Crystallization and preliminary X-ray data analysis of β-alanine synthase fromDrosophila melanogaster

Abstract

-Alanine synthase catalyzes the last step in the reductive degradation pathway for uracil and thymine, which represents the main clearance route for the widely used anticancer drug 5-fluorouracil. Crystals of the recombinant enzyme from Drosophila melanogaster, which is closely related to the human enzyme, were obtained by the hanging-drop vapour-diffusion method. They diffracted to 3.3 Å at a synchrotron-radiation source, belong to space group C2 (unit-cell parameters a = 278.9, b = 95.0, c = 199.3 Å , = 125.8 ) and contain 8-10 molecules per asymmetric unit.

The Crystal Structure of β-Alanine Synthase from Drosophila melanogaster Reveals a Homooctameric Helical Turn-Like Assembly

“…39 However, no solution could be found at first due to the large number of molecules (8)(9)(10) expected in the asymmetric unit, the low resolution and completeness of the original data, and the low sequence identity of DmβAS to the most similar search models [21% to N-carbamyl-D-amino acid amidohydrolases (DCases) and 28% to the Pyrococcus horikoshii PH0642 protein]. Eventually, a dimeric model of the latter protein [Protein Data Bank (PDB) ID: 1j31] 34 with an adjusted and truncated sequence was probed in PHASER 40 and led to a promising solution.…”

“…Analysis by analytical size-exclusion chromatography suggests an oligomer composed of eight or more polypeptide chains. 39 In the crystal structure, DmβAS exists as a homooctamer in the shape of an almost complete helical turn ( Fig. 2c and d).…”

“…In fact, addition of NCβA is required because DmβAS could thus far not be crystallized in the absence of the substrate. 39 Residual electron density not associated with protein residues is observed in the active sites of all but the corner subunits in both nat1 and nat2. It has an elongated shape stretching out from Cys234 toward Arg237 and His238 in some of the active sites, whereas in others, it is spherical and located at approximately equal distance from these three residues.…”

“…The latter seems more likely considering the required long equilibration period before appearance of crystals (ca 30 days) and the overall peculiar crystallization behavior. 39 …”

“…39 It is thus far unknown whether the octamer observed in the crystal structure is the physiologically most significant or most populated state or if much larger quaternary assemblies are formed in vivo. The octamer is built up from tightly interlocked dimeric units.…”