beta-Alanine Synthase : One reaction, two folds and mechanisms

beta-alanine synthase (betaAS) is the third enzyme in the reductive pyrimidine catabolic pathway which is responsible for the breakdown of pyrimidine bases, including several anti-cancer drugs in higher organisms. We have solved the high resolution structures of two beta-alanine synthases that perform the same reaction but have two very different folds and utilize different reaction mechanisms. The fold of the homodimeric betaAS from the yeast Saccharomyces kluyveri (SkbetaAS) identifies it as a member of the Acy1/M20 family of metallopeptidases. Its subunit consists of two domains, of which the larger harbors a di-zinc centre crucial for catalysis, while the smaller domain mediates dimerization. We determined the structure of a productive substrate complex, the first for a dimeric member of the Acy1/M20 family. With this structure we show that a conformational change from an open state to a closed state is required for catalysis. Additionally the results of our site-directed mutagenesis studies, performed to identify residues involved in substrate binding and catalysis are discussed. These results support the role of E159 as a catalytic base and identify R322 as a key substrate-binding residue. We have also characterized the structure of betaAS from Drosophila melanogaster (DmbetaAS) which has been identified as a member of the nitrilase superfamily, with a characteristic alphabetabetaalpha-sandwich fold and a conserved Cys-Glu-Lys catalytic triad. We have determined the threedimensional structure in two different space groups, in P21212 to 2.8 Å and in C2 to 3.3 Å resolution, respectively. The first crystal form has four molecules in the asymmetric unit, forming a homotetramer in a dimer-ofdimers arrangement. This homotetramer is assembled into an octamer with the molecule from an adjacent asymmetric unit in a helical turn-like assembly. The same octameric formation is found in the second crystal form, which has all eight monomers present in the asymmetric unit