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Vitamin D homeostasis in the immune system is the focus of this review. The production of both the activating (25- and 1[alpha]-hydroxylase) and the metabolizing (24-hydroxylase) enzymes by cells of the immune system itself, indicates that 1,25[(OH).sub.2][D.sub.3] can be produced locally in immune reaction sites. Moreover, the strict regulation of these enzymes by immune signals is highly suggestive for an autocrine/paracrine role in the immune system, and opens new treatment possibilities.

Background. It is hypothesized that free radical damage contributes to aging. Age-related decline in activity of the antioxidant enzyme glutathione peroxidase (GPx) may contribute to increased free radicals. We hypothesized that GPx activity decreases with age in a population of older women with disability. Methods. Whole blood GPx activity was measured in baseline stored samples from participants in the Women's Health and Aging Study I, a cohort of disabled community-dwelling older women. Linear regression was used to determine cross- sectional associations between GPx activity and age, adjusting for hemoglobin, coronary disease, diabetes, selenium, and body mass index. Results. Six hundred one participants had complete demographic, disease, and laboratory information. An inverse association was observed between GPx and age (regression coefficient = -2.9, p < .001), indicating that for each 1-year increase in age, GPx activity decreased by 2.9 [micro]mol/min/L. This finding remained significant after adjustment for hemoglobin, coronary disease, diabetes, and selenium, but not after adjustment for body mass index and weight loss. Conclusion, This is the first study to examine the association between age and GPx activity in an older adult cohort with disability and chronic disease. These findings suggest that, after age 65, GPx activity declines with age in older women with disability. This decline does not appear to be related to diseases that have been previously reported to alter GPx activity. Longitudinal examination of GPx activity and other antioxidant enzymes in diverse populations of older adults will provide additional insight into age- and disease-related changes in these systems. Key Words: Glutathione peroxidase--Oxidative stress--Aging--Older adult.

The synthesis of trifluoromethyl phenyl benzamides and their effect on histone acetyltransferase (HAT) activity of p300 is reported. It is found that pentadecyl hydrocarbon chain of (N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide) is required to activate the HAT under certain context.

The development of menadione chemistry had led to choice of 6-[2'-(3'-methyl)-1',4'-naphthoquinolyl]hexanoic acid, called [M.sub.5] as a potent reversible and uncompetitive inhibitor of both human and Plasmodium falciparum glutathione reductases. The synthesis and kinetic characterization of a fluoromethyl-[M.sub.5] analogue that acts as a mechanism-based inhibitor of both enzymes is described.

Neutrophil NADPH oxidase plays a key role in host defense and in inflammation by releasing large amounts of superoxide and other ROSs. Proinflammatory cytokines such as GM-CSF and TNF-[alpha] prime [...]

Many enveloped viruses exploit the class E vacuolar protein-sorting (VPS) pathway to bud from cells, and use peptide motifs to recruit specific class E VPS factors. Homologous to E6AP COOH terminus (HECT) ubiquitin ligases have been implicated as cofactors for PPXY motif-dependent budding, but precisely which members of this family are responsible, and how they access the VPS pathway is unclear. Here, we show that PPXY-dependent viral budding is unusually sensitive to inhibitory fragments derived from specific HECT ubiquitin ligases, namely WWP1 and WWP2. We also show that WWP1, WWP2, or Itch ubiquitin ligase recruitment promotes PPXY-dependent virion release, and that this function requires that the HECT ubiquitin ligase domain be catalytically active. Finally, we show that several mammalian HECT ubiquitin ligases, including WWP1, WWP2, and Itch are recruited to class E compartments induced by dominant negative forms of the class E VPS ATPase, VPS4. These data indicate that specific HECT ubiquitin ligases can link PPXY motifs to the VPS pathway to induce viral budding.

Byline: Wioletta BAaszczak (1), Jadwiga Sadowska (1), Cristina M. Rosell (2), Jozef Fornal (1) Keywords: [alpha]-Amylase; Microstructure; Dough; Bread crumb; Staling; Texture Abstract: The influence of fungal and bacterial [alpha]-amylases on the texture and microstructure of dough and bread was investigated. Loaf specific volume, crumb porosity and texture properties were accepted as bread structure and texture characteristics. The microstructure analysis of dough and bread using light and scanning microscopy methods was performed. The changes in loaf specific volume, crumb porosity and texture properties showed different anti-staling activity of the enzymes used. The simultaneous action of enzyme addition and the fermentation process evoked significant changes in the microstructure of dough. The character of these changes depended on the kind of enzyme used. A substantial effect of both amylases on starch behaviour during bread baking and staling was also observed. Author Affiliation: (1) Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-747, Olsztyn, Poland (2) Instituto de Agroquimica y Technologia de Alimentos (IATA-CSIC), Burjassot, Valencia, Spain Article History: Registration Date: 23/06/2004 Received Date: 11/03/2004 Online Date: 04/08/2004

Byline: N. L. Vasilenko (1), G. A. Nevinsky (1) Keywords: DNA repair; repair enzyme structure; function mechanisms; protein complexes Abstract: DNA repair is of paramount importance to protect the integrity of the cell genome. Over the last decade, DNA repair enzymes have been intensely studied owing to their role in mutagenesis, carcinogenesis, and aging. This led to discoveries of novel enzymes, protein factors as well as their intermolecular complexes involved in the repair process. Alternative pathways of cell response to the same type of damage were identified. Data on the tertiary structure of a number of enzymes and mechanisms of their molecular interaction were obtained. This paper is an overview of latest advances in the research of cell response to DNA damage via direct, base and nucleotide excision, and mismatch DNA repair. Author Affiliation: (1) Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of Sciences, Novosibirsk, 630090, Russia Article History: Registration Date: 12/10/2004

Synechococcus sp. strain RF-1 is a unicellular freshwater cyanobacterium that fixes [N.sub.2] aerobically and exhibits a circadian rhythm for nitrogenase activity under a light-dark regimen. Synechococcus sp. strain RF-1 also utilizes nitrate, nitrite, or ammonium for growth. Under the diazotrophic growth, the nitrate uptake in Synechococcus sp. strain RF-1 was induced by nitrate or nitrite but repressed by ammonium. In contrast, a prominent nitrate reductase (NR) activity was detected in diazotrophically grown cells using the reduced methyl viologen assay. The NR activity was not inhibited by ammonium and only slightly enhanced by nitrate. The different expression patterns of nitrate uptake and NR in Synechococcus sp. strain RF-1 were reflected in general at the transcript level determined by reverse transcriptase PCR. Under both nitrate-induced and uninduced conditions, the in situ NR activity exhibited similar biphasic kinetics for nitrate. The recombinant NR encoded by the narB gene of Synechococcus sp. strain RF-1, expressed in E. coli, also showed the biphasic kinetics with similar pH and temperature profiles. By in-gel NR activity assay, the recombinant NarB was found to exist as a single form. Both the high- and low-affinity NR activities of the recombinant NarB showed the same thermostability. When modified at the N terminus by a polyhistidine tag, the recombinant NR activity was shifted from biphasic to hyperbolic kinetics and showed only a single [K.sub.m] for nitrate, indicating the functional importance of the NarB N-terminal structure in NR kinetics.

The enzyme 2-phosphoglycolate phosphatase from Escherichia coli, encoded by the gph gene, was purified and characterized. The enzyme was highly specific for 2-phosphoglycolate and showed good catalytic efficiency ([k.sub.cat]/[K.sub.m]), which enabled the conversion of this substrate even at low intracellular concentrations. A comparison of the structural and functional features of this enzyme with those of 2-phosphoglycolate phosphatases of different origins showed a high similarity of the sequences, implying the use of the same catalytic mechanism. Western blot analysis revealed constitutive expression of the gph gene, regardless of the carbon source used, growth stage, or oxidative stress conditions. We showed that this housekeeping enzyme is involved in the dissimilation of the intracellular 2-phosphoglycolate formed in the DNA repair of 3'-phosphoglycolate ends. DNA strand breaks of this kind are caused by agents such as the radiomimetic compound bleomycin. The differential response between a 2-phosphoglycolate phosphatase-deficient mutant and its parental strain after treatment with bleomycin allowed us to connect the intracellular formation of 2-phosphoglycolate with the production of glycolate, which is subsequently incorporated into general metabolism. We thus provide evidence for a salvage function of 2-phosphoglycolate phosphatase in the metabolism of a two-carbon compound generated by the cellular DNA repair machinery.

MurG is an essential glycosyltransferase that forms the glycosidic linkage between N-acetyl muramyl pentapeptide and N-acetyl glucosamine in the biosynthesis of the bacterial cell wall. This enzyme is a member of a major superfamily of NDP-glycosyltransferases for which no x-ray structures containing intact substrates have been reported. Here we present the 2.5-[Angstrom] crystal structure of Escherichia coli MurG in complex with its donor substrate, UDP-GlcNAc. Combined with genomic analysis of other superfamily members and site-specific mutagenesis of E. coli MurG, this structure sheds light on the molecular basis for both donor and acceptor selectivity for the superfamily. This structural analysis suggests that it will be possible to evolve new glycosyltransferases from prototypical superfamily members by varying two key loops while maintaining the overall architecture of the family and preserving key residues.

Research reveals that horseradish peroxidase exhibits high pressure stability and an unfolding midpoint of 12.0 plus or minus 0.1 kbar. The enzyme's stability is attributed to a rigid core. Data show that although calcium removal and disulfide bonds reduction affect the protein folding and stability, the residual stability is still above averageof other proteins. Heme substitution does not affect the enzyme.

Results reveal that the complex formation by the NADPH:FMN oxidoreductase with luciferase occurs at a 1:1 molar ratio of the monomers of the apoenzyme or holoenzyme. However, no complex formation is observed between luciferase and oxidoreductase dimers.

The catalytic mechanism of NADPH:protochlorophyllide oxidoreductase involves two steps that do not require light and convert light-driven reaction products into photoactive intermediate and Chlide. Data indicate that the dark reactions occur at or above the 'glass transition' temperature of proteins, indicating that domain movement and/or structural reorganization of the protein is required for catalysis.

Research reveals that LytG is an autolysin synthesized during vegetative growth of Bacillus subtilis. Peptide analysis suggest that LytG is a glucosaminidase involved in the structural organization of peptidoglycan during bacterial growth. The enzyme mediates cell division, lysis, and cell motility.

This article delineates the mechanism of action of thymidylate synthase, an enzyme involved in the de novo synthesis of dTMP nucleotide essential for DNA synthesis. The enzyme utilizes CH(sub)2H(sub)4folate, a cofactor which serves as both the one-carbon donor to the substrate dUMP and a reductant in a dynamic alignment and realignment of reactants.

This article discusses the structure-function aspects of thymidylate synthase, an enzyme involved in the de novo pathway for synthesis of the DNA nucleotide dTMP. Crystallographic data are interpreted to address how the progressive conformational changes in the enzyme are coupled to function such as sequestration and multistep reorientation required for the enzyme activity.

The crystal structure of the human SET7/9 large fragment reveals a beta-domain and conserved SET domains at the N-terminus and the mutagenesis studies identify the involvement of two amino acid residues at the C-terminus in the catalysis. Further, the binding of the AdoMet cofactor suggests the involvement of invariant residues as well in catalysis.

The genetic and physiologic role played by SET domain enzymes is examined using a Rubisco large subunit methyltransferase-S-adenosylhomocysteine and a HEPES ion complex. Results show an all-beta structure for the SET domain whose conserved regions bind S-adenosylmethionine and lysine substrate. The catalysis is mediated by amino acid tyrosine at the conserved site.

Results demonstrate that amino acid substitutions in KdpA protein, involved in potassium binding and translocation, renders KdpFABC complex totally incapable of selecting cations in Escherichia coli.

Results reveal that N-isopropylammelide aminohydrolase reacts with a variety of s-triazine substrates cleaving the side chain carbon-nitrogen bonds. It also reacts with s-triazine pesticides. The enzyme contains a five-coordinate metal center and the activity is dependent on the zinc moiety.

Research indicates that the HisZ and HisG proteins, constituents of the HisZG heteromeric ATP-phosphoribosyl transferase, exhibit a simple monomer-dimer structures, while the holoenzyme presents an octamer with four subunits each of HisZ and HisG. Furthermore, the sedimentation properties of the complex depend on the substrates and inhibitors present.

Substrate binding of the human mitochondrial branched chain aminotransferase with substrate amino acids, isoleucine ketimine and pyridoxamine phosphate, does not involve domain or loop closure as seen in other proteins. Furthermore, cysteine residues 315 and 318 are important for the enzyme structure and form a thiol-thiolate hydrogen bond.

Transglutaminase 2 (TG2) is an inducible transamidating acyltransferase that catalyzes [Ca.sup.2+]-dependent protein modifications. It acts as a G protein in transmembrane signalling and as a cell surface adhesion mediator, this distinguishes it from other members of the transglutaminase family. The sequence motifs and domains revealed in the recent TG2 structure, can each be assigned distinct cellular functions, including the regulation of cytoskeleton, cell adhesion and cell death. Ablation of TG2 in mice results in impaired wound healing, autoimmunity and diabetes, reflecting the number and variety of TG2 functions. An important role for the enzyme in the pathogenesis of coeliac disease, fibrosis and neurodegenerative disorders has also been demonstrated, making TG2 an important therapeutic target.

Site-specific mutagenesis reveals that the mutant phenylalanine dehydrogenase, in which asparagine residue 145 is substituted, exhibits more than 50-fold discrimination between L-phenylalanine and L-tyrosine with a higher specificity for the former. Data indicate that the specificity increases are due to large decreases in K(sub)m values.

The enzymatic base flipping by uracil DNA glycosylase is facilitated by a pyrene nucleotide wedge place opposite a uracil base in duplex DNA so as to preorganize the target base in an extrahelical conformation. Data show that a lock-and-key mechanism facilitates the enzyme mediated uracil recognition by the pyrene wedge substrate.

The crystal structure of the nonprocessive cellulase Cel9M from Clostridium cellulolyticum exhibits a dockerin and a catalytic module that interact with the substrate. Comparison of Cel9M with the other structurally related 9 family cellulases suggest differences in the substrate activity between the variants.

Results reveal that the structure of Bacillus subtilis nitric oxide synthase resembles that of mammalian nitric oxide synthase oxygenase domain but without the N-terminal beta-hairpin hook and the zinc-binding region, suggesting different binding properties for pterin side chains in the former. Data indicate that the heme pocket recognizes L-Arg and N-(omega)-hydroxy-L-Arg from which the enzyme produces nitrogen oxides.

The synthesis of the catecholic siderophore bacillibactin is accomplished by the nonribosomal peptide synthetase (NRPS) encoded by the dhb operon. DhbE is responsible for the initial step in bacillibactin synthesis, the activation of the aryl acid 2,3-dihydroxybenzoate (DHB). The stand-alone adenylation (A) domain DhbE, the structure of which is presented here, exhibits greatest homology to other NRPS A-domains, acyl-CoA ligases and luciferases. It's structure is solved in three different states, without the ligands ATP and DHB (native state), with the product DHB-AMP (adenylate state) and with the hydrolyzed product AMP and DHB (hydrolyzed state). The 59.9-kDa protein folds into two domains, with the active site at the interface between them. In contrast to previous proposals of a major reorientation of the large and small domains on substrate binding, we observe only local structural rearrangements. The structure of the phosphate binding loop could be determined, a motif common to many adenylate-forming enzymes, as well as with bound DHB-adenylate and the hydrolyzed product DHB*AMP. Based on the structure and amino acid sequence alignments, an adapted specificity conferring code for aryl acid activating domains is proposed, allowing assignment of substrate specificity to gene products of previously unknown function. adenylation domain | nonribosomal peptide synthesis | X-ray crystal structure | antibiotic biosynthesis | siderophore formation

The induction of photolysis of tryptophan residues requires a direct contact between the excited tryptophan and the disulfide bond in the goat alpha-lactalbumin. The tryptophan-mediated photoreduction of a disulfide bond forms a single free cysteine with a second cysteine forming a thioether linkage with the indole group of tryptophan.

Results demonstrate that there is no basic nitrogen atom involvement in the high-affinity binding of cytochrome P450 2D6 to substrates. Data indicate that some substrates bind through a negative charge either by electrostatic interaction or by hydrogen-bonding while others do not share this property.

The digestion of Bowtie junctions by RuvC reveals that they function as symmetric immobile junctions and that RuvC cleaves antiparallel junctions more efficiently than parallel juctions, indicating that the antiparallel junctions are better substrates for RuvC.

The role of the DNA flexibility in enzyme-DNA interactions is tested by kinetically analyzing the effects of the high efficiency AUA/TAT and low efficiency GUG/CAC sequences of the uracil DNA glycosylase. Data indicate that the differences in the enzyme's efficiency are largely due to differences in the binding and not catalysis.

The mutation of a single amino acid change at the RNase H catalytic site of HIV-reverse transcriptase is examined with respect to its role in the binding of nucleic acid substrates at different concentrations of magnesium. Results reveal that alterations of the magnesium-binding residues affect enzyme-template stability and show enhanced enzyme-DNA binding.

Results reveal that the nonsubstrate ligand binding site is not the intersubunit cleft in the cysteine 112 glutathiolated glutathione S-transferase A1-1 or in the Cys 112-modified enzyme. Data indicate that lithocholic acid and estradiol disulfate ligands act as competitive inhibitors of the native enzyme and bind to the hydrophobic substrate site.

Crystal structure analysis of the complex between phospholipase A(sub)2 and aristolochic acid at 1.7 angstrom resolution reveals the specific binding of aristolochic acid to the enzyme and the attendant conformational changes in the enzyme leading to the inhibitory action of prostaglandins synthesis. Arachidonic acid is an intermediate in the biosynthesis of prostaglandins.

Results reveal that replacement of the conserved amino acid residues does not decrease the reactivity of the active-site Cys161 toward iodoacetamide or the rate of transfer of acyl residues to Cys161. Details of the catalytic mechanism of the beta-ketoacyl synthase domain with respect to the animal fatty acid synthase complex are presented.

Results show that the water-soluble domain of the nitric oxide reductase subunit C exhibits a alpha-helical structure and contains a partially solvent-exposed heme. The heme iron is coordinated by histidine and methionine ligands in both. Data suggest that the soluble domain of subunit C interacts with the catalytic subunit B.

The human glutaminyl cyclase expressed in the secretory pathway of Pichia pastoris contain disulfide bond. Treatment with dithiothreitol inactivates the enzyme as well as changes conformation of the enzyme. This enzyme differs from that expressed in Escherichia coli.

Results reveal that six residues of the Nudix box sequence of the Nudix family hydrolytic enzyme GDP-mannose mannosyl hydrolase participate in the catalysis. Data indicate that the mechanism of action of GDP-mannose mannosyl hydrolase involves base catalysis.

Results show that the interaction of acetylcholinesterase with different molecules of the anti-Alzheimer drug (-)-huperzine A and its homologues occurs at the 'anionic' subsite of the active site of the enzyme. Data indicate that rearrangement of the amino acid residues of the 'oxyanion' hole occurs due to a peptide flip.

The X-ray crystallographic analysis of an apo, a binary complex, and a tertiary complex of human formaldehyde dehydrogenase, with the respective substrates bound, show that the binding of the first substrate, alcohol or enzyme, leads to only minor changes in the enzyme structure. Data suggest that the substrate binding follows a random mechanism.

Results show that the conformational changes that mediate the active site assembly and metal binding by EcoRV endonuclease are modulated by the electrostatic effects in the DNA cleavage region of the enzyme. Kinetic and crystallographic analyses of the charged active site mutants reveal that site-specific mutations do not affect enzyme activity.

The conformational change in the 12S subunit of transcarboxylase is triggered by substrate binding and involves an increase in alpha-helix with varying number of MM-CoA ligands bound per 12S hexamer. Results indicate that the -COO- group on MM-CoA ligand mediates the cooperative conformational change in 12S.