Abstract
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.
