The enzymes of the mandelate pathway convert R-mandelamide to benzoate; these include mandelamide hydrolase (MAH), mandelate racemase (MR), S-mandelate dehydrogenase, benzoylformate decarboxylase (BFD), and two nicotinamide-dependent dehydrogenases. The primary sequence information in the data bases has increased dramatically in recent years and thus extensive information regarding the primary sequence relationships of all of the enzymes in the pathway is now available. In addition, high resolution x-ray structures are now available for MR and BFD. In the proposed project period the primary sequence and three-dimensional structure information of these enzymes will be used to better understand the mechanisms of the reactions catalyzed by MR, BFD, and MAH. The proposed studies on MR will provide information about the importance of active site electrophilic catalysts in stabilizing the enolic intermediate, including divalent metal ions, the (epsilon-ammonium group of Lys-164, and the carboxylic acid group of Glu-317; and 2) restructure the active site such that the imidazolium group of His-297 can be replaced by the (epsilon-amino group of Lys. In vitro recombination strategies will be used to """"""""shuffle"""""""" the genes of MR and three other members of the enolase superfamily, galactonate dehydratase from E. coli, glucarate dehydratase from P. putida, and N-acylamino acid racemase from Amycolaptosis, so that new proteins that catalyze the racemization of mandelate and the dehydration of galactonate may be constructed. The proposed studies on BFD will examine the importance of active site amino acid functional groups in catalysis. Although the first steps in the mechanisms of the reactions catalyzed by BFD and the structurally homologous pyruvate decarboxylase and pyruvate oxidase are similar, potential acid/base catalysts are not conserved. Finally, MAH is a member of an amidase/esterase superfamily that has not been structurally characterized. Further characterization of MAH will yield insights into the origins of enantiomer-specific catalysis, since MAH is a stereo-random catalyst but other members of the family are stereospecific catalysts.
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