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Entamoeba histolytica, a protozoan intestinal parasite, is the causative agent of human amebiasis. Amebiasis is the fourth leading cause of death and the third leading cause of morbidity due to protozoan infections worldwide(1), resulting in ~70,000 deaths annually. E. histolytica has been listed by the National Institutes of Health as a category B priority biodefense pathogen in the United States. Treatment relies on metronidazole(2), which has adverse effects(3), and potential resistance of E. histolytica to the drug is an increasing concern(4,5). To facilitate drug screening for this anaerobic protozoan, we developed and validated an automated, high-throughput screen (HTS). This screen identified auranofin, a US Food and Drug Administration (FDA)-approved drug used therapeutically for rheumatoid arthritis, as active against E. histolytica in culture. Auranofin was ten times more potent against E. histolytica than metronidazole. Transcriptional profiling and thioredoxin reductase assays suggested that auranofin targets the E. histolytica thioredoxin reductase, preventing the reduction of thioredoxin and enhancing sensitivity of trophozoites to reactive oxygen-mediated killing. In a mouse model of amebic colitis and a hamster model of amebic liver abscess, oral auranofin markedly decreased the number of parasites, the detrimental host inflammatory response and hepatic damage. This new use of auranofin represents a promising therapy for amebiasis, and the drug has been granted orphan-drug status from the FDA.

An extended bioinformatic analysis has described the genome-based link for the absence of GHS biosynthesis, the presence of the type III pantothenate kinase (PanK) and the presence of the coenzyme A-disulfide reductase (CoADR)-like enzymes in a number of bacteria and spirochetes. The results have indicated that the type III PanK in the spore-forming Bacillus anthracis plays a vital role in the novel thiol/disulfide redox biology of this category A biodefense pathogen.

Research has been conducted on the flavoprotein alpha-glycerophosphate oxidases. Enzyme-monitored turnover analyses of the streptococcal GlpOs have been carried out.

Results show that RofA activates the prtF gene which encodes the fibronectin-binding adhesin protein F in Streptococcus pyogenes. Data point out that RofA specifically binds to two distinct sites on DNA, a 17-basepair site adjacent to the rofA promoter and a 40-basepair site adjacent to the prtF promoter.

This review documents the applications of cysteine-sulfenic acid (Cys-SOH) derivative introduction into diverse proteins and its impact on proteins' function and stabilization. The role of Cys-SOH in various cellular processes and in the industrial production of chemicals is also discussed.

Research conducted on alkaline phosphatase protein of Enterococcus faecalis, its identification and characterization is discussed. Alkaline phosphatase (AP) activity has been quantified, and minimal cell activity has been shown where cytoplasm retains AP.

A study was conducted to analyze the flavin adenine dinucleotide of tetrameric NADH peroxidase from Enterococcus faecalis and three mutant enzymes using time-resolved polarized fluorescence. The energy transfer between the flavins in various subunits within dimeric flavoproteins were determined by time-resolved fluorescence anisotropy analysis ofthe bound flavin cofactor. Results indicated that fluorescence lifetime heterogeneity was not limited to glutathione reductase.

The substitution of the His 10 of flavoprotein NADH peroxidase with Gln (H10Q) or alanine (H10A) have no influence on the rate of enzyme reduction by NADH, and remains rate limiting in turnover. The oxidized form of H10A mutant, Cys42-sulfenic acid, exhibits increased long-wavelength absorbance bands. The 50-fold hike in the second order rate constant for H2O2 inactivation of H10Q mutant peroxidase reduces the free energy of activation.

A study using kinetic and spectroscopic techniques was conducted to examine the kinetic mechanism of the catalytic activity of NADH peroxidase derived from Enterococcus faecalis. The results show that the catalytic mechanism involves rapid formation of a complex of oxidized enzyme and NADH, a rate-limiting hydride transfer step that produces two-electron-reduced NADH peroxidase and rapid formation of an NADH peroxidase-NADH intermediate.

A study was conducted on the crystallography of NADH peroxidase Cys42Ala and Cys42Ser mutants. They were found to have a three-dimensional structure almost identical to that of the wild-type enzyme. An activity decrease factor of around 2,500 was observed upon removal of the sulfhydryl group of Cys 42 during comparisons of the wild-type with the two target mutant enzymes.

Residues Ser38 and Cys42 align with the two cysteines of the redox-active disulfides found in glutathione reductase, lipoamide dehydrogenase, mercuric reductase, and trypanothione reductase in the L40C mutation. However,the formation of the Cys40-Cys42 disulfide bond involved a movement of Cys42-SG to achieve a new position 5.9 angstroms from the flavin-C(4a) position, preventing the disulfide bond from linking with the flavin as a redox center.

Examination of the kinetic and redox characteristics of the flavoprotein NADH peroxidase C42S and C42A mutants without the cysteine-sulfenic acid redox center by anaerobic stopped-flow analysis reveals that peroxidase activity is completely abolished by the substitution of Ser and Ala for Cys42 in the enterococcal NADH peroxidase. Site-directed mutagenesis helps form the recombinant peroxidase having Ser and Ala instead of Cys42. Kinetic data reveal the function of Cys42 in the catalytic redox cycle of wild-type NADH peroxidase.

A series of binding subsites were discovered in barnase that induces large rate enhancements for the extracelllular ribonuclease. Ribonucleotide substrates composed of phosphate, nucleosides and guanosine were synthesized. Guanosine was found to be specifically attached to the BO primary base site of barnase. A 1000-fold rate increase in enzyme kinetics was enhanced by the phosphate at the P +2 site. The +1 subsite showed stronger specificity for purines than pyrimidines. The catalytic efficiency of the enzyme was influenced by longer substrate length from dinucleotide monophosphate to dinucleotide

NADH peroxidase from cell strains of Streptococcus faecalis and alkyl hydroxyperoxide reductase containing flavoprotein from Salmonella typhirium was analyzed using chemical and spectroscopic methods. Based on findings, NADH peroxidase was found to contain a non-flavin redox center as well. This disproved a prior hypothesis connecting these enzymes to the flavoprotein disulfide reductase family since they were identified as a stable derivative of cysteine-sulfenic acid. This protein-sulfenic acid is vital to catalytic processes and oxidative stress in bacterial cells.

The first two enzymes are characterized for the bacillithiol (Cys-GlcN-malate (BSH)) biosynthetic pathway in Bacillus anthracis, which is combined to produce [alpha]-D-glucosaminyl L-malate (GlcN-malate) from UDP-GlcNAc and L-malate. The Bacillus anthracis bshA locus is identified in a transposon-site hybridization study as needed for growth, sporulation or germination, which has shown that the biosynthesis of BSH has represented a target for the development of novel antimicrobials with broad-spectrum activity against Gram-positive pathogens like Bacillus anthracis.

The crystal structure characterization at 2.10 [Angstrome] resolution for the Bacillus anthracis coenzyme A-disulfide reductase isoform (BaCoADR-RHD) containing a C-terminal rhodanese homology domain (RHD) which is the first structural representative of the multidomain proteins class of the rhodanese superfamily is reported. The results indicated that the BaCoADR-RHD isoform does not catalyze the reduction of coenzyme A-disulfide, but both enzymes conserved the Cys-SSCoA redox center.

The characterization of the stability, the [super 15]N backbone relaxation, and the hydrogen- deuterium exchange properties of reduced [NTD-[(SH).sub.2]] and oxidized (NTD-[S.sub.2]) N-terminal domain of AhpF (NTD) from Salmonella typhimurium. The significantly increased folded-state dynamic fluctuations observed in the catalytic 'a' fold for residues in NTD-[S.sub.2] compared to NTD-[(SH).sub.2] demonstrated the oxidation of the active site disulfide which resulted in minor increase in stability but dramatic increase in conformational heterogeneity in residues primarily in the redox active 'a' fold.

The p[K.sub.a] of the cysteinyl residues of Salmonella typhimurium AhpC (StAhpC) is assessed by using different approaches. The studies have shown that the p[K.sub.a] value of AhpC has balanced the need for a deprotonated thiol with the fact that thiolates with higher p[K.sub.a] values are stronger nucleophiles.