ABSTRACT
Dna2 protein plays an important role in Okazaki fragment maturation on the lagging strand and also participates in DNA repair in Eukarya. Herein, we report the first biochemical characterization of a Dna2 homologue from Archaea, the hyperthermophile Pyrococcus horikoshii (Dna2Pho). Dna2Pho has both a RecB-like nuclease motif and seven conserved helicase motifs similar to Dna2 from Saccharomyces cerevisiae. Dna2Pho has single-stranded (ss) DNA-stimulated ATPase activity, DNA helicase activity (5' to 3' direction) requiring ATP, and nuclease activity, which prefers free 5'-ends of ssDNA as substrate. These activities depend on MgCl(2) concentrations. Dna2Pho requires a higher concentration of MgCl(2) for the nuclease than helicase activity. Both the helicase and nuclease activities of Dna2Pho were inhibited by substrates with RNA segments at the 5'-end of flap DNA, whereas the nuclease activity of Dna2 from S. cerevisiae was reported to be stimulated by RNA segments in the 5'-tail (Bae, S.-H., and Seo, Y. S. (2000) J. Biol. Chem. 38022-38031).
Subject(s)
Adenosine Triphosphatases/metabolism , Archaeal Proteins/metabolism , DNA Helicases/metabolism , DNA/metabolism , Deoxyribonucleases/metabolism , Pyrococcus/enzymology , Saccharomyces cerevisiae Proteins , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/chemistry , Adenosine Triphosphate/pharmacology , Amino Acid Sequence , Base Sequence , DNA Helicases/antagonists & inhibitors , DNA Helicases/chemistry , DNA Primers , Deoxyribonucleases/antagonists & inhibitors , Deoxyribonucleases/chemistry , Magnesium Chloride/pharmacology , Molecular Sequence DataABSTRACT
A gene encoding the L-aspartate oxidase homologue was identified via genome sequencing in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3. We succeeded in expressing the encoding gene in Escherichia coli and purified the product to homogeneity. Characterization of the protein revealed that it is the most thermostable L-aspartate oxidase detected so far. In addition to the oxidase activity, the enzyme catalyzed L-aspartate dehydrogenation in the presence of an artificial electron acceptor such as phenazine methosulfate, 2,6-dichlorophenol-indophenol, and ferricyanide. L-Aspartate oxidase is known to function as the first enzyme in the de novo NAD biosynthetic pathway in prokaryotes. By a similarity search in public databases, the genes that encode the homologue of all other enzymes involved in the pathway were identified in the P. horikoshii OT-3 genome. This suggests that P. horikoshii OT-3 may use the de novo NAD biosynthetic pathway under anaerobic conditions.
Subject(s)
Amino Acid Oxidoreductases/metabolism , Genome, Archaeal , NAD/biosynthesis , Pyrococcus/enzymology , Amino Acid Oxidoreductases/chemistry , Amino Acid Oxidoreductases/genetics , Amino Acid Sequence , Cloning, Molecular , Enzyme Stability , Escherichia coli Proteins , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Pyrococcus/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , TemperatureABSTRACT
A gene homologous to D-ribose-5-phosphate isomerase (EC 5.3.1.6) was found in the genome of Pyrococcus horikoshii. D-ribose-5-phosphate isomerase (PRI) is of particular metabolic importance since it catalyzes the interconversion between the ribose and ribulose forms involved in the pentose phosphate cycle and in the process of photosynthesis. The gene consisting of 687 bp was overexpressed in Escherichia coli, and the resulting enzyme showed activity at high temperatures with an optimum over 90 degrees C. The crystal structures of the enzyme, free and in complex with D-4-phosphoerythronic acid inhibitor, were determined. PRI is a tetramer in the crystal and in solution, and each monomer has a new fold consisting of two alpha/beta domains. The 3D structures and the characterization of different mutants indicate a direct or indirect catalytic role for the residues E107, D85, and K98.
Subject(s)
Aldose-Ketose Isomerases/chemistry , Pyrococcus/enzymology , Aldose-Ketose Isomerases/antagonists & inhibitors , Aldose-Ketose Isomerases/metabolism , Amino Acid Sequence , Binding Sites , Circular Dichroism , Crystallography, X-Ray , Hot Temperature , Molecular Sequence Data , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, Tertiary , Pyrococcus/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Sequence AlignmentABSTRACT
Introns in protein-coding genes are ubiquitous in eukaryotic cells, but pre-mRNA splicing has yet to be reported in archaeal and its viral genomes. We present evidence of introns in genes encoding a homolog of eukaryotic Cbf5p (centromere-binding factor 5; a subunit of a small nucleolar ribonucleoprotein) in three Archaea; Aeropyrum pernix, Sulfolobus solfataricus and Sulfolobus tokodaii. Splicing of pre-mRNAs in vivo was demonstrated by reverse transcriptase-mediated polymerase chain reaction. The exon-intron boundaries of these genes are predicted to be folded into a structure similar to the bulge-helix-bulge motif, suggesting that splicing of these pre-mRNAs probably depends on the splicing system elucidated for archaeal pre-tRNAs and rRNAs.
