BBMB Research Seminars
March 13, 2008
Nathaniel Ginder
Iowa State University
1414 Molecular Biology Buidling
4:10 p.m.
The Structure and Enzymatic Properties of SAICAR Synthetase
SAICAR synthetase catalyzes the eighth step in bacterial de novo purine nucleotide biosynthesis:
ATP + l-aspartate + CAIR à ADP + Pi + SAICAR
Enzymes involved in nucleotide synthesis are common targets for chemotheropeutic and antimicrobial drugs. They work by reducing the rate of growth of rapidly proliferating cells, thus preventing tumor growth and microbial fecundity.
l-Alanosine is a natural product of Streptomyces alanosinicus with antiviral and antitumor activities. It is an l-aspartate analogue that acts as a substrate in vitro and in vivo for SAICAR synthetase. The product of the reaction is a potent inhibitor of adenylosuccinate synthetase and adenylosuccinate lyase and is the compound responsible for l-alanosine toxicity. Methylthioadenosine phosphorylase (MTAP) is an important salvage enzyme for adenine nucleotides. The gene is located close to the loci of tumor suppressors genes and is often co-deleted with them in tumors. For instance, approximately 30% of all T-cell acute lymphocytic leukemia lack an intact salvage pathway. l-Alanosine is toxic to cell lines of such cancers at concentrations below those that poison normal cells. l-alanosine may be effective as a chemotherapeutic agent in combination with other
We have determined the kinetic mechanisms for Escherichia coli SAICAR synthetase and human SAICAR synthetase. We have also identified two new magnesium binding sites in the active site and demonstrated how they interact with CAIR. The binding of CAIR and metals orders a loop that is disordered in the unligated state. Site-directed mutagenesis experiments reveal a role for this loop in aspartate binding. Recent crystallographic experiments demonstrate the mode of binding of aspartate.
Crystal structures of CAIR analogs reveal phosphoryl intermediates that likely mimic those of a putative phosphoryl intermediate. Positional Isotope Exchange (PIX) experiments in the presence of gamma-O18-ATP and CAIR show isotope scrambling at the gamma-phosphoryl position of ATP. This is also indicative of the formation of a phosphoryl intermediate. The understanding of the mechanism of SAICAR synthetase can give insights into the novel construction of inhibitors that can act as antimicrobial or chemotherapeutic agents.
Ginder N. D., Binkowski D. J., Fromm H. J., and Honzatko R. B. (2006) Nucleotide complexes of Escherichia coli phosphoribosylaminoimidazole succinocarboxamide synthetase. J Biol Chem. 281:20680-8.
Ginder N. D., Binkowski D. J., Chen X., Nix J. C., Fromm H. J., and Honzatko R. B. (2008) Entrapment of Phosphoryl Intermediates by SAICAR Synthetase. Prepared manuscript.
Binkowski D. J., Ginder N. D., Fromm H. J., and Honzatko R. B. (2008) Linkage of Function in Human AIR Carboxylase/SAICAR Synthetase. Prepared Manuscript.
