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Yuh-Ju Sun

Publications and source records attributed to Yuh-Ju Sun.

11 recordsLinked to original sources
PubMed2006

Kinetic and structural properties of inorganic pyrophosphatase from the pathogenic bacterium Helicobacter pylori.

Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of pyrophosphate (PPi) to orthophosphate (Pi) and controls the level of PPi in cells. PPase plays an essential role in energy conservation and provides the energy for many biosynthetic pathways. The Helicobacter pylori pyrophosphatase (HpPPase) gene was cloned, expressed, purified, and found to have a molecular weight of 20 kDa. The K(m) and V (max) of HpPPase were determined as 214.4 microM and 594 micromol Pi min(-1) mg(-1), respectively. PPi binds Mg(2+) to form a true substrate that activates the enzyme. However, free PPi could be a potent inhibitor for HpPPase. The effects of the inhibitors NaF, ATP, iminodiphosphate, and N-ethylmaleimide on HpPPase activity were evaluated. NaF showed the highest inhibition of the enzyme. Crystal structures of HpPPase and the PPi-HpPPase complex were determined. HpPPase comprises three alpha-helices and nine beta-strands and folds as a barrel structure. HpPPase forms a hexamer in both the solution and crystal states, and each monomer has its own PPi-binding site. The PPi binding does not cause a significant conformational change in the PPi-HpPPase complex, which might represent an inhibition state for HpPPase in the absence of a divalent metal ion.

Amino Acid Sequence↗
PubMed2006

Complexed crystal structure of replication restart primosome protein PriB reveals a novel single-stranded DNA-binding mode.

PriB is a primosomal protein required for replication restart in Escherichia coli. PriB stimulates PriA helicase activity via interaction with single-stranded DNA (ssDNA), but the molecular details of this interaction remain unclear. Here, we report the crystal structure of PriB complexed with a 15 bases oligonucleotide (dT15) at 2.7 A resolution. PriB shares structural similarity with the E.coli ssDNA-binding protein (EcoSSB). However, the structure of the PriB-dT15 complex reveals that PriB binds ssDNA differently. Results from filter-binding assays show that PriB-ssDNA interaction is salt-sensitive and cooperative. Mutational analysis suggests that the loop L45 plays an important role in ssDNA binding. Based on the crystal structure and biochemical analyses, we propose a cooperative mechanism for the binding of PriB to ssDNA and a model for the assembly of the PriA-PriB-ssDNA complex. This report presents the first structure of a replication restart primosomal protein complexed with DNA, and a novel model that explains the interactions between a dimeric oligonucleotide-binding-fold protein and ssDNA.

Amino Acid Sequence↗
PubMed2006

Comparative immunoproteomics of identification and characterization of virulence factors from Helicobacter pylori related to gastric cancer.

Helicobacter pylori is an important risk factor of gastric cancer (GC). Although many H. pylori virulence factors have been reported, the pathogenic mechanism by which H. pylori infection causes GC remains unclear. The aims of this study were to identify GC-related antigens from H. pylori and characterize their roles in the development of GC. As GC and duodenal ulcer (DU) are considered clinically divergent, we compared two-dimensional immunoblots of an acid-glycine extract of H. pylori probed with serum samples from 15 patients with GC and 15 with DU to find GC-related antigens, which were subsequently identified by mass spectrometry. Many protein spots were recognized by more than one serum, and 24 of these were better recognized by GC sera. The proteins showing higher frequency of recognition in GC group are threonine synthase, rod shape-determining protein, S-adenosylmethionine synthetase, peptide chain release factor 1, DNA-directed RNA polymerase alpha subunit, co-chaperonin GroES (monomeric and dimeric forms), response regulator OmpR, and membrane fusion protein. Of these proteins, GroES was identified as a dominant GC-related antigen with a much higher seropositivity of GC samples (64.2%, n = 95) compared with 30.9% for gastritis (n = 94) and 35.5% for DU (n = 124). GroES seropositivity was more commonly associated with antral GC than with non-antral GC (odds ratio = 2.7; 95% confidence interval, 1.1-6.7). In peripheral blood mononuclear cells, GroES stimulated production of interleukin (IL)-8, IL-6, granulocyte macrophage colony-stimulating factor, IL-1beta, tumor necrosis factor-alpha, cyclooxygenase-2, and prostaglandin E(2). Moreover when incubated with gastric epithelial cells, GroES induced expression of IL-8, cell proliferation, and up-regulation of c-jun, c-fos, and cyclin D1 but caused down-regulation of p27(Kip1). We conclude that GroES of H. pylori is a novel GC-associated virulence factor and may contribute to gastric carcinogenesis via induction of inflammation and promotion of cell proliferation.

Antigens, Bacterial↗
PubMed2006

Crystal structure of the human FOXK1a-DNA complex and its implications on the diverse binding specificity of winged helix/forkhead proteins.

Interleukin enhancer binding factor (ILF) is a human transcription factor and a new member of the winged helix/forkhead family. ILF can bind to purine-rich regulatory motifs such as the human T-cell leukemia virus-long terminal region and the interleukin-2 promoter. Here we report the 2.4 A crystal structure of two DNA binding domains of ILF (FOXK1a) binding to a 16-bp DNA duplex containing a promoter sequence. Electrophoretic mobility shift assay studies demonstrate that two ILF-DNA binding domain molecules cooperatively bind to DNA. In addition to the recognition helix recognizing the core sequences through the major groove, the structure shows that wing 1 interacts with the minor groove of DNA, and the H2-H3 loop region makes ionic bonds to the phosphate group, which permits the recognition of DNA. The structure also reveals that the presence of the C-terminal alpha-helix in place of a typical wing 2 in a member of this family alters the orientation of the C-terminal basic residues (RKRRPR) when binding to DNA outside the core sequence. These results provide a new insight into how the DNA binding specificities of winged helix/forkhead proteins may be regulated by their less conserved regions.

Amino Acid Sequence↗
PubMed2005

Cloning and characterization of spermidine synthase and its implication in polyamine biosynthesis in Helicobacter pylori strain 26695.

The HP0832 (speE) gene of Helicobacter pylori strain 26695 codes for a putative spermidine synthase, which belongs to the polyamine biosynthetic pathway. Spermidine synthase catalyzes the production of spermidine from putrescine and decarboxylated S-adenosylmethionine (dcSAM), which serves as an aminopropyl donor. The deduced amino acid sequence of the HP0832 gene shares less than 20% sequence identity with most spermidine synthases from mammalian cells, plants and other bacteria. In this study, the HP0832 open reading frame (786 bp) was cloned into the pQE30 vector and overexpressed in Escherichia coli strain SG13009. The resulting N-terminally 6xHis-tagged HP0832 protein (31.9 kDa) was purified by Ni-NTA affinity chromatography at a yield of 15 mg/L of bacteria culture. Spermidine synthase activity of the recombinant protein was confirmed by the appearance of spermidine after incubating the enzyme with putrescine and dcSAM. Substrate specificity studies have shown that spermidine could not replace putrescine as the aminopropyl acceptor. Endogenous spermidine synthase of H. pylori was detected with an antiserum raised against the recombinant HP0832 protein. H. pylori strain 26695 contains putrescine and spermidine at a molar ratio of 1:3, but no detectable spermine or norspermidine was observed, suggesting that the spermidine biosynthetic pathway may provide the main polyamines in H. pylori strain 26695.

Amino Acid Sequence↗
PubMed2004

Lipid binding in rice nonspecific lipid transfer protein-1 complexes from Oryza sativa.

Nonspecific lipid transfer proteins (nsLTPs) facilitate the transfer of phospholipids, glycolipids, fatty acids and steroids between membranes, with wide-ranging binding affinities. Three crystal structures of rice nsLTP1 from Oryza sativa, complexed with myristic (MYR), palmitic (PAL) or stearic acid (STE) were determined. The overall structures of the rice nsLTP1 complexes belong to the four-helix bundle folding with a long C-terminal loop. The nsLTP1-MYR and the nsLTP1-STE complexes bind a single fatty acid while the nsLTP1-PAL complex binds two molecules of fatty acids. The C-terminal loop region is elastic in order to accommodate a diverse range of lipid molecules. The lipid molecules interact with the nsLTP1-binding cavity mainly with hydrophobic interactions. Significant conformational changes were observed in the binding cavity and the C-terminal loop of the rice nsLTP1 upon lipid binding.

Amino Acid Sequence↗
PubMed2003

Crystallization and preliminary X-ray diffraction analysis of thermophilic imidase from pig liver.

Imidase is an enzyme, also known as dihydropyrimidinase (EC 3.5.2.2), hydantoinase, dihydropyrimidine hydrase or dihydropyrimidine amidohydrolase, that catalyzes the reversible hydrolysis of 5,6-dihydrouracil to 3-ureidopropionate and many other imides. Substrate specificity, metal content and amino-acid sequence all differ significantly between bacterial and mammalian imide-hydrolyzing enzymes. In this study, a thermophilic imidase was isolated from pig liver and crystallized. Two kinds of imidase crystals were grown by the hanging-drop vapour-diffusion method using polyethylene glycol MME 5000 and 2-propanol as precipitants. One belongs to the triclinic P(1) space group, with unit-cell parameters a = 96.35, b = 96.87, c = 154.87 A, alpha = 82.10, beta = 72.54, gamma = 77.19 degrees, and the other belongs to the orthorhombic C222(1) space group, with unit-cell parameters a = 113.92, b = 157.22, c = 156.21 A.

Amidohydrolases↗
PubMed2003

Constrained multiple sequence alignment tool development and its application to RNase family alignment.

In this paper, we design a heuristic algorithm of computing a constrained multiple sequence alignment (CMSA for short) for guaranteeing that the generated alignment satisfies the user-specified constraints that some particular residues should be aligned together. If the number of residues needed to be aligned together is a constant alpha, then the time-complexity of our CMSA algorithm for aligning K sequences is O(alphaKn(4)), where n is the maximum of the lengths of sequences. In addition, we have built up such a CMSA software system and made several experiments on the RNase sequences, which mainly function in catalyzing the degradation of RNA molecules. The resulting alignments illustrate the practicability of our method.

Algorithms↗
PubMed2002

Crystal structure of pea Toc34, a novel GTPase of the chloroplast protein translocon.

Toc34, a 34-kDa integral membrane protein, is a member of the Toc (translocon at the outer-envelope membrane of chloroplasts) complex, which associates with precursor proteins during protein transport across the chloroplast outer membrane. Here we report the 2.0 A resolution crystal structure of the cytosolic part of pea Toc34 in complex with GDP and Mg2+. In the crystal, Toc34 molecules exist as dimers with features resembling those found in a small GTPase in complex with a GTPase activating protein (GAP). However, gel filtration experiments revealed that dimeric and monomeric forms of Toc34 coexisted in phosphate saline buffer solution at pH 7.2. Mutation of Arg 128, an essential residue for dimerization, to an Ala residue led to the formation of an exclusively monomeric species whose GTPase activity is significantly reduced compared to that of wild type Toc34. These results, together with a number of structural features unique to Toc34, suggest that each monomer acts as a GAP on the other interacting monomer.

Amino Acid Motifs↗
PubMed2002

Constrained multiple sequence alignment tool development and its application to RNase family alignment.

In this paper, we design an algorithm of computing a constrained multiple sequence alignment (CMSA for short) for guaranteeing that the generated alignment satisfies the user-specified constraints that some particular residues should be aligned together. If the number of residues needed to be aligned together is a constant alpha, then the time-complexity of our CMSA algorithm for aligning K sequences is O (alphaKn4), where n is the maximum of the lengths of sequences. In addition, we have build up such a CMSA software system and made several experiments on the RNase sequences, which mainly function in catalyzing the degradation of RNA molecules. The resulting alignments illustrate the practicability of our method.

Algorithms↗