Glutathione S-transferase (GST) is a superfamily 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. Class pi GST (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, 2001; US Patent Number 6610660, 2003; and European Patent Number EP 0 929 538 B1, 2004). Using structure-based approach, GSTP specificity has been achieved with two structural modifications of the prodrug.? ? 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. 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), and obtained a wealth of structural information for structure-based design of novel antimicrobial agents.? ? RNA POLYMERASE-ASSOCIATED TRANSCRIPTION FACTORS? ? Stringent starvation protein A (SspA) of Escherichia coli is a transcriptional activator for the lytic development of phage P1, and is essential for stationary phase-induced acid tolerance. We have determined the crystal structure of Yersinia pestis SspA, which is 83% identical to E. coli SspA in amino acid sequence and is functionally complementary in supporting the lytic growth of phage P1 and acid resistance of an E. coli sspA mutant. The structure reveals that SspA assumes the characteristic fold of glutathione S-transferase (GST). However, SspA lacks GST activity and does not bind glutathione. Functional roles of the structural features of SspA were investigated by assessing the ability of deletion and site-directed mutants to confer acid resistance of E. coli and to activate transcription from a phage P1 late promoter, thereby supporting the lytic growth of phage P1. The results indicate that a surface pocket is important for both transcriptional activation of the phage P1 late promoter and acid resistance of E. coli. The size, shape, and property of the pocket suggest that it mediates protein-protein interactions.? ? RNA-PROCESSING PROTEINS? ? Members of the Ribonuclease III (RNase III) family are double-stranded (ds) RNA-specific endoribonucleases, characterized by a signature motif (ERLEFLGDS) in their active centers and a two-base 3' overhang in their products. While Dicer, which produces small interfering RNAs, is currently considered the most important member, the founding member was bacterial RNase III. Facilitating dsRNA processing, RNase IIIs are post-transcriptional regulators of gene expression. 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 processing enzyme that cleaves dsRNA, or as a regulatory protein that binds dsRNA without cleaving it.?
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