Glucose-inhibited Division Protein Research Articles

Pharmacological assessment of Ru(II) complex with GidA protein- A novel topoisomerase II inhibitor towards cancer therapeutics

The interactions of ruthenium(II) complex with Glucose inhibited division protein A (GidA protein) was studied through various spectroscopic techniques with the ultimate goal of preparing adducts with good selectivity for cancer cells. In all the cases, formation of a tight metal-protein conjugate was observed. The influence of pH, reducing agents and chelators on the formation of adduct was analysed by UV- visible spectroscopy. While there was no effect on the addition of sodium ascorbate, some alterations on some selected bands were seen on the UV-visible spectra on the addition of EDTA. The adduct was stable in the pH range of 5-8. Addition of ruthenium(II) complex effectively quenched the intrinsic fluorescence of GidA and it occurred through static quenching. The effect of ruthenium(II) complex on the conformation of GidA has been examined by analyzing CD spectrum. Though, there was some conformational changes observed in the presence of ruthenium(II) complex, α- helix in the secondary structure of GidA retained its identity. Molecular docking of ruthenium(II) complex with GidA also indicated that GidA docks through hydrophobic interaction. The stable semisynthetic complex (ruthenium(II) complex with GidA) was checked for topoisomerase II inhibition. Relaxation and decatenation assay proved topoisomerase II inhibition of semisynthetic complex. Communicated by Ramaswamy H. Sarma

LC_Glucose-Inhibited Division Protein Is Required for Motility, Biofilm Formation, and Stress Response in Lysobacter capsici X2-3.

Glucose-inhibited division protein (GidA) plays a critical role in the growth, stress response, and virulence of bacteria. However, how gidA may affect plant growth-promoting bacteria (PGPB) is still not clear. Our study aimed to describe the regulatory function of the gidA gene in Lysobacter capsici, which produces a variety of lytic enzymes and novel antibiotics. Here, we generated an LC_GidA mutant, MT16, and an LC_GidA complemented strain, Com-16, by plasmid integration. The deletion of LC_GidA resulted in an attenuation of the bacterial growth rate, motility, and biofilm formation of L. capsici. Root colonization assays demonstrated that the LC_GidA mutant showed reduced colonization of wheat roots. In addition, disruption of LC_GidA showed a clear diminution of survival in the presence of high temperature, high salt, and different pH conditions. The downregulated expression of genes related to DNA replication, cell division, motility, and biofilm formation was further validated by real-time quantitative PCR (RT–qPCR). Together, understanding the regulatory function of GidA is helpful for improving the biocontrol of crop diseases and has strong potential for biological applications.

GidA, a tRNA Modification Enzyme, Contributes to the Growth, and Virulence of Streptococcus suis Serotype 2

Glucose-inhibited division protein (GidA), is a tRNA modification enzyme functioning together with MnmE in the addition of a carboxymethylaminomethyl group to position 5 of the anticodon wobble uridine of tRNA. Here, we report a GidA homolog from a Chinese isolate SC-19 of the zoonotic Streptococcus suis serotype 2 (SS2). gidA disruption led to a defective growth, increased capsule thickness, and reduced hemolytic activity. Moreover, the gidA deletion mutant (ΔgidA) displayed reduced mortality and bacterial loads in mice, reduced ability of adhesion to and invasion in epithelial cells, and increased sensitivity to phagocytosis. The iTRAQ analysis identified 372 differentially expressed (182 up- and 190 down-regulated) proteins in ΔgidA and SC-19. Numerous DNA replication, cell division, and virulence associated proteins were downregulated, whereas many capsule synthesis enzymes were upregulated by gidA disruption. This is consistent with the phenotypes of the mutant. Thus, GidA is a translational regulator that plays an important role in the growth, cell division, capsule biosynthesis, and virulence of SS2. Our findings provide new insight into the regulatory function of GidA in bacterial pathogens.

Purification of GidA protein, a novel topoisomerase II inhibitor produced by Streptomyces flavoviridis

The presence of topoisomerase II inhibition activities in the intracellular extract of Streptomyces flavoviridis was investigated. One active compound inhibiting relaxation activity of topoisomerase II was determined to be a protein. This active principle was purified to homogeneity by gel filtration followed by ion exchange chromatography. The apparent molecular mass was 42 kDa as determined by SDS-PAGE. MALDI TOF peptide mass fingerprinting analysis confirmed this topoisomerase II inhibitor, as glucose-inhibited division protein A (GidA) by MOWSE score of 72. The effects of purified GidA protein on DNA relaxation and decatenation by topoisomerase II were investigated. It inhibited topoisomerase II activity and acted as a topoisomerase poison that significantly stabilized the covalent DNA-topoisomerase II reaction intermediate "cleavable complex", as observed with etoposide. Collectively, these findings indicate that GidA is a potent inhibitor of topoisomerase II enzyme, which can be exploited for rational drug design in human carcinomas.

An intact SAM‐dependent methyltransferase fold is encoded by the human endothelin‐converting enzyme‐2 gene

A recent survey of protein expression patterns in patients with Alzheimer's disease (AD) has identified ece2 (chromosome: 3; Locations: 3q27.1) as the most significantly downregulated gene within the tested group. ece2 encodes endothelin-converting enzyme ECE2, a metalloprotease with a role in neuropeptide processing. Deficiency in the highly homologous ECE1 has earlier been linked to increased levels of AD-related {beta}-amyloid peptide in mice, consistent with a role for ECE in the degradation of that peptide. Initially, ECE2 was presumed to resemble ECE1, in that it comprises a single transmembrane region of {approx}20 residues flanked by a small amino-terminal cytosolic segment and a carboxy-terminal lumenar peptidase domain. The carboxy-terminal domain has significant sequence similarity to both neutral endopeptidase, for which an X-ray structure has been determined, and Kell blood group protein. After their initial discovery, multiple isoforms of ECE1 and ECE2 were discovered, generated by alternative splicing of multiple exons. The originally described ece2 transcript, RefSeq NM{_}174046, contains the amino-terminal cytosolic portion followed by the transmembrane region and peptidase domain (Fig. 1, isoform B). Another ece2 transcript, available from the Mammalian Gene Collection under MGC2408 (Fig. 1, isoform C), RefSeq accession NM{_}032331, is predicted to be translated into a 255 residue peptidemore » with low but detectable sequence similarity to known S-adenosyl-L-methionine (SAM)-dependent methyltransferases (SAM-MTs), such as the hypothetical protein TT1324 from Thermus thermophilis, PDB code 2GS9, which shares 30% amino acid sequence identity with ECE2 over 138 residues of the sequence. Intriguingly, another 'elongated' ece2 transcript (Fig. 1, isoform A) (RefSeq NM{_}014693) contains an amino-terminal portion of the putative SAM-MT domain, the transmembrane domain, and the protease domain. This suggests the possibility for coexistence of the putative SAM-MT and protease domains in a single polypeptide and their transmembrane interplay. Although sequence conservation across the SAM-MT family is weak, the structural fold is highly conserved. The most conserved part of this fold is the SAM-binding subdomain, which is shared between MGC2408 and hypothetical protein TT1324. Typically, the SAM-binding subdomain is flanked by a variable Nterminal extension and, at the C-terminus, by a substrate- binding subdomain, which varies enormously in size but preserves a conserved topology with three antiparallel b-strands. The 'elongated' transcript of ece2 lacks this substrate-binding subdomain. To test the hypothesis that the 255 residue ece2 gene product MGC2408 represents a complete SAM-MT fold, we have determined a crystal structure of this protein in the presence of SAH.« less

Crystallization and preliminary X-ray crystallographic studies of the small form of glucose-inhibited division protein A from Thermus thermophilus HB8.

Glucose-inhibited division protein A (GidA) acts in tRNA modification. It has been suggested that GidA is involved in the biosynthesis of the hypermodified nucleotide 5-methylaminomethyl-2-thiouridine in the wobble position of bacterial tRNAs, which stabilizes codon-anticodon interactions. Thermus thermophilus HB8 has a putative small gidA gene in addition to the normal gidA gene. The crystallization and preliminary X-ray crystallographic studies of the product of this small gidA gene (GidA(small)) are reported here. The crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 78.51, c = 66.10 A and one monomer per asymmetric unit. The crystals were found to diffract X-rays to beyond 1.65 A resolution.

Crystal structure of the small form of glucose-inhibited division protein A fromThermus thermophilusHB8

Crystal structure of the small form of glucose-inhibited division protein A from<i>Thermus thermophilus</i>HB8

Crystal structure of the Escherichia coli glucose-inhibited division protein B (GidB) reveals a methyltransferase fold.

Crystal structure of the Escherichia coli glucose-inhibited division protein B (GidB) reveals a methyltransferase fold.

Cloning and characterization of a Helicobacter bizzozeronii urease gene cluster.

The urease gene cluster from Helicobacter bizzozeronii was cloned and sequenced. A genomic library was constructed in a lambda-ZAPII vector using TSP5091-digested H. bizzozeronii chromosomal DNA. Four overlapping recombinant bacteriophages carrying the H. bizzozeronii urease genes were identified by using a fragment of H. bizzozeronii ureB as a probe. Sequence analysis of two clones (pHB1 and pHB3) revealed seven open reading frames encoding proteins with predicted masses of 26.5, 60.3, 21.7, 19.5, 28.6, 21.7 and 29.6 kDa representing the structural genes, Urease A and B and its accessory genes, urease I, E, F, G and H, respectively. In addition, three open reading frames upstream of the ureA gene encoding a putative tRNA transferase, a putative Glucose inhibited division protein B (GidB) and a protein with unknown function were also identified. A clone (pHB5) containing a complete urease gene cluster was constructed. The homologue analysis revealed that UreA polypeptide exhibited 64-90% identity to that of Helicobacter heilmanii, Helicobacter felis, Helicobacter pylori, Helicobacter mustelae and Helicobacter hepaticus. UreB polypeptides exhibited 76.8-96% identity to that of H. heilmanii, H. felis, H. pylori, H. mustelae and H. hepaticus. The UreI, E, F, G and H also showed 44-86% identity to that of H. pylori. Among these accessory genes, UreE had a lowest percentage identity to that of H. pylori.

GidA is an FAD‐binding protein involved in development of Myxococcus xanthus

A gene encoding a homologue of the Escherichia coli GidA protein (glucose-inhibited division protein A) lies immediately upstream of aglU, a gene encoding a WD-repeat protein required for motility and development in Myxococcus xanthus. The GidA protein of M. xanthus shares about 48% identity overall with the small (approximately equal to 450 amino acid) form of GidA from eubacteria and about 24% identity overall with the large (approximately equal to 620 amino acid) form of GidA from eubacteria and eukaryotes. Each of these proteins has a conserved dinucleotide-binding motif at the N-terminus. To determine if GidA binds dinucleotide, the M. xanthus gene was expressed with a His6 tag in E. coli cells. Purified rGidA is a yellow protein that absorbs maximally at 374 and 450 nm, consistent with FAD or FMN. Thin-layer chromatography (TLC) showed that rGidA contains an FAD cofactor. Fractionation and immunocytochemical localization show that full length GidA protein is present in the cytoplasm and transported to the periplasm of vegetative-grown M. xanthus cells. In cells that have been starved for nutrients, GidA is found in the cytoplasm. Although GidA lacks an obvious signal sequence, it contains a twin arginine transport (Tat) motif, which is conserved among proteins that bind cofactors in the cytoplasm and are transported to the periplasm as folded proteins. To determine if GidA, like AglU, is involved in motility and development, the gidA gene was disrupted. The gidA- mutant has wild-type gliding motility and initially is able to form fruiting bodies like the wild type when starved for nutrients. However, after several generations, a stable derivative arises, gidA*, which is indistinguishable from the gidA- parent on vegetative medium, but is no longer able to form fruiting bodies. The gidA* mutant releases a heat-stable, protease-resistant, small molecular weight molecule that acts in trans to inhibit aggregation and gene expression of wild-type cells during development.

Chromosome mapping of the sweet potato little leaf phytoplasma reveals genome heterogeneity within the phytoplasmas.

To further understand the genomic diversity and genetic architecture of phytoplasmas, a physical and genetic map of the sweet potato little leaf (SPLL) strain V4 phytoplasma chromosome was determined. PFGE was used to determine the size of the SPLL-V4 genome, which was estimated to be 622 kb. A physical map was prepared by two-dimensional reciprocal digestions using the restriction endonucleases BssHII, Smal, Eagl and I-Ceul. Sixteen cleavage sites were located on the map. Southern hybridizations of digested SPLL-V4 chromosomal DNA were done using random clones and PCR-amplified genes as probes. This confirmed fragment positions and located the two rRNA operons and the linked fus/tuf genes encoding elongation factors G and Tu, respectively, on the physical map. An inversion of one of the rRNA operons was observed from hybridization data. Sequence analysis of one of the random clones identified a gid gene encoding a glucose-inhibited division protein. Digestions of the tomato big bud (TBB) phytoplasma chromosome with the same four enzymes revealed genome heterogeneity when compared to the closely related SPLL-V4, and a preliminary chromosome size for the TBB phytoplasma of 662 kb was estimated. This mapping information has revealed that significant genome diversity exists within the phytoplasmas.