CARB 3 |
| Many members of the mechanistically diverse enolase superfamily have unknown functions. We are using two strategies to assign their functions: 1) experimental screening of libraries of potential substrates, and 2) in silico prediction using homology models and docking of libraries of potential substrates. Using the first strategy, we have discovered several acid sugar dehydratases, including L fuconate dehydtratase, D tartrate dehydratase, and enzymes that catalyze the dehydration of both galactarate and L talarate as well as their interconversion by epimerization. L Talarate is a previously unreported metabolite, but Salmonella typhimurium LT2, whose genome encodes the bifunctional galactarate/L-talarate dehydratase, can utilize L talarate as carbon source; disruption of the gene encoding the dehydratase abolishes the ability to utilize L talarate. Using the in silico strategy, we predicted that members of a divergent orthologous group would catalyze the racemization of N succinyl Arg and N succinyl Lys as well as L,L dipeptides in which Arg or Lys are at the epimerizable, C terminal position. We experimentally verified these predictions, discovering that N-succinyl Arg and N succinyl Lys are the preferred substrates. We solved the structure of this N succinylamino acid racemase; the predicted and experimental structures of liganded complexes are in excellent agreement. This success suggests that computational approaches provide a viable strategy for the successful assignment of function to members of the enolase superfamiliy as well as other enzymes discovered in genome projects. Supported by GM-71790. |
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Wolfrom/Isbell/New Investigator Award Symposium
8:30 AM-11:50 AM, Sunday, August 19, 2007 BCEC -- 162B, Oral
Division of Carbohydrate Chemistry |