Structural insights into the octamerization of glycerol dehydrogenase.

Glycerol dehydrogenase (GDH) catalyzes glycerol oxidation to dihydroxyacetone in a NAD+-dependent manner. As an initiator of the oxidative pathway of glycerol metabolism, a variety of functional and structural studies of GDH have been conducted previously. Structural studies revealed intriguing features of GDH, like the flexible β-hairpin and its significance.

Deglycosylation of eukaryotic-expressed flagellin restores adjuvanticity.

Flagellin, the TLR5 agonist, shows potent adjuvant activities in diverse vaccines and immunotherapies. Vibrio vulnificus flagellin B expressed in eukaryotic cells (eFlaB) could not stimulate TLR5 signaling. Enzymatic deglycosylation restored eFlaB's TLR5 stimulating functionality, suggesting that glycosylation interferes with eFlaB binding to TLR5.

Fumarase activity in NAD-dependent malic enzyme, MaeA, from Escherichia coli.

NAD-dependent malic enzymes catalyze NAD reduction to NADH while converting malate to pyruvate and CO. In this study, NAD was reduced to NADH by MaeA, NAD-dependent malic enzyme from Escherichia coli, when fumarate was used as substrate. This suggested that MaeA catalyzed the conversion of fumarate to malate and then malate to pyruvate.

Structural and functional insights into the flexible β-hairpin of glycerol dehydrogenase.

During glycerol metabolism, the initial step of glycerol oxidation is catalysed by glycerol dehydrogenase (GDH), which converts glycerol to dihydroxyacetone in a NAD -dependent manner via an ordered Bi-Bi kinetic mechanism. Structural studies conducted with GDH from various species have mainly elucidated structural details of the active site and ligand binding. However, the structure of the full GDH complex with both cofactor and substrate bound is not determined, and thus, the structural basis of the kinetic mechanism of GDH remains unclear.

Fluorescence Spectroscopic Analysis of ppGpp Binding to cAMP Receptor Protein and Histone-Like Nucleoid Structuring Protein.

The cyclic AMP receptor protein (CRP) is one of the best-known transcription factors, regulating about 400 genes. The histone-like nucleoid structuring protein (H-NS) is one of the nucleoid-forming proteins and is responsible for DNA packaging and gene repression in prokaryotes. In this study, the binding of ppGpp to CRP and H-NS was determined by fluorescence spectroscopy.

Determination of glutathione-binding to proteins by fluorescence spectroscopy.

Glutathione (GSH) is the most abundant non-protein thiol and its cellular concentration has been reported as 17 mM in Escherichia coli. This study introduces a label-free method to determine the binding affinity of GSH to proteins, utilizing the intrinsic fluorescence of proteins; the dissociation constants of GSH for d-arabinose 5-phosphate isomerase KdsD, fumarase C, malate dehydrogenase, and RNA polymerase subunit α have been determined as 96 ± 8, 246 ± 42, 292 ± 78, and 296 ± 97 μM, respectively. The dissociation constants, less than 2% of the cellular concentration of GSH, suggests that protein-GSH interactions are strong enough to make all of the GSH-binding sites occupied fully.

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3.

Mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas systems use a "Cascade" ribonucleoprotein complex to guide RNA specifically to complementary sequence in invader double-stranded DNA (dsDNA), a process called "interference." After target recognition by Cascade, formation of an R-loop triggers recruitment of a Cas3 nuclease-helicase, completing the interference process by destroying the invader dsDNA.

Molecular characterization of vulnibactin biosynthesis in Vibrio vulnificus indicates the existence of an alternative siderophore.

Vibrio vulnificus is a halophilic estuarine bacterium that causes fatal septicemia and necrotizing wound infections in humans. Virulent V. vulnificus isolates produce a catechol siderophore called vulnibactin, made up of one residue of 2, 3-dihydroxybenzoic acid (2, 3-DHBA) and two residues of salicylic acid (SA).

Contribution of six flagellin genes to the flagellum biogenesis of Vibrio vulnificus and in vivo invasion.

Vibrio vulnificus is a halophilic pathogenic bacterium that is motile due to the presence of a single polar flagellum. V. vulnificus possesses a total of six flagellin genes organized into two loci (flaFBA and flaCDE).

Structures of the γ-class carbonic anhydrase homologue YrdA suggest a possible allosteric switch.

The YrdA protein shows high sequence similarity to γ-class carbonic anhydrase (γ-CA) proteins and is classified as part of the γ-CA protein family. However, its function has not been fully elucidated as it lacks several of the conserved residues that are considered to be necessary for γ-CA catalysis. Interestingly, a homologue of γ-CA from Methanosarcina thermophila and a β-carboxysomal γ-CA from a β-cyanobacterium have shown that these catalytic residues are not always conserved in γ-CAs.

Effect of Salmonella treatment on an implanted tumor (CT26) in a mouse model.

The use of bacteria has contributed to recent advances in targeted cancer therapy especially for its tumor-specific accumulation and proliferation. In this study, we investigated the molecular events following bacterial therapy using an attenuated Salmonella Typhimurium defective in ppGpp synthesis (ΔppGpp), by analyzing those proteins differentially expressed in tumor tissues from treated and untreated mice. CT26 murine colon cancer cells were implanted in BALB/c mice and allowed to form tumors.

Crystal structure of a minimal nitroreductase, ydjA, from Escherichia coli K12 with and without FMN cofactor.

Nitroreductases (NTR) are enzymes that reduce hazardous nitroaromatic compounds and are of special interest due to their potential use in bioremediation and their activation of prodrugs in directed anticancer therapies. We elucidated the crystal structures of ydjA from Escherichia coli (Ec_ydjA), one of the smallest NTRs, in its flavin mononucleotide (FMN)-bound and cofactor-free forms. The alpha+beta mixed monomeric Ec_ydjA forms a homodimeric structure through the interactions of the long central helices and the extended regions at both termini.

High-resolution structure of ybfF from Escherichia coli K12: a unique substrate-binding crevice generated by domain arrangement.

Esterases are one of the most common enzymes and are involved in diverse cellular functions. ybfF protein from Escherichia coli (Ec_ybfF) belongs to the esterase family for the large substrates, palmitoyl coenzyme A and malonyl coenzyme A, which are important cellular intermediates for energy conversion and biomolecular synthesis. To obtain molecular information on ybfF esterase, which is found in a wide range of microorganisms, we elucidated the crystal structures of Ec_ybfF in complexes with small molecules at resolutions of 1.

Crystallization and preliminary X-ray diffraction analysis of ydjA, a minimal nitroreductase from Escherichia coli K12.

Nitroreductases that reduce hazardous nitroaromatic compounds are of interest because of their central role in nitroaromatic toxicity, their potential use in bioremediation and their utility in activating prodrugs in directed anticancer therapies. To provide the molecular background to the enzymatic mechanism of the ydjA nitroreductase, which is one of the smallest nitroreductases, the ydjA gene from Escherichia coli K12 was cloned and expressed and the expressed protein Ec_ydjA was purified. Ec_ydjA was crystallized from 20%(w/v) polyethylene glycol 1000, 0.

Crystallization and preliminary X-ray diffraction analysis of ybfF, a new esterase from Escherichia coli K12.

The product of the recently discovered ybfF gene, which belongs to the esterase family, does not show high sequence similarity to other esterases. To provide the molecular background to the enzymatic mechanism of the ybfF esterase, the ybfF protein from Escherichia coli K12 (Ec_ybfF) was cloned, expressed and purified. The Ec_ybfF protein was crystallized from 60% Tacsimate and 0.

A zinc finger-containing glycine-rich RNA-binding protein, atRZ-1a, has a negative impact on seed germination and seedling growth of Arabidopsis thaliana under salt or drought stress conditions.

Despite the fact that glycine-rich RNA-binding proteins (GRPs) have been implicated in the responses of plants to changing environmental conditions, the reports demonstrating their biological roles are severely limited. Here, we examined the functional roles of a zinc finger-containing GRP, designated atRZ-1a, in Arabidopsis thaliana under drought or salt stress conditions. Transgenic Arabidopsis plants overexpressing atRZ-1a displayed retarded germination and seedling growth compared with the wild-type plants under salt or dehydration stress conditions.

Functional characterization of a glycine-rich RNA-binding protein 2 in Arabidopsis thaliana under abiotic stress conditions.

Although glycine-rich RNA-binding protein 2 (GRP2) has been implicated in plant responses to environmental stresses, the function and importance of GRP2 in stress responses are largely unknown. Here, we examined the functional roles of GRP2 in Arabidopsis thaliana under high-salinity, cold or osmotic stress. GRP2 affects seed germination of Arabidopsis plants under salt stress, but does not influence seed germination and seedling growth of Arabidopsis plants under osmotic stress.

Cold shock domain proteins and glycine-rich RNA-binding proteins from Arabidopsis thaliana can promote the cold adaptation process in Escherichia coli.

Despite the fact that cold shock domain proteins (CSDPs) and glycine-rich RNA-binding proteins (GRPs) have been implicated to play a role during the cold adaptation process, their importance and function in eukaryotes, including plants, are largely unknown. To understand the functional role of plant CSDPs and GRPs in the cold response, two CSDPs (CSDP1 and CSDP2) and three GRPs (GRP2, GRP4 and GRP7) from Arabidopsis thaliana were investigated. Heterologous expression of CSDP1 or GRP7 complemented the cold sensitivity of BX04 mutant Escherichia coli that lack four cold shock proteins (CSPs) and is highly sensitive to cold stress, and resulted in better survival rate than control cells during incubation at low temperature.

Activating transcription factor-2 mediates transcriptional regulation of gluconeogenic gene PEPCK by retinoic acid.

All-trans-retinoic acid (RA) is known to increase the rate of transcription of the PEPCK gene upon engagement of the RA receptor (RAR). RA also mediates induction of specific gene transcription via several signaling pathways as a nongenomic effect. Here we show that RA upregulation of PEPCK promoter activity requires the cAMP response element (CRE)-1 in addition to the RA-response element and that activating transcription factor-2 (ATF-2) binds the CRE element to mediate this effect.

Ebselen has dehydroascorbate reductase and thioltransferase-like activities.

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a seleno-organic compound, has been reported to mimic glutathione peroxidase (GPX). Since bovine erythrocyte GPX showed dehydroascorbic acid (DHA) reductase and thioltransferase (TTase) activities, ebselen was also examined for DHA reductase and TTase-like activities. Evidence is reported that, in the presence of GSH, ebselen catalyzed the in vitro reduction of DHA to L-ascorbic acid in a dose-dependent manner.