Acidophilic character of yeast PID261/BUD32, a putative ancestor of eukaryotic protein kinases

Yeast piD261/Bud32 and its homologues are present in eukaryotes and in archaea but not in bacteria and are believed to make up a primordial branch of the eukaryotic protein kinase superfamily. Here, we show that, at variance with the majority of Ser/Thr protein kinases which recognize phosphoacceptor sites specified by basic and/or proline residues, piD261 phosphorylates in vitro a number of acidic proteins and peptides, and it recognizes seryl residues specified by carboxylic side chains. These data suggest that recognition of acidic sites might have been a primordial trait of protein kinases, which was modified during evolution to cope with the increasing complexity of protein phosphorylation in eukaryotes.     

A thermostable shikimate 5-dehydrogenase from the archaeon Archaeoglobus fulgidus

Shikimate 5-dehydrogenase (SKDH; EC 1.1.1.25) catalyzes the reversible reduction of 3-dehydroshikimate to shikimate and is a key enzyme in the aromatic amino acid biosynthesis pathway. The shikimate 5-dehydrogenase gene, aroE, from Archaeoglobus fulgidus was cloned and overexpressed in Escherichia coli. The recombinant enzyme purified as a homodimer and yielded a maximum specific activity of 732 U/mg at 87 degrees C (with NADP+ as coenzyme). Apparent Km values for shikimate, NADP+, and NAD+ were estimated at 0.17+/-0.03 mM, 0.19+/-0.01 mM, and 11.4+/-0.4 mM, respectively. The half-life of the A. fulgidus SKDH is 2 h at the assay temperature (87 degrees C) and 17 days at 60 degrees C. Addition of 1 M NaCl or KCl stabilized the enzyme's half-life to approximately 70 h at 87 degrees C and approximately 50 days at 60 degrees C. This work presents the first kinetic analysis of an archaeal SKDH.     

Computational identification of a new SelD-like family that may participate in sulfur metabolism in hyperthermophilic sulfur-reducing archaea

Background

Selenium (Se) and sulfur (S) are closely related elements that exhibit similar chemical properties. Some genes related to S metabolism are also involved in Se utilization in many organisms. However, the evolutionary relationship between the two utilization traits is unclear.

Results

In this study, we conducted a comparative analysis of the selenophosphate synthetase (SelD) family, a key protein for all known Se utilization traits, in all sequenced archaea. Our search showed a very limited distribution of SelD and Se utilization in this kingdom. Interestingly, a SelD-like protein was detected in two orders of Crenarchaeota: Sulfolobales and Thermoproteales. Sequence and phylogenetic analyses revealed that SelD-like protein contains the same domain and conserved functional residues as those of SelD, and might be involved in S metabolism in these S-reducing organisms. Further genome-wide analysis of patterns of gene occurrence in different thermoproteales suggested that several genes, including SirA-like, Prx-like and adenylylsulfate reductase, were strongly related to SelD-like gene. Based on these findings, we proposed a simple model wherein SelD-like may play an important role in the biosynthesis of certain thiophosphate compound.

Conclusions

Our data suggest novel genes involved in S metabolism in hyperthermophilic S-reducing archaea, and may provide a new window for understanding the complex relationship between Se and S metabolism in archaea.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-908) contains supplementary material, which is available to authorized users.     

Enzymatic and structural characterization of type II isopentenyl diphosphate isomerase from hyperthermophilic archaeon Thermococcus kodakaraensis

Enzymatic and thermodynamic characteristics of type II isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase (Tk-IDI) from Thermococcus kodakaraensis, which catalyzes the interconversion of IPP and DMAPP, were examined. FMN was tightly bound to Tk-IDI, and the enzyme required NADPH and Mg2+ for the isomerization in both directions. The melting temperature (Tm), the change of enthalpy (deltaH(m)), and the heat capacity change (deltaC(p)) of Tk-IDI were 88.0 degrees C, 444 kJ mol(-1), and 13.2 kJ mol(-1) K(-1), respectively, indicating that Tk-IDI is fairly thermostable. Kinetic parameters dramatically changed when the temperature crossed 80 degrees C even though its native overall structure was stably maintained up to 90 degrees C, suggesting that local conformational change would occur around 80 degrees C. This speculation was supported by the result of the circular dichroism analysis that showed the shift of the alpha-helical content occurred at 80 degrees C.     

Distinct tRNA modifications in the thermo-acidophilic archaeon, Thermoplasma acidophilum

Thermoplasma acidophilum is a thermo-acidophilic archaeon. We purified tRNA^Leu (UAG) from T. acidophilum using a solid-phase DNA probe method and determined the RNA sequence after determining via nucleoside analysis and m⁷G-specific aniline cleavage because it has been reported that T. acidophilum tRNA contains m⁷G, which is generally not found in archaeal tRNAs. RNA sequencing and liquid chromatography–mass spectrometry revealed that the m⁷G modification exists at a novel position 49. Furthermore, we found several distinct modifications, which have not previously been found in archaeal tRNA, such as 4-thiouridine9, archaeosine13 and 5-carbamoylmethyuridine34. The related tRNA modification enzymes and their genes are discussed.     

Crystal structure of archaeal chromatin protein Alba2-double-stranded DNA complex from Aeropyrum pernix K1

All thermophilic and hyperthermophilic archaea encode homologs of dimeric Alba (Sac10b) proteins that bind cooperatively at high density to DNA. Here, we report the 2.0 Å resolution crystal structure of an Alba2 (Ape10b2)-dsDNA complex from Aeropyrum pernix K1. A rectangular tube-like structure encompassing duplex DNA reveals the positively charged residues in the monomer-monomer interface of each dimer packing on either side of the bound dsDNA in successive minor grooves. The extended hairpin loop connecting strands β3 and β4 undergoes significant conformational changes upon DNA binding to accommodate the other Alba2 dimer during oligomerization. Mutational analysis of key interacting residues confirmed the specificity of Alba2-dsDNA interactions.     

Thermophilic anaerobic digestion of sewage sludge: focus on the influence of the start-up. A review

The thermophilic anaerobic digestion (TAD) of sewage sludge has often been found to be less stable than mesophilic treatment. In comparison to mesophilic digesters, thermophilic reactors treating sludge are generally characterized by relatively high concentrations of volatile fatty acids (VFA) in the effluent along with poor effluent quality, indicating a lower level of process stability. However, reviewing the literature related to the procedure for obtaining a thermophilic inoculum, it seems that most of the problems associated with the instability and the accumulation of organic intermediates are the result of the manner in which the thermophilic sludge has been obtained. In this paper, the different options available for obtaining an anaerobic digester operating at thermophilic temperature (55°C) have been reviewed. In this light, rapid heating to the target temperature followed by the development of thermophilic microorganisms, which can be determined by VFA dropping to ≤500?mg acetic acid L^?1 before increasing the organic loading rate (OLR), has been determined the most suitable means of establishing TAD.     

Using Homolog Groups to Create a Whole-Genomic Tree of Free-Living Organisms: An Update

Genomic trees have been constructed based on the presence and absence of families of protein-encoding genes observed in 27 complete genomes, including genomes of 15 free-living organisms. This method does not rely on the identification of suspected orthologs in each genome, nor the specific alignment used to compare gene sequences because the protein-encoding gene families are formed by grouping any protein with a pairwise similarity score greater than a preset value. Because of this all inclusive grouping, this method is resilient to some effects of lateral gene transfer because transfers of genes are masked when the recipient genome already has a homolog (not necessarily an ortholog) of the incoming gene. Of 71 genes suspected to have been laterally transferred to the genome of Aeropyrum pernix, only approximately 7 to 15 represent genes where a lateral gene transfer appears to have generated homoplasy in our character dataset. The genomic tree of the 15 free-living taxa includes six different bacterial orders, six different archaeal orders, and two different eukaryotic kingdoms. The results are remarkably similar to results obtained by analysis of rRNA. Inclusion of the other 12 genomes resulted in a tree only broadly similar to that suggested by rRNA with at least some of the differences due to artifacts caused by the small genome size of many of these species. Very small genomes, such as those of the two Mycoplasma genomes included, fall to the base of the Bacterial domain, a result expected due to the substantial gene loss inherent to these lineages. Finally, artificial ``partial genomes' were generated by randomly selecting ORFs from the complete genomes in order to test our ability to recover the tree generated by the whole genome sequences when only partial data are available. The results indicated that partial genomic data, when sampled randomly, could robustly recover the tree generated by the whole genome sequences. Received: 30 May 2001 / Accepted: 10 October 2001