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Assigning valid functions to proteins identified in genome projects is challenging: overprediction and database annotation errors are the principal concerns1.We and others2 are developing computation-guided strategies for functional discovery with 'metabolite docking' to experimentally derived3 or homology-based4 three-dimensional structures. Bacterial metabolic pathways often are encoded by 'genome neighbourhoods' (gene clusters and/or operons), which can provide important clues for functional assignment. We recently demonstrated the synergy of docking and pathway context by 'predicting' the intermediates in the glycolytic pathway in Escherichia coli5. Metabolite docking to multiple binding proteins and enzymes in the same pathway increases the reliability of in silico predictions of substrate specificities because the pathway intermediates are structurally similar. Here we report that structure-guided approaches for predicting the substrate specificities of several enzymes encoded by a bacterial gene cluster allowed the correct prediction of the in vitro activity of a structurally characterized enzyme of unknown function (PDB2PMQ), 2-epimerization of trans-4-hydroxy-L-proline betaine (tHyp-B) and cis-4-hydroxy-D-proline betaine (cHyp-B), and also the correct identification of the catabolic pathway in which Hyp-B 2-epimerase participates. The substrate-liganded pose predicted by virtual library screening (docking) was confirmed experimentally. The enzymatic activities in the predicted pathway were confirmed by in vitro assays and genetic analyses; the intermediates were identified by metabolomics; and repression of the genes encoding the pathway by high salt concentrations was established by transcriptomics, confirming the osmolyte role of tHyp-B. This study establishes the utility of structure-guided functional predictions to enable the discovery of new metabolic pathways.
Wehave applied a structure-guided strategy usingmetabolite docking to multiple proteins and enzymes in a metabolic pathway to discover a previously undocumented reaction, 4R-hydroxyproline betaine 2-epimerase (Hyp-B 2-epimerase; Fig. 1a), as well as the catabolic pathway by which tHyp-B is converted to a-ketoglutarate. The crystallographically determined unliganded structure of the uncharacterized 'target' as well as homology models for a binding protein and a second enzyme encoded by its genome neighbourhood were used to predict the Hyp-B 2-epimerase activity as well as those of downstream enzymes of the pathway.
The marine bacterium Pelagibaca bermudensis encodes an uncharacterized member of the enolase superfamily (National Center for Biotechnology Information GI number 114543141) in which two lysine residues of the TIM-barrel domain are positioned to function as acid- base catalysts6-8. The NewYork SGXResearch Consortiumdetermined its...