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In Bacillus anthracis, the novel type III pantothenate kinase ([PanK.sub.Ba]; encoded by coaX) catalyzes the first committed step in coenzyme A biosynthesis. We have demonstrated by analyzing the growth characteristics of a conditional coaX mutant that [PanK.sub.Ba] is an essential enzyme, thus contributing to its validation as a new antimicrobial target.

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.

The kinetic and redox properties of the NADH peroxidase R 303M mutant have been studied. Correlation with the crystal structure is discussed. The crystal structure of the Streptococcus pyogenes HADH oxidase C44S mutant, a close structural and mechanistic homologue of Npx, shows that Arg 303 of the peroxidase is replaced with Val314. The Val residue is conserved in the five known Nox sequences as either Val or Leu. It appears that Arg303 has a very important role in determining peroxidase function vs that of oxidase.

Research has been conducted on the Enterococcus faecalis bkd gene cluster. The investigation of the physiological role of the pathway for the branched-chain alpha-keto acid catabolism is discussed.

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

Research has been conducted on the heme-deficient gram-positive bacterium Streptococcus pyogenes. The analysis of the gene products that are important in the aerobic metabolism is presented.

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.

The authors discuss physiology, cloning and characterization of bkd gene cluster from Enterococcus faecalis containing genetic determinants that encode the enzymes constituting the branched-chain alpha-keto acid dehydrogenase complex. Results indicate the energy generation function of the E. faecalis system.

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.

A study was conducted to characterize the Enterococcus faecalis Nox C42S mutant. An Applied Photophysics DX.17MV stopped-flow spectrophotometer was used to carry out rapid-reaction analyses. Dissolved oxygen solutions for stopped-flow work were determined by bubbling buffer in glass syringes. Results confirmed a C(4a)-peroxyflavin intermediate in the oxygen reaction of the mutant. Findings also showed that the replacement of Cys42 with Ser leads to an altered O2 reduction stoichiometry.

Recent studies have shown that the flavoprotein reduced peroxidase from Enterococcus faecalis 10C1 contains, aside from FAD, a cysteine-sulfenic redox center. To characterize this redox center, an expression protocol was initially developed which yields [3-13C]Cys42-labeled protein in 100 mg quantities. Using 13C nuclear magnetic resonance analysis, the study provided additional evidences in support of the existence of the cysteine-sulfenic redox center. It also added significantly to the the structure-based understanding of protein-sulfenic acid stabilization and function.

An investigation of the crystal structure of the flavoprotein NADH peroxidase through reduced exposure to ambient oxygen demonstrates the existence of the Cys42-sulfenic acid as the functional non-flavin redox center of the peroxidase. The Cys42-sulfenic acid atoms Calpha-Cbeta-Sgamma-O exhibit a planar arrangement stacked parallel to the si face of the FAD isoalloxazine. The sulfenyl oxygen atom is located just 3.3 Angstrom from FAD-C4A and His 10-Nepsilon2 forms a hydrogen bond with the sulfenic acid oxygen. The sulfenic acid FAD charge-transfer interaction is examined.

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.

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 kinetic, redox properties and 2.30 Angstrom resolution structural characterization of Bacillus anthracis coenzyme A-disulfide reductase (CoADR, BACoADR) is described. The results from the NAD(P)H-reduced BACoADR structures provide insight into the reduced form of Cys42-SH/CoASH) of the Cys42-SSCoA redox center.

The structural and functional divergence between [alpha]-glycerophosphate oxidase (GlpO) from Streptococcus sp. and the bacterial and mitochondrial [alpha]-glycerophosphate dehydrogenases (GlpDs) is demonstrated.

The multiwavelength anomalous dispersion data was used to solve the crystal structure of Staphylococcus aureus coenzyme A-disulfide reductase (CoADR) and refine it at a resolution of 1.54 Angstrom. The bioinformatics analyses indicate that CoADR enzymes are broadly distributed in both bacterial and archaeal kingdoms, suggesting an even broader significance for the CoASH/CoAS-disulfide redox system in prokaryotic thiol/disulfide homeostasis.

The first structure of a glycerol kinase from a Gram-positive organism, Enterococcus casseliflavus is determined to 2.8 Armstrong resolution in the presence of glycerol and to 2.5 Armstrong resolution in the absensce of substrate. The mechanism of catalysis employed in the glycerol metabolism pathway is well understood by the structures of the Enterococcus Casseliflavus apo and complex glycerol kinase forms.

We analyzed 69 eastern Tennessee wildlife samples for Baylisascaris spp. during 2011. The prevalence of Baylisascaris spp. in raccoons (Procyon lotor) was 16% (8/49), an increase compared to previous surveys in this region. One Virginia opossum (Didelphis virginiana) had eggs in its feces, indicating that opossums can play a role in Baylisascaris spp. transmission. [ABSTRACT FROM AUTHOR]