Christopher D. Putnam

Evolution and mechanism from structures of an ADP-ribosylating toxin and NAD complex.

A member of the Bacillus-produced vegetative insecticidal proteins (VIPs) possesses high specificity against the major insect pest, corn rootworms, and belongs to a class of binary toxins and regulators of biological pathways distinct from classical A-B toxins. The 1.5 Å resolution crystal structure of the enzymatic ADP-ribosyltransferase component, VIP2, from Bacillus cereus reveals structurally homologous N- and C-terminal α/β domains likely representing the entire class of binary toxins and implying evolutionary relationships between families of ADP-ribosylating toxins. The crystal structure of the kinetically trapped VIP2–NAD complex identifies the NAD binding cleft within the C-terminal enzymatic domain and provides a structural basis for understanding the targeting and catalysis of the medically and environmentally important binary toxins. These structures furthermore provide specific experimental results to help resolve paradoxes regarding the specific mechanism of ADP-ribosylation of actin by implicating ground state destabilization and nicotinamide product sequestration as the major driving forces for catalysis.

Lessons learned from structural results on uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG) functions as a sentry guarding against uracil in DNA. UDG initiates DNA base excision repair (BER) by hydrolyzing the uracil base from the deoxyribose. As one of the best studied DNA glycosylases, a coherent and complete functional mechanism is emerging that combines structural and biochemical results. This functional mechanism addresses the detection of uracil bases within a vast excess of normal DNA, the features of the enzyme that drive catalysis, and coordination of UDG with later steps of BER while preventing the release of toxic intermediates. Many of the solutions that UDG has evolved to overcome the challenges of policing the genome are shared by other DNA glycosylases and DNA repair enzymes, and thus appear to be general.

DNA damage recognition and repair pathway coordination revealed by the structural biochemistry of DNA repair enzymes

Cells have evolved distinct mechanisms for both preventing and removing mutagenic and lethal DNA damage. Structural and biochemical characterization of key enzymes that function in DNA repair pathways are illuminating the biological and chemical mechanisms that govern initial lesion detection, recognition, and excision repair of damaged DNA. These results are beginning to reveal a higher level of DNA repair coordination that ensures the faithful repair of damaged DNA. Enzyme-induced DNA distortions allow for the specific recognition of distinct extrahelical lesions, as well as tight binding to cleaved products, which has implications for the ordered transfer of unstable DNA repair intermediates between enzymes during base excision repair.

The food of sweet and bitter fancy.

The MAP30 ribosomal inactivating protein structure has been determined by NMR spectroscopy. This anti-HIV and anti-cancer protein is an RNA and DNA glycosylase as well as a DNA apurinic/apyrimidinic (AP) lyase.