Comparative analyses of the two proliferating cell nuclear antigens from the hyperthermophilic archaeon, Thermococcus kodakarensis.

The DNA sliding clamp is a multifunctional protein involved in cellular DNA transactions. In Archaea and Eukaryota, proliferating cell nuclear antigen (PCNA) is the sliding clamp. The ring-shaped PCNA encircles double-stranded DNA within its central hole and tethers other proteins on DNA.

Evolutionary Analysis of Functional Divergence among Chemokine Receptors, Decoy Receptors, and Viral Receptors.

Chemokine receptors (CKRs) function in the inflammatory response and in vertebrate homeostasis. Decoy and viral receptors are two types of CKR homologs with modified functions from those of the typical CKRs. The decoy receptors are able to bind ligands without signaling.

Biochemical and genetical analyses of the three mcm genes from the hyperthermophilic archaeon, Thermococcus kodakarensis.

In eukaryotes, the replicative DNA helicase 'core' is the minichromosome maintenance (Mcm) complex (MCM), forming a heterohexameric complex consisting of six subunits (Mcm2-7). Recent studies showed that the CMG (Cdc45-MCM-GINS) complex is the actual helicase body in the replication fork progression complex. In Archaea, Thermococcus kodakarensis harbors three genes encoding the Mcm homologs on its genome, contrary to most archaea, which have only one homolog.

Functional expression of single-chain antibody to leukotriene C(4).

Leukotriene C(4) (LTC(4)) is synthesized by binding of glutathione to LTA(4), an epoxide derived from arachidonic acid, and further metabolized to LTD(4) and LTE(4). We previously prepared a monoclonal antibody with a high affinity and specificity to LTC(4). To explore the structure of the antigen-binding site of a monoclonal antibody against LTC(4) (mAbLTC), we isolated full-length cDNAs for heavy and light chains of mAbLTC.

Computational and experimental analyses of furcatin hydrolase for substrate specificity studies of disaccharide-specific glycosidases.

Disaccharide-specific glycosidases (diglycosidases) are unique glycoside hydrolases, as their substrate specificities differ from those of monosaccharide-specific beta-glycosidases (monoglycosidases), in spite of similarities in their sequences and reaction mechanisms. Diglycosidases selectively hydrolyse the beta-glycosidic bond between glycone and aglycone of disaccharide glycosides, but do not cleave the bond between two saccharides, and barely hydrolyse monosaccharide glycosides. We analysed the substrate recognition mechanisms of diglycosidases by computational and experimental methods, using furcatin hydrolase (FH) (EC 3.

Recruitment of thioredoxin-like domains into prostaglandin synthases.

A variety of prostaglandin (PG) synthases with different evolutionary origins have been identified. These enzymes catalyze reduction and oxidation reactions. However, despite the similarity in their reactions, thioredoxin-like proteins were not found in the PG synthesis pathway until recently.

Molecular characterization of a novel type of prostamide/prostaglandin F synthase, belonging to the thioredoxin-like superfamily.

Prostaglandin F (PGF) ethanolamide (prostamide F) synthase, which catalyzed the reduction of prostamide H(2) to prostamide F(2alpha), was found in mouse and swine brain. The enzyme was purified from swine brain, and its amino acid sequence was defined. The mouse enzyme consisted of a 603-bp open reading frame coding for a 201-amino acid polypeptide with a molecular weight of 21,669.

A study of archaeal enzymes involved in polar lipid synthesis linking amino acid sequence information, genomic contexts and lipid composition.

Cellular membrane lipids, of which phospholipids are the major constituents, form one of the characteristic features that distinguish Archaea from other organisms. In this study, we focused on the steps in archaeal phospholipid synthetic pathways that generate polar lipids such as archaetidylserine, archaetidylglycerol, and archaetidylinositol. Only archaetidylserine synthase (ASS), from Methanothermobacter thermautotrophicus, has been experimentally identified.

Identification of cryptochrome DASH from vertebrates.

A new type of cryptochrome, CRY-DASH, has been recently identified. The CRY-DASH proteins constitute the fifth subfamily of the photolyase/cryptochrome family. CRY-DASHs have been identified from Synechocystis sp.

How does a topological inversion change the evolutionary constraints on membrane proteins?

The members of the aquaporin family and those of the ClC chloride ion channel family consist of two-fold tandem repeats. The orientation of the N-terminal domain against membrane is opposite to that of the C-terminal domain. Several lines of evidence suggest that the extracellular and the cytoplasmic environments impose different evolutionary constraints on proteins (e.

Analysis of membrane stereochemistry with homology modeling of sn-glycerol-1-phosphate dehydrogenase.

Different enantiomeric isomers, sn-glycerol-1-phosphate and sn-glycerol-3-phosphate, are used as the glycerophosphate backbones of phospholipids in the cellular membranes of Archaea and the remaining two kingdoms, respectively. In Archaea, sn-glycerol-1-phosphate dehydrogenase is involved in the generation of sn-glycerol-1-phosphate, while sn-glycerol-3-phosphate dehydrogenase synthesizes the enantiomer in Eukarya and Bacteria. The coordinates of sn-glycerol-3-phosphate dehydrogenase are available, although neither the tertiary structure nor the reaction mechanism of sn-glycerol-1-phosphate dehydrogenase is known.

Archaeal-type rhodopsins in Chlamydomonas: model structure and intracellular localization.

Phototaxis in the unicellular green alga Chlamydomonas reinhardtii is mediated by rhodopsin-type photoreceptor(s). Recent expressed sequence tag database from the Kazusa DNA Research Institute has provided the basis for unequivocal identification of two archaeal-type rhodopsins in it. Here we demonstrate that one is located near the eyespot, wherein the photoreceptor(s) has long been thought to be enriched, along with the results of bioinformatic analyses.

Identification of a new cryptochrome class. Structure, function, and evolution.

Cryptochrome flavoproteins, which share sequence homology with light-dependent DNA repair photolyases, function as photoreceptors in plants and circadian clock components in animals. Here, we coupled sequencing of an Arabidopsis cryptochrome gene with phylogenetic, structural, and functional analyses to identify a new cryptochrome class (cryptochrome DASH) in bacteria and plants, suggesting that cryptochromes evolved before the divergence of eukaryotes and prokaryotes. The cryptochrome crystallographic structure, reported here for Synechocystis cryptochrome DASH, reveals commonalities with photolyases in DNA binding and redox-dependent function, despite distinct active-site and interaction surface features.

Two novel genes expressed in Xenopus germ line: characteristic features of putative protein structures, their gene expression profiles and their possible roles in gametogenesis and embryogenesis.

We compared the secondary spermatogonia and the primary spermatocytes of Xenopus for the proteins in their microsomal fractions and identified a newly synthesized protein (94 kDa) and three other proteins (99, 85, and 72 kDa) which increased their amount after entering the meiotic phase. These four proteins were used as antigens to produce polyclonal antibody which was found to react with the four proteins as well as two other proteins (208 and 60 kDa). Immunoscreening of Xenopus testis cDNA library with this polyclonal antibody yielded two cDNA clones (Xmegs and Xtr) encoding novel proteins.

Expansion of the zinc metallo-hydrolase family of the beta-lactamase fold.

Recently, the zinc metallo-hydrolase family of the beta-lactamase fold has grown quite rapidly, accompanied by the accumulation of sequence and structure data. The variety of the biological functions of the family is higher than expected. In addition, the members often have mosaic structures with additional domains.

Biochemical analysis of replication factor C from the hyperthermophilic archaeon Pyrococcus furiosus.

Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are accessory proteins essential for processive DNA synthesis in the domain Eucarya. The function of RFC is to load PCNA, a processivity factor of eukaryotic DNA polymerases delta and epsilon, onto primed DNA templates. RFC-like genes, arranged in tandem in the Pyrococcus furiosus genome, were cloned and expressed individually in Escherichia coli cells to determine their roles in DNA synthesis.

Molecular evolution of the myeloperoxidase family.

Animal myeloperoxidase and its relatives constitute a diverse protein family, which includes myeloperoxidase, eosinophil peroxidase, thyroid peroxidase, salivary peroxidase, lactoperoxidase, ovoperoxidase, peroxidasin, peroxinectin, cyclooxygenase, and others. The members of this protein family share a catalytic domain of about 500 amino acid residues in length, although some members have distinctive mosaic structures. To investigate the evolution of the protein family, we performed a comparative analysis of its members, using the amino acid sequences and the coordinate data available today.

Hjc resolvase is a distantly related member of the type II restriction endonuclease family.

Hjc resolvase is an archaeal enzyme involved in homologous DNA recombination at the Holliday junction intermediate. However, the structure and the catalytic mechanism of the enzyme have not yet been identified. We performed database searching using the amino acid sequence of the enzyme from Pyrococcus furiosus as a query.

Mutational analysis of the Pyrococcus furiosus holliday junction resolvase hjc revealed functionally important residues for dimer formation, junction DNA binding, and cleavage activities.

The Holliday junction cleavage protein, Hjc resolvase of Pyrococcus furiosus, is the first Holliday junction resolvase to be discovered in Archaea. Although the archaeal resolvase shares certain biochemical properties with other non-archaeal junction resolvases, no amino acid sequence similarity has been identified. To investigate the structure-function relationship of this new Holliday junction resolvase, we constructed a series of mutant hjc genes using site-directed mutagenesis targeted at the residues conserved among the archaeal orthologs.

Molecular analysis of zebrafish photolyase/cryptochrome family: two types of cryptochromes present in zebrafish.

Background: Cryptochromes (CRY), members of the DNA photolyase/cryptochrome protein family, regulate the circadian clock in animals and plants. Two types of animal CRYs are known, mammalian CRY and Drosophila CRY. Both CRYs participate in the regulation of circadian rhythm, but they have different light dependencies for their reactions and have different effects on the negative feedback loop which generates a circadian oscillation of gene expression.

Domain analysis of an archaeal RadA protein for the strand exchange activity.

Archaeal RadA, like eukaryotic Rad51 and bacterial RecA, promotes strand exchange between DNA strands with homologous sequences in vitro and is believed to participate in the homologous recombination in cells. The amino acid sequences of the archaeal RadA proteins are more similar to the eukaryotic Rad51s rather than the bacterial RecAs, and the N-terminal region containing domain I is conserved among the RadA and Rad51 proteins but is absent from RecA. To understand the structure-function relationship of RadA, we divided the RadA protein from Pyrococcus furiosus into two parts, the N-terminal one-third (RadA-n) and the residual C-terminal two-thirds (RadA-c), the latter of which contains the central core domain (domain II) of the RecA/Rad51 family proteins.

Bacterial cryptochrome and photolyase: characterization of two photolyase-like genes of Synechocystis sp. PCC6803.

Photolyase is a DNA repair enzyme that reverses UV-induced photoproducts in DNA in a light-dependent manner. Recently, photolyase homologs were identified in higher eukaryotes. These homologs, termed crypto-chromes, function as blue light photoreceptors or regulators of circadian rhythm.

Mutational analysis of the functional motifs of RuvB, an AAA+ class helicase and motor protein for holliday junction branch migration.

Escherichia coli RuvB protein, together with RuvA, promotes branch migration of Holliday junctions during homologous recombination and recombination repair. The RuvB molecular motor is an intrinsic ATP-dependent DNA helicase with a hexameric ring structure and its architecture has been suggested to be related to those of the members of the AAA+ protein class. In this study, we isolated a large number of plasmids carrying ruvB mutant genes and identified amino acid residues important for the RuvB functions by examining the in vivo DNA repair activities of the mutant proteins.