X-RAY CRYSTAL-STRUCTURE OF GLYCINAMIDE RIBONUCLEOTIDE SYNTHETASE FROMESCHERICHIA-COLI

Glycinamide ribonucleotide synthetase (GAR-syn) catalyzes the second s tep of the de novo purine biosynthetic pathway; the conversion of phos phoribosylamine, glycine, and ATP to glycinamide ribonucleotide (GAR), ADP, and Pi. OAR-syn containing an N-terminal polyhistidine tag was e xpressed as the SeMet incorporated protein for crystallographic studie s. In addition, the protein as isolated contains a Pro294Leu mutation. This protein was crystallized, and the structure solved using multipl e-wavelength anomalous diffraction (MAD) phase determination and refin ed to 1.6 Angstrom resolution. GAR-syn adopts an alpha/beta structure that consists of four domains labeled N, A, B, and C. The N, A, and C domains are clustered to form a large central core structure whereas t he smaller B domain is extended outward. Two hinge regions, which migh t readily facilitate interdomain movement, connect the B domain and th e main core. A search of structural databases showed that the structur e of GAP-syn is similar to D-alanine:D-alanine ligase, biotin carboxyl ase, and glutathione synthetase, despite low sequence similarity. Thes e four enzymes all utilize similar ATP-dependent catalytic mechanisms even though they catalyze different chemical reactions. Another ATP-bi nding enzyme with low sequence similarity but unknown function, synaps in Ia, was also found to share high structural similarity with OAR-syn . Interestingly, the GAP-syn N domain shows similarity to the N-termin al region of glycinamide ribonucleotide transformylase and several din ucleotide-dependent dehydrogenases. Models of ADP and GAR binding were generated based on structure and sequence homology. On the basis of t hese models, the active site lies in a cleft between the large domain and the extended B domain. Most of the residues that facilitate ATP bi nding belong to the A or B domains. The N and C domains appear to be l argely responsible for substrate specificity. The structure of OAR-syn allows modeling studies of possible channeling complexes with PPRP am idotransferase.