Thermostable endoglucanase gene derived by amplification from the genomic DNA of a cellulose-enriched mixed culture from mudspring water of Mt. Makiling, Laguna, Philippines.

Culture-independent molecular-based approaches can be used to identify genes of interest from environmental sources that have desirable properties such as thermo activity. For this study, a putative thermo stable endoglucanase gene was identified from a mixed culture resulting from the inoculation of Brock-CMcellulose (1%) broth with mudspring water from Mt. Makiling, Laguna, Philippines that had been incubated at 90 °C. Genomic DNA was extracted from the cellulose-enriched mixed culture and endo1949 forward and reverse primers were used to amplify the endoglucanase gene, which was cloned into pCR-script plasmid vector. Blastn alignment of the sequenced insert revealed 99.69% similarity to the glycosyl hydrolase, sso1354 (CelA1; Q97YG7) from Saccharolobus solfataricus. The endoglucanase gene (GenBank accession number MK984682) was determined to be 1,021 nucleotide bases in length, corresponding to 333 amino acids with a molecular mass of ~ 37 kDa. The endoglucanase gene was inserted into a pET21 vector and transformed in E. coli BL21 for expression. Partially purified recombinant Mt. Makiling endoglucanase (MM-Engl) showed a specific activity of 187.61 U/mg and demonstrated heat stability up to 80 °C. The thermo-acid stable endoglucanase can be used in a supplementary hydrolysis step to further hydrolyze the lignocellulosic materials that were previously treated under high temperature-dilute acid conditions, thereby enhancing the release of more glucose sugars for bioethanol production.

Malonic semialdehyde reductase, succinic semialdehyde reductase, and succinyl-coenzyme A reductase from Metallosphaera sedula: enzymes of the autotrophic 3-hydroxypropionate/4-hydroxybutyrate cycle in Sulfolobales.

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates during autotrophic CO(2) fixation in various members of the Crenarchaea. In this cycle, as determined using Metallosphaera sedula, malonyl-coenzyme A (malonyl-CoA) and succinyl-CoA are reductively converted via their semialdehydes to the corresponding alcohols 3-hydroxypropionate and 4-hydroxybutyrate. Here three missing oxidoreductases of this cycle were purified from M. sedula and studied. Malonic semialdehyde reductase, a member of the 3-hydroxyacyl-CoA dehydrogenase family, reduces malonic semialdehyde with NADPH to 3-hydroxypropionate. The latter compound is converted via propionyl-CoA to succinyl-CoA. Succinyl-CoA reduction to succinic semialdehyde is catalyzed by malonyl-CoA/succinyl-CoA reductase, a promiscuous NADPH-dependent enzyme that is a paralogue of aspartate semialdehyde dehydrogenase. Succinic semialdehyde is then reduced with NADPH to 4-hydroxybutyrate by succinic semialdehyde reductase, an enzyme belonging to the Zn-dependent alcohol dehydrogenase family. Genes highly similar to the Metallosphaera genes were found in other members of the Sulfolobales. Only distantly related genes were found in the genomes of autotrophic marine Crenarchaeota that may use a similar cycle in autotrophic carbon fixation.

3-hydroxypropionyl-coenzyme A dehydratase and acryloyl-coenzyme A reductase, enzymes of the autotrophic 3-hydroxypropionate/4-hydroxybutyrate cycle in the Sulfolobales.

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates in autotrophic CO(2) fixation in various Crenarchaea, as studied in some detail in Metallosphaera sedula. This cycle and the autotrophic 3-hydroxypropionate cycle in Chloroflexus aurantiacus have in common the conversion of acetyl-coenzyme A (CoA) and two bicarbonates via 3-hydroxypropionate to succinyl-CoA. Both cycles require the reductive conversion of 3-hydroxypropionate to propionyl-CoA. In M. sedula the reaction sequence is catalyzed by three enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the CoA- and MgATP-dependent formation of 3-hydroxypropionyl-CoA. The next two enzymes were purified from M. sedula or Sulfolobus tokodaii and studied. 3-Hydroxypropionyl-CoA dehydratase, a member of the enoyl-CoA hydratase family, eliminates water from 3-hydroxypropionyl-CoA to form acryloyl-CoA. Acryloyl-CoA reductase, a member of the zinc-containing alcohol dehydrogenase family, reduces acryloyl-CoA with NADPH to propionyl-CoA. Genes highly similar to the Metallosphaera CoA synthetase, dehydratase, and reductase genes were found in autotrophic members of the Sulfolobales. The encoded enzymes are only distantly related to the respective three enzyme domains of propionyl-CoA synthase from C. aurantiacus, where this trifunctional enzyme catalyzes all three reactions. This indicates that the autotrophic carbon fixation cycles in Chloroflexus and in the Sulfolobales evolved independently and that different genes/enzymes have been recruited in the two lineages that catalyze the same kinds of reactions.

Deficiency of the tRNATyr:Psi 35-synthase aPus7 in Archaea of the Sulfolobales order might be rescued by the H/ACA sRNA-guided machinery.

Up to now, Psi formation in tRNAs was found to be catalysed by stand-alone enzymes. By computational analysis of archaeal genomes we detected putative H/ACA sRNAs, in four Sulfolobales species and in Aeropyrum pernix, that might guide Psi 35 formation in pre-tRNA(Tyr)(GUA). This modification is achieved by Pus7p in eukarya. The validity of the computational predictions was verified by in vitro reconstitution of H/ACA sRNPs using the identified Sulfolobus solfataricus H/ACA sRNA. Comparison of Pus7-like enzymes encoded by archaeal genomes revealed amino acid substitutions in motifs IIIa and II in Sulfolobales and A. pernix Pus7-like enzymes. These conserved RNA:Psi-synthase- motifs are essential for catalysis. As expected, the recombinant Pyrococcus abyssi aPus7 was fully active and acted at positions 35 and 13 and other positions in tRNAs, while the recombinant S. solfataricus aPus7 was only found to have a poor activity at position 13. We showed that the presence of an A residue 3' to the target U residue is required for P. abyssi aPus7 activity, and that this is not the case for the reconstituted S. solfataricus H/ACA sRNP. In agreement with the possible formation of Psi 35 in tRNA(Tyr)(GUA) by aPus7 in P. abyssi and by an H/ACA sRNP in S. solfataricus, the A36G mutation in the P. abyssi tRNA(Tyr)(GUA) abolished Psi 35 formation when using P. abyssi extract, whereas the A36G substitution in the S. solfataricus pre-tRNA(Tyr) did not affect Psi 35 formation in this RNA when using an S. solfataricus extract.

Archaeal phylogeny based on proteins of the transcription and translation machineries: tackling the Methanopyrus kandleri paradox.

BACKGROUND: Phylogenetic analysis of the Archaea has been mainly established by 16S rRNA sequence comparison. With the accumulation of completely sequenced genomes, it is now possible to test alternative approaches by using large sequence datasets. We analyzed archaeal phylogeny using two concatenated datasets consisting of 14 proteins involved in transcription and 53 ribosomal proteins (3,275 and 6,377 positions, respectively). RESULTS: Important relationships were confirmed, notably the dichotomy of the archaeal domain as represented by the Crenarchaeota and Euryarchaeota, the sister grouping of Sulfolobales and Aeropyrum pernix, and the monophyly of a large group comprising Thermoplasmatales, Archaeoglobus fulgidus, Methanosarcinales and Halobacteriales, with the latter two orders forming a robust cluster. The main difference concerned the position of Methanopyrus kandleri, which grouped with Methanococcales and Methanobacteriales in the translation tree, whereas it emerged at the base of the euryarchaeotes in the transcription tree. The incongruent placement of M. kandleri is likely to be the result of a reconstruction artifact due to the high evolutionary rates displayed by the components of its transcription apparatus. CONCLUSIONS: We show that two informational systems, transcription and translation, provide a largely congruent signal for archaeal phylogeny. In particular, our analyses support the appearance of methanogenesis after the divergence of the Thermococcales and a late emergence of aerobic respiration from within methanogenic ancestors. We discuss the possible link between the evolutionary acceleration of the transcription machinery in M. kandleri and several unique features of this archaeon, in particular the absence of the elongation transcription factor TFS.

Dynamics of the binuclear center of the quinol oxidase from Acidianus ambivalens.

We have investigated the kinetic and thermodynamic properties of carbon monoxide binding to the fully reduced quinol oxidase (cytochrome aa(3)) from the hyperthermophilic archaeon Acidianus ambivalens. After flash photolysis of CO from heme a(3), the complex recombines with an apparent rate constant of approximately 3 s(-1), which is much slower than with the bovine cytochrome c oxidase (approximately 80 s(-1)). Investigation of the CO-recombination rate as a function of the CO concentration shows that the rate saturates at high CO concentrations, which indicates that CO must bind transiently to Cu(B) before binding to heme a(3). With the A. ambivalens enzyme the rate reached 50% of its maximum level (which reflects the dissociation constant of the Cu(B)(CO) complex) at approximately 13 microM CO, which is a concentration approximately 10(3) times smaller than for the bovine enzyme (approximately 11 mM). After CO dissociation we observed a rapid absorbance relaxation with a rate constant of approximately 1.4 x 10(4) s(-1), tentatively ascribed to a heme-pocket relaxation associated with release of CO after transient binding to Cu(B). The equilibrium constant for CO transfer from Cu(B) to heme a(3) was approximately 10(4) times smaller for the A. ambivalens than for the bovine enzyme. The approximately 10(3) times smaller Cu(B)(CO) dissociation constant, in combination with the approximately 10(4) times smaller equilibrium constant for the internal CO transfer, results in an apparent dissociation constant of the heme a(3)(CO) complex which is "only" about 10 times larger for the A. ambivalens ( approximately 4 x 10(-3) mM) than for the bovine (0.3 x 10(-3) mM) enzyme. In summary, the results show that while the basic mechanism of CO binding to the binuclear center is similar in the A. ambivalens and bovine (and R. sphaeroides) enzymes, the heme-pocket dynamics of the two enzymes are dramatically different, which is discussed in terms of the different structural details of the A. ambivalens quinol oxidase and adaptation to different living conditions.

The alcohol dehydrogenase gene: distribution among Sulfolobales and regulation in Sulfolobus solfataricus.

The distribution of the alcohol dehydrogenase gene (adh) among different Archaea was investigated by Southern blot analysis revealing the potentiality of the adh gene as a specific marker for the genus Sulfolobus. Moreover, the in vivo expression of the adh gene from a new isolate of Sulfolobus solfataricus, G theta, was studied to investigate gene regulation in Archaea. Primer extension analysis allowed the identification of a single initiation site and the TATA box element. Comparison of the G theta adh promoter with the corresponding Ssadh (adh from S. solfataricus) and RC3adh (adh from Sulfolobus RC3) also revealed the presence of two putative regulatory inverted repeats at the 5' of the TATA element. Northern blot analysis and enzymatic assays demonstrated that the transcription and expression of the G theta adh gene is regulated by different carbon and energy sources or by the natural substrate of the ADH enzyme.

Gene analysis of trehalose-producing enzymes from hyperthermophilic archaea in Sulfolobales.

The genes encoding new trehalose-producing enzymes from S. acidocaldarius ATCC33909 were cloned to analyze the distribution of these genes in Sulfolobales. Comparison of the amino acid sequences with S. solfataricus KM1 showed approximately 50% similarity. Southern analysis suggest that homologues of the trehalose-producing enzyme genes exist widely in sulfolobales and strains in Sulfolobales were classified into three kinds of genotypes.