CRYSTAL-STRUCTURES OF NATIVE AND RECOMBINANT YEAST FUMARASE

Crystal structures for both native and recombinant forms of yeast fuma rase from Saccharomyces cerevisiae have been completed to moderate res olution by two separate laboratories. The recombinant form was obtaine d by the construction of an expression plasmid for Escherichia coli. D espite a high level of amino acid sequence similarity, purification of the eukaryotic enzyme from the wild-type prokaryotic enzyme was feasi ble. The crystal structure of the native form, NY-fumarase, encompasse s residues R22 through M484, while the recombinant form, RY-fumarase, consists of residues S27 through L485. Both crystal structures lack th e N-terminal translocation segment. Each subunit of the homotetrameric protein has three domains. The active site is formed by segments from each of three polypeptide chains. The results of these studies on the eukaryotic proteins are unique, since the recombinant form was done i n the absence of dicarboxylic acid and has an unoccupied active site. As a comparison, native fumarase was crystallized in the presence of t he competitive inhibitor, mese-tartrate. Mese-tartrate occupies a posi tion close to that of the bound citrate molecule found in the active s ite of the E. coli enzyme. This inhibitor participates in hydrogen bon ding to an active-site water molecule. The independent determination o f the two structures provides further evidence that an active-site wat er molecule may play an active role in the fumarase-catalyzed reaction . (C) 1998 Academic Press.