O2-ARYL DIAZENIUMDIOLATES Glutathione S-transferase (GST) is a family of enzymes that catalyze glutathione conjugation with electrophilic compounds. In preneoplastic and neoplastic cells, specific forms of GSTs are expressed at high levels and to participate in the cells' resistance to anticancer drugs. Isozyme GSTP is of particular importance in biological resistance to alkylating agents. Therapeutic strategies aimed at inhibiting GSTP to extend the efficacy of alkylating agents have been unsuccessful. However, GSTP-activated prodrugs have shown great potential in targeting specific cancer cells. We have developed a new type of anticancer agents, O2-aryl diazeniumdiolates, which kill cancer cells by releasing nitric oxide intracellularly (Australia Patent Number 733590; US Patent Number 6610660; and European Patent Number 04009529.1-2101). Two lead compounds have since been developed. FOLATE PATHWAY ENZYMES 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a key enzyme in the folate biosynthetic pathway, catalyzing the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). Folate cofactors are essential for life. Mammals derive folates from their diets, whereas most microorganisms must synthesize folate de novo. Therefore, HPPK is an ideal target for the development of novel antimicrobial agents. HPPK contains 158 amino acid residues and is thermostable, which makes it an excellent model system for the mechanistic study of the enzymatic pyrophosphoryl transfer. We have mapped out the trajectory of HPPK-catalyzed reaction by determining the three-dimensional structures of apo-HPPK (ligand-free enzyme), HPPK-MgATPanalog (binary complex of HPPK with one substrate), HPPK-MgATPanalog-HP (ternary complex of HPPK with both substrates), HPPK-AMP-HPPP (ternary complex of HPPK with both products), and HPPK-HPPP (binary complex of HPPK with one product). RNA-PROCESSING PROTEINS G Protein ERA is involved in the maturation of ribosomal RNA (rRNA) and is a key component that couples the regulation of growth and cell cycle in bacteria. ERA homologues identified in human and mouse appear to play a role in the regulation of apoptosis. We have determined the crystal structure of ligand-free ERA (apo-ERA) at 2.4- resolution previously. The three-dimensional structure revealed a two-domain arrangement of ERA: an N-terminal domain that resembles p21 Ras and a C-terminal domain that is unique. Structure-based topological search of the C-domain fails to reveal any meaningful match, although sequence analysis suggests that it contains a KH-domain. KH-domains are RNA-binding motifs that usually occur in tandem repeats and exhibit low sequence similarity except for the well-conserved segment VIGxxGxxIK. We identified a beta-alpha-alpha-beta fold that contains the VIGxxGxxIK sequence and is shared by the C-domain of ERA and the KH-domain. We proposed that this beta-alpha-alpha-beta fold is the RNA-binding motif, the minimum structural requirement for RNA-binding. ERA dimerizes in crystal. The dimer formation involves a significantly distorted switch II region, which may relate to how ERA protein regulates downstream events. Ribonuclease III (RNase III) shows specificity toward double-stranded RNA (dsRNA). It is conserved in bacteria, worms, flies, plants, fungi, and mammals. Bacterial RNase III, containing an endonuclease domain (endoND) and a dsRNA-binding domain (dsRBD), can affect RNA structure and gene expression in either of two ways: as a dsRNA-processing enzyme that cleaves dsRNA, or as a dsRNA-binding protein that binds but does not cleave dsRNA. We have determined the endoND structure of Aquifex aeolicus RNase III (Aa-RNase III) and modeled a catalytic complex of full-length Aa-RNase III with dsRNA. We have also determined the crystal structure of a mutant Aa-RNase III, which binds but does not cleave dsRNA, in complex with dsRNA, revealing the architecture of a non-catalytic assembly. The major differences between the two functional forms of RNase III-dsRNA are the conformation of the protein and the orientation and location of dsRNA. Every member of the RNase III family contains one or two copies of the endoND and one copy of the dsRBD. Therefore, the information derived from our structures sheds light on the structure and function of other RNase III proteins, which has gained added importance with the recent discovery of the RNase III ortholog """"""""Dicer"""""""" in RNA interference, a broad class of RNA silencing phenomena found in fungi, plants, and animals.
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