Thermostable aminopeptidase from Pyrococcus horikoshii

From the genome sequence data of the thermophilic archaeon Pyrococcus horikoshii, an open reading frame was found which encodes a protein (332 amino acids) homologous with an endoglucanase from Clostridium thermocellum (42% identity), deblocking aminopeptidase from Pyrococcus furiosus (42% identity) and an aminopeptidase from Aeromonas proteolytica (18% identity). This gene was cloned and expressed in Escherichia coli, and the characteristics of the expressed protein were examined. Although endoglucanase activity was not detected, this protein was found to have aminopeptidase activity to cleave the N-terminal amino acid from a variety of substrates including both N-blocked and non-blocked peptides. The enzyme was stable at 90 degrees C, with the optimum temperature over 90 degrees C. The metal ion bound to this enzyme was calcium, but it was not essential for the aminopeptidase activity. Instead, this enzyme required the cobalt ion for activity. This enzyme is expected to be useful for the removal of N(alpha)-acylated residues in short peptide sequence analysis at high temperatures.     

Specific interactions of three proliferating cell nuclear antigens with replication-related proteins in Aeropyrum pernix

Proliferating cell nuclear antigen (PCNA) is a well-known multifunctional protein involved in eukaryotic and archaeal DNA transactions. The homotrimeric PCNA ring encircles double-stranded DNA within its central hole and tethers many proteins on DNA. Plural genes encoding PCNA-like proteins have been found in the genome sequence of crenarchaeal organisms . We describe here the biochemical properties of the three PCNAs, PCNA1, PCNA2 and PCNA3, from the hyperthermophilic archaeon, Aeropyrum pernix . PCNA2 can form a trimeric structure by itself, and it also forms heterotrimeric structures with PCNA1 and PCNA3. However, neither PCNA1 nor PCNA3 can form homotrimers. The DNA synthesis activity of DNA polymerase I and II, the endonuclease activity of FEN1, and the nick-sealing activity of DNA ligase were stimulated by the complex of PCNA2 and 3 or PCNA1, 2 and 3. These results suggest that the heterotrimeric PCNA at least including PCNA2 and 3 function as the clamp in the replisome. However, PCNA2 is the most abundant in the cells throughout the growth stages among the three PCNAs, and therefore, PCNA2 may perform multitasks by changing complex composition.     

A nicotinamide mononucleotide adenylyltransferase with unique adenylyl group donor specificity from a hyperthermophilic archaeon, Pyrococcus horikoshii OT-3

A gene encoding a nicotinamide mononucleotide (NMN) adenylyltransferase (NMNAT, EC 2.7.7.1) homologue was identified via genome sequencing in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3. The gene encoded a protein of 186 amino acids with a molecular weight of 21,391. The deduced amino acid sequence of the gene showed 59% identities to the NMNAT from Methanococcus jannaschii. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified to homogeneity. Characterization of the enzyme revealed that it is an extremely thermostable NMNAT; the activity was not lost after incubation at 80 °C for 30 min. The native molecular mass was estimated to be 77 kDa. The K_m values for ATP and NMN were calculated to be 0.056 and 0.061 mM, respectively. The optimum temperature of the reaction was estimated to be around 90 °C. The adenylyl group donor specificity was examined by high-performance liquid chromatography (HPLC). At 70 °C, ATP was a prominent donor. However, above 80 °C, a relatively small, but significant, NMNAT activity was detected when ATP was replaced by ADP or AMP in the reaction mixture. To date, an NMNAT that utilizes ADP or AMP as an adenylyl group donor has not been found. The present study provides interesting information in which a di- or mono-phosphate nucleotide can be utilized by adenylyltransferase at high temperature.     

Substrate specificity of archaeon Sulfolobus tokodaii biotin protein ligase

Biotin protein ligase (BPL) is an enzyme mediating biotinylation of a specific lysine residue of the carboxyl carrier protein (BCCP) of biotin-dependent enzymes. We recently found that the substrate specificity of BPL from archaeon Sulfolobus tokodaii is totally different from those of many other organisms, in reflection of a difference in the local sequence of BCCP surrounding the canonical lysine residue. There is a conserved glycine residue in the biotin-binding site of Escherichia coli BPL, but this residue is replaced with alanine in S. tokodaii BPL. To test the notion that this substitution dictates the substrate specificity of the latter enzyme, this residue, Ala-43, was converted to glycine. The K(m) values of the resulting mutant, A43G, for substrates, were smaller than those of the wild type, suggesting that the residue in position 43 of BPL plays an important role in substrate binding.     

Structure and regulation of rat long-chain acyl-CoA synthetase

Complementary DNAs encoding rat long-chain acyl-CoA synthetase have been isolated. The cDNAs were identified using synthetic oligonucleotide probes based on partial amino acid sequences of lysyl endopeptidase peptides of the purified enzyme. Rat long-chain acyl-CoA synthetase is predicted to contain 699 amino acid residues and to have a calculated molecular weight of 78,177. Significant sequence similarity was found between parts of long-chain acyl-CoA synthetase and firefly luciferase. Based on the similarity of the reaction mechanisms of the two enzymes, we propose a function for the similar region. The long-chain acyl-CoA synthetase mRNA is expressed in liver, heart, and epididymal adipose tissues and, to a much lesser extent, in brain, small intestine, and lung. The level of long-chain acyl-CoA synthetase mRNA is increased 7-8-fold in rat liver by feeding a diet high in carbohydrate or fat, consistent with the physiological significance of the enzyme in fatty acid metabolism.     

Complete Sequence and Gene Organization of the Genome of a Hyper-thermophilic Archaebacterium, Pyrococcus horikoshii OT3

The complete sequence of the genome of a hyper-thermophilicarchaebacterium, Pyrococcus horikoshii OT3, has been determinedby assembling the sequences of the physical map-based contigsof fosmid clones and of long polymerase chain reaction (PCR)products which were used for gap-filling. The entire lengthof the genome was 1,738,505 bp. The authenticity of the entiregenome sequence was supported by restriction analysis of longPCR products, which were directly amplified from the genomicDNA. As the potential protein-coding regions, a total of 2061open reading frames (ORFs) were assigned, and by similaritysearch against public databases, 406 (19.7%) were related togenes with putative function and 453 (22.0%) to the sequencesregistered but with unknown function. The remaining 1202 ORFs(58.3%) did not show any significant similarity to the sequencesin the databases. Sequence comparison among the assigned ORFsin the genome provided evidence that a considerable number ofORFs were generated by sequence duplication. By similarity search,11 ORFs were assumed to contain the intein elements. The RNAgenes identified were a single 16S-23S rRNA operon, two 5S rRNAgenes and 46 tRNA genes including two with the intron structure.All the assigned ORFs and RNA coding regions occupied 91.25%of the whole genome. The data presented in this paper are availableon the internet at http://www.nite.go.jp.     

A unique biotin carboxyl carrier protein in archaeon Sulfolobus tokodaii

Biotin carboxyl carrier protein (BCCP) is one subunit or domain of biotin-dependent enzymes. BCCP becomes an active substrate for carboxylation and carboxyl transfer, after biotinylation of its canonical lysine residue by biotin protein ligase (BPL). BCCP carries a characteristic local sequence surrounding the canonical lysine residue, typically -M-K-M-. Archaeon Sulfolobus tokodaii is unique in that its BCCP has serine replaced for the methionine C-terminal to the lysine. This BCCP is biotinylated by its own BPL, but not by Escherichia coli BPL. Likewise, E. coli BCCP is not biotinylated by S. tokodaii BPL, indicating that the substrate specificity is different between the two organisms.