GENOMIC ENZYMOLOGY: THE ENOLASE SUPERFAMILY AND OMPDC SUPRAFAMILY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our studies focus on understanding the structural bases for divergent evolution in groups of homologous enzymes that catalyze different reactions. We are interested in the enolase superfamily, a group of mechanistically diverse enzymes that share enolization of a carboxylate anion substrate, and the orotidine 5-monophosphate (OMPDC) suprafamily, a group of functionally distinct enzymes that share no discernible mechanistic attributes. The members of both groups share the (b/a)8-fold, the most commonly observed fold in structurally characterized enzymes. We use the MINRMS program developed at the Computer Graphics Laboratory to superimpose the structures of members of both groups so that we can identify those regions of the structures that might be associated with a particular divergent function. We use CHIMERA to visualize these superpositions. In the enolase superfamily the active sites of o-succinylbenzoate synthase (OSBS) and the L-Ala-D/L-Glu epimerase are homologous despite their different reactions. However, these differ in the identity of the group at the end of the eighth b-strand in the barrel, a Gly in OSBS and an Asp in the epimerase. The structural superpositions we performed suggested that mutation of the Asp in the epimerase to a Gly might enlarge the active site and allow the substrate for the OSBS reaction to bind and perhaps undergo dehydration. We have performed this mutagenesis experiment and determined that the mutant epimerase is, in fact, able to catalyze (low levels) of the OSBS reaction. We are now extending those studies by using directed evolution to identify additional mutations that will allow enhancement of the designed OSBS activity. One such substitution has been found and we are searching for others. We also are using the MINRMS program as well as GCG and CLUSTALW to compare and contrast the active sites of divergent, but homologous, OMP decarboxylases to guide site-directed and mechanistic experiments to establish the chemical mechanisms of the catalyzed reaction.