The enzymes of the mandelate pathway convert R-mandelamide to benzoate via a total of six enzymes. In the past project period, we have cloned and sequenced the genes for five of these enzymes, mandelamide hydrolase (MAH), mandelate racemase (MR), S-mandelate dehydrogenase, benzoylformate decarboxylase, and an NAD+/NADP+-dependent benzaldehyde dehydrogenase (NAD+-/NADP+-BDH). The primary sequence of each of these enzymes is homologous to enzymes catalyzing mechanistically related reactions. We have purified but have not yet cloned the gene for an NADP+-dependent BDH. In addition, we have obtained a high resolution x-ray structure for MR and used this as the basis for investigations of the mechanism of the reaction using site-directed mutagenesis and for determining the structure of NM irreversibly inhibited by an active site-directed inhibitor. The current Research Plan is divided into four broad areas, each of which utilizes the synergistic experimental methods of molecular biology, x-ray crystallography, and mechanistic enzymology: 1) We propose to continue studying the mechanism of the reaction catalyzed by MR by determining the free energy profile for the reaction. The functions of specific amino acid functional groups in the active site will be investigated by both site-directed (Glu 317 and Asp 270) and unnatural mutagenesis. 2) We propose to determine whether an omega-loop in the active site of MDH determines both the identity of the electron acceptor in the second half-reaction (oxidation of FMNH2) and the chemistry by which products are produced. This will be accomplished by the construction of chimeric enzymes in which the omega-loop in MDH is replaced by omega-loops from homologous enzymes which catalyze different second half-reactions. 3) We propose to purify MAH so that we can begin to characterize the mechanism of amide hydrolysis. Using x-ray crystallography, we will determine whether NM contains the newly recognized alpha/beta hydrolase fold. 4) We propose to clone and sequence the gene for the NADP+-dependent BDH so that the evolutionary relationship between the two BDHs can be determined. This project capitalizes upon the diverse talents of four laboratories and will hopefully provide significant advances in understanding the relationships between structure and catalysis in several mechanistically distinct and biomedically important classes of enzyme-catalyzed reactions.
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