylalanine (Phe177), and the position typically occupied by the ligand carboxylate is occupied by the indole group of Trp215. (C) Ligand concentration vs thermal denaturation stabilization of Csal_0678 for various 4 and 5 atom ligands similar to ethanolamine. (D) Genomic environment of the ethanolamine utilization pathway of Chromohalobacter salexigens and Agrobacterium tumefaciens. (E) Details of the chemical transformation of ethanolamine to glycine and the genes previous annotations.