Sequence analysis of glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 and comparison of the enzymatic characteristics of native and recombinant GDHs

The gdhA gene encoding glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned and sequenced. Phylogenetic analysis was performed on an alignment of 25 GDH sequences including KOD1-GDH, and two protein families were distinguished, as previously reported. KOD1-GDH was classified as new member of the hexameric GDH Family II. The gdhA gene was expressed in Escherichia coli, and recombinant KOD1-GDH was purified. Its enzymatic characteristics were compared with those of the native KOD1-GDH. Both enzymes had a molecular mass of 47 300 Da and were shown to be functional in a hexameric form (284 kDa). The N-terminal amino acid sequences of native KOD1-GDH and the recombinant GDH were VEIDPFEMAV and MVEIDPFEMA, respectively, indicating that native KOD1-GDH does not retain the initial methionine at the N-terminus. The recombinant GDH displayed enzyme characteristics similar to those of the native GDH, except for a lower level of thermostability, with a half-life of 2 h at 100° C, compared to 4 h for the native enzyme purified from KOD1. Kinetic studies suggested that the reaction is biased towards glutamate production. KOD1-GDH utilized both coenzymes NADH and NADPH, as do most eukaryal GDHs. Received: 6 May 1997 / Accepted: 23 September 1997     

Characterization of two intein homing endonucleases encoded in the DNA polymerase gene of Pyrococcus kodakaraensis strain KOD1.

Two intein endonucleases, denoted PI- Pko I and PI- Pko II, in the DNA polymerase gene of the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 were expressed in Escherichia coli and the recombinant endonucleases were characterized. Both endonucleases were thermostable and cleaved their intein-less DNA sequences leaving four base 3'-hydroxyl overhangs. PI-Pko I exhibited 22 times higher specific activity than PI-Pko II and the activity of PI-Pko II was enhanced at higher potassium ion concentrations (1 M). Recognition sequences were also determined using synthetic oligonucleotides inserted into plasmid pUC19. It was shown that DNA sequences of 19 and 16 bp are needed for cleavage by PI-Pko I and PI-Pko II, respectively. PI-Pko II could cleave the downstream junction region between intein-encoding and mature DNA polymerase regions and cleavage by PI-Pko II could be detected even when chromosomal DNA of P.kodakaraensis KOD1 was used as substrate. Therefore, it is suggested that these endonucleases are switching endonucleases whose function lies in the rearrangement of chromosomal DNA.     

Feasibility study for decomposition of gelatin layers on X-ray films by thermostable alkaline protease from alkaliphilic Bacillus sp.

Enzymatic decomposition of gelatin layers on X-ray films and repeated utilization of enzyme for potential industrialization were investigated using thermostable alkaline protease from the alkaliphilic Bacillus sp. B21-2. The decomposition of gelatin layers at 50 °C with the mutant enzyme (Ala187 was replaced by Pro) was higher than those of the wild-type and other mutant enzymes. In the repeated experiment for every 60 min (20 U ml^–1, 50 °C), the mutant enzyme could be satisfactorily used five times while three times for the wild-type enzyme.     

Identification of the gene encoding esterase, a homolog of hormone-sensitive lipase, from an oil-degrading bacterium, strain HD-1.

The gene encoding an esterase (HDE) was cloned from an oil-degrading bacterium, strain HD-1. HDE is a member of the hormone-sensitive lipase family and composed of 317 amino acid residues with a molecular weight of 33,633. The HDE-encoding gene was expressed in Escherichia coli, and the recombinant protein was purified and characterized. Amino acid sequence analysis indicated that the methionine residue was removed from its NH(2)-terminus. The good agreement of the molecular weights estimated by SDS-PAGE (35,000) and gel filtration (38,000) suggests that it acts in a monomeric form. HDE showed hydrolytic activity towards p-nitrophenyl esters of fatty acids with an acyl chain length of 2 to 14 and tributyrin, whereas it showed little hydrolytic activity towards p-nitrophenyl oleate (C(18)), tricaprylin and triolein. Determination of the kinetic parameters for the hydrolyses of the p-nitrophenyl substrates from C(2) to C(14) indicated that HDE shows a relatively broad substrate specificity. However, comparison of the k(cat)/K(m) values indicated that the C(10)-C(14) substrates are the most preferred ones. Such a preference for substrates with long acyl chains may be a characteristic of HDE.     

Hyperthermostable protein structure maintained by intra and inter-helix ion-pairs in archaeal O6-methylguanine-DNA methyltransferase.

The crystal structure of O6-methylguanine-DNA methyltransferase (EC 2.1.1.63) of hyperthermophilic archaeon Pyrococcuskodakaraensis strain KOD1 (Pk -MGMT) was determined by single isomorphous replacement method with anomalous scattering (SIRAS) at 1.8 A resolution. The archaeal protein is extremely thermostable and repairs alkylated DNA by suicidal alkyl transfer from guanine O6 to its own cysteine residue. Archaea constitute the third primary kingdom of living organisms, sharing characteristics with procaryotic and eucaryotic cells. They live in various extreme environments and are thought to include the most ancient organisms on the earth. Structural studies on hyperthermophilic archaeal proteins reveal the structural features essential for stability under the extreme environments in which these organisms live, and will provide the structural basis required for stabilizing various mesophilic proteins for industrial applications. Here, we report the crystal structure of Pk-MGMT and structural comparison of Pk-MGMT and methyltransferase homologue from Escherichia coli (AdaC, C-terminal fragment of Ada protein). Analyses of solvent-accessible surface area (SASA) reveals a large discrepancy between Pk-MGMT and AdaC with respect to the property of the ASA. In the Pk-MGMT structure, the intra-helix ion-pairs contribute to reinforce stability of alpha-helices. The inter-helix ion-pairs exist in the interior of Pk-MGMT and stabilize internal packing of tertiary structure. Furthermore, structural features of helix cappings, intra and inter-helix ion-pairs are found around the active-site structure in Pk-MGMT.     

The HA proteins of botulinum toxin disrupt intestinal epithelial intercellular junctions to increase toxin absorption

The type B botulinum neurotoxin (BoNT) elicits flaccid paralysis and death in humans by intoxicating peripheral nerves after oral absorption. Here, we examine the function of the haemagglutinin (HA), a non-toxic component of the large 16S BoNT complex. We find that the HA acts in the intestine to disrupt epithelial barrier function by opening intercellular tight and adherens junctions. This allows transport of BoNT and other large solutes into the systemic circulation and explains how the type B BoNT complexes are efficiently absorbed. In vitro , HA appears to act on the epithelial cell via the basolateral membrane only, suggesting the possibility of another step in the absorptive process. These studies show that the 16S BoNT complex is a multifunctional protein assembly equipped with the machinery to efficiently breach the intestinal barrier and act systemically on peripheral nerves.     

Endothelial dysfunction in the early- and late-stage type-2 diabetic Goto-Kakizaki rat aorta

Hodgkin and Reed-Sternberg (H-RS) cells of classical Hodgkin lymphoma (cHL) present an impaired expression of immunoglobulin genes, but escape apoptotic death. We investigated whether nitric oxide synthases (NOS) are expressed by H-RS cells, studied their association with EBV status and the expression of apoptotic proteins, and investigated their relationship to the clinical outcome of 171 patients. NOS1 and NOS2 were expressed in a large number of cases, whereas NOS3 expression was not detected. Positive associations were found between NOS1 and p53, bax and NOS2, bcl-2 and NOS2, bax and p53, and between bax and fasL. Inverse correlations were established between EBV and NOS2 and between EBV and bcl-2. A shorter overall survival (OS) was associated with strong expression of NOS2. In conclusion, NOS are expressed by H-RS cells of cHL.     

Systematic approaches to using the FOX hunting system to identify useful rice genes

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23 000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis . This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.     

HvLsi1 is a silicon influx transporter in barley

Most plants accumulate silicon in their bodies, and this is thought to be important for resistance against biotic and abiotic stresses; however, the molecular mechanisms for Si uptake and accumulation are poorly understood. Here, we describe an Si influx transporter, HvLsi1, in barley. This protein is homologous to rice influx transporter OsLsi1 with 81% identity, and belongs to a Nod26-like major intrinsic protein sub-family of aquaporins. Heterologous expression in both Xenopus laevis oocytes and a rice mutant defective in Si uptake showed that HvLsi1 has transport activity for silicic acid. Expression of HvLsi1 was detected specifically in the basal root, and the expression level was not affected by Si supply. There was a weak correlation between Si uptake and the expression level of HvLsi1 in eight cultivars tested. In the seminal roots, HvLsi1 is localized on the plasma membrane on the distal side of epidermal and cortical cells. HvLsi1 is also located in lateral roots on the plasma membrane of hypodermal cells. These cell-type specificity of localization and expression patterns of HvLsi1 are different from those of OsLsi1. These observations indicate that HvLsi1 is a silicon influx transporter that is involved in radial transport of Si through the epidermal and cortical layers of the basal roots of barley.