The primary goals of our Section are to address the structure and function of biomolecular systems with anticancer and antimicrobial significance and to explore the feasibility of designing drugs/prodrugs to target such biomolecules. In our efforts to achieve these goals, we have established collaborations within NIH as well as with extramural experts in genetics, molecular biology, protein chemistry, enzymology, carcinogenesis, and medicinal chemistry. These collaborations have greatly extended our range of experiments. Glutathione S-transferase: Rational Design of Electrophilic Diazeniumdiolates for Pharmacologic Delivery of Nitric OxideMany tumors become drug resistant by overexpressing the detoxification enzyme glutathione S-transferase (GST). Of the three GST major isoforms u alpha, mu, and pi u pi is the predominant form in cancer cells. We are attempting to design agents that will overcome this drug resistance by generating nitric oxide (NO) selectively in the active site of GST-pi, which could increase the effectiveness of anticancer therapies. Our comparison of the active sites and transition-state analogs of the three isozymes revealed a potential strategy for achieving isozyme selectivity. Application of this strategy has resulted in a pi-selective NO donor. If our planned cytotoxicity studies show that this donor or subsequent NO donors improve the potency of electrophilic anticancer agents toward cells overexpressing GST-pi, the means of overcoming drug resistance in some clinically important tumor types may be forthcoming.6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase: Mechanism of Pyrophosphoryl Transfer6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is the first 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. In contrast, most microorganisms must synthesize folate de novo. Therefore, HPPK is an ideal target for the development of novel antimicrobial agents, which are urgently needed to fight the worldwide crisis of antibiotic resistance. HPPK contains 158 amino acid residues and is thermostable, which makes it an excellent model system for the study of the pyrophosphoryl transfer mechanism, of which little is known. We have determined the crystal structures of apo-HPPK at 1.50 +, of the binary complex with MgADP at 1.50 +, and of the ternary complex with both HP and MgAMPCPP at 1.25 +. Our analysis of these structures will provide essential information on the reaction mechanism of pyrophosphoryl transfer and critical knowledge for the design of novel antimicrobial molecules.Era Protein: GTPase-Dependent Cell Cycle RegulatorEra is an essential GTPase found in every bacterium sequenced to date. Highly conserved Era homologs are also found in eukaryotes, such as mouse and human. The Era homolog may be a candidate for a tumor suppressor, because it is located in a chromosomal region where loss of heterozygosity is often associated with various types of cancer. In bacteria, Era has a regulatory role in cell cycle control by coupling cell growth rate with cytokinesis. Cell division is signaled when a threshold of Era activity is reached. Artificially reducing the expression or impairing the activity of Era results in bacterial cell cycle arrest at a predivisional two-cell stage. The arrest lasts until Era activity accumulates to the threshold level, allowing another cell cycle to start. Because the synthesis of Era itself is positively correlated with growth rate, the cell division rate is thus coordinately maintained. We have determined the crystal structure of Era from Escherichia coli at 2.4 + resolution, which reveals a two-domain arrangement: an N-terminal domain that resembles p21 Ras and a unique C-terminal domain that contains an RNA-binding motif. The crystal structure determination of Era in complex with GDP and with a GTP analog is in progress. Our analysis of these structures will provide insight into the conformational changes of the protein during GTP hydrolysis, which may be part of the signaling pathway of this cell cycle regulator.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010326-01
Application #
6419874
Study Section
(LCB)
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2000
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Dabrazhynetskaya, Alena; Brendler, Therese; Ji, Xinhua et al. (2009) Switching protein-DNA recognition specificity by single-amino-acid substitutions in the P1 par family of plasmid partition elements. J Bacteriol 191:1126-31
Tu, Chao; Tropea, Joseph E; Austin, Brian P et al. (2009) Structural basis for binding of RNA and cofactor by a KsgA methyltransferase. Structure 17:374-85
Gan, Jianhua; Shaw, Gary; Tropea, Joseph E et al. (2008) A stepwise model for double-stranded RNA processing by ribonuclease III. Mol Microbiol 67:143-54
Shaw, Gary; Gan, Jianhua; Zhou, Yan Ning et al. (2008) Structure of RapA, a Swi2/Snf2 protein that recycles RNA polymerase during transcription. Structure 16:1417-27
Ji, Xinhua (2008) The mechanism of RNase III action: how dicer dices. Curr Top Microbiol Immunol 320:99-116
Ji, Xinhua; Pal, Ajai; Kalathur, Ravi et al. (2008) Structure-Based Design of Anticancer Prodrug PABA/NO. Drug Des Devel Ther 2:123-130
Tu, Chao; Tan, Yu Hong; Shaw, Gary et al. (2008) Impact of low-frequency hotspot mutation R282Q on the structure of p53 DNA-binding domain as revealed by crystallography at 1.54 angstroms resolution. Acta Crystallogr D Biol Crystallogr 64:471-7
Blaszczyk, Jaroslaw; Li, Yue; Gan, Jianhua et al. (2007) Structural basis for the aldolase and epimerase activities of Staphylococcus aureus dihydroneopterin aldolase. J Mol Biol 368:161-9
Gan, Jianhua; Wu, Yan; Prabakaran, Ponraj et al. (2007) Structural and biochemical analyses of shikimate dehydrogenase AroE from Aquifex aeolicus: implications for the catalytic mechanism. Biochemistry 46:9513-22
Saavedra, Joseph E; Srinivasan, Aloka; Buzard, Gregory S et al. (2006) PABA/NO as an anticancer lead: analogue synthesis, structure revision, solution chemistry, reactivity toward glutathione, and in vitro activity. J Med Chem 49:1157-64

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