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Jeffrey H Miller - One of the best experts on this subject based on the ideXlab platform.
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genome sequence of the hyperthermophilic crenarchaeon Pyrobaculum aerophilum
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Sorel Fitzgibbon, Heidi Ladner, Melvin I Simon, Jeffrey H MillerAbstract:We determined and annotated the complete 2.2-megabase genome sequence of Pyrobaculum aerophilum, a facultatively aerobic nitrate-reducing hyperthermophilic (T-opt = 100 degrees C) crenarchaeon. Clues were found suggesting explanations of the organism's surprising intolerance to sulfur, which may aid in the development of methods for genetic studies of the organism. Many interesting features worthy of further genetic studies were revealed. Whole genome computational analysis confirmed experiments showing that P. aerophilum (and perhaps all crenarchaea) lack 5' untranslated regions in their mRNAs and thus appear not to use a ribosome-binding site (Shine-Dalgarno)-based mechanism for translation initiation at the 5' end of transcripts. Inspection of the lengths and distribution of mononucleotide repeat-tracts revealed some interesting features. For instance, it was seen that mononucleotide repeat-tracts of Gs (or Cs) are highly unstable, a pattern expected for an organism deficient in mismatch repair. This result, together with an independent study on mutation rates, suggests a "mutator" phenotype.
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biochemical characterization of uracil processing activities in the hyperthermophilic archaeon Pyrobaculum aerophilum
Journal of Biological Chemistry, 2001Co-Authors: Alessandro A. Sartori, Sorel Fitzgibbon, Jeffrey H Miller, Primo Schar, Josef JiricnyAbstract:Abstract Deamination of cytosine to uracil and 5-methylcytosine to thymine represents a major mutagenic threat particularly at high temperatures. In double-stranded DNA, these spontaneous hydrolytic reactions give rise to G·U and G·T mispairs, respectively, that must be restored to G·C pairs prior to the next round of DNA replication; if left unrepaired, 50% of progeny DNA would acquire G·C → A·T transition mutations. The genome of the hyperthermophilic archaeon Pyrobaculum aerophilum has been recently shown to encode a protein, Pa-MIG, a member of the endonuclease III family, capable of processing both G·U and G·T mispairs. We now show that this latter activity is undetectable in crude extracts of P. aerophilum. However, uracil residues in G·U mispairs, in A·U pairs, and in single-stranded DNA were efficiently removed in these extracts. These activities were assigned to a ∼22-kDa polypeptide named Pa-UDG (P. aerophilum uracil-DNA glycosylase). The recombinantPa-UDG protein is highly thermostable and displays a considerable degree of homology to the recently described uracil-DNA glycosylases from Archaeoglobus fulgidus andThermotoga maritima. Interestingly, neitherPa-MIG nor Pa-UDG was inhibited by UGI, a generic inhibitor of the UNG family of uracil glycosylases. Yet a small fraction of the total uracil processing activity present in crude extracts of P. aerophilum was inhibited by this peptide. This implies that the hyperthermophilic archaeon possesses at least a three-pronged defense against the mutagenic threat of hydrolytic deamination of cytosines in its genomic DNA.
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biochemical characterization of uracil processing activities in the hyperthermophilic archaeon Pyrobaculum aerophilum
Journal of Biological Chemistry, 2001Co-Authors: Alessandro A. Sartori, Sorel Fitzgibbon, Jeffrey H Miller, Primo Schar, Josef JiricnyAbstract:Deamination of cytosine to uracil and 5-methylcytosine to thymine represents a major mutagenic threat particularly at high temperatures. In double-stranded DNA, these spontaneous hydrolytic reactions give rise to G.U and G.T mispairs, respectively, that must be restored to G.C pairs prior to the next round of DNA replication; if left unrepaired, 50% of progeny DNA would acquire G.C --> A.T transition mutations. The genome of the hyperthermophilic archaeon Pyrobaculum aerophilum has been recently shown to encode a protein, Pa-MIG, a member of the endonuclease III family, capable of processing both G.U and G.T mispairs. We now show that this latter activity is undetectable in crude extracts of P. aerophilum. However, uracil residues in G.U mispairs, in A.U pairs, and in single-stranded DNA were efficiently removed in these extracts. These activities were assigned to a approximately 22-kDa polypeptide named Pa-UDG (P. aerophilum uracil-DNA glycosylase). The recombinant Pa-UDG protein is highly thermostable and displays a considerable degree of homology to the recently described uracil-DNA glycosylases from Archaeoglobus fulgidus and Thermotoga maritima. Interestingly, neither Pa-MIG nor Pa-UDG was inhibited by UGI, a generic inhibitor of the UNG family of uracil glycosylases. Yet a small fraction of the total uracil processing activity present in crude extracts of P. aerophilum was inhibited by this peptide. This implies that the hyperthermophilic archaeon possesses at least a three-pronged defense against the mutagenic threat of hydrolytic deamination of cytosines in its genomic DNA.
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leaderless transcripts of the crenarchaeal hyperthermophile Pyrobaculum aerophilum
Journal of Molecular Biology, 2001Co-Authors: Malgorzata M Slupska, Sorel Fitzgibbon, John Besemer, Angela G. King, Mark Borodovsky, Jeffrey H MillerAbstract:Abstract We mapped transcription start sites for ten unrelated protein-encoding Pyrobaculum aerophilum genes by primer extension and S 1 nuclease mapping. All of the mapped transcripts start at the computationally predicted translation start codons, two of which were supported by N-terminal protein sequencing. A whole genome computational analysis of the regions from −50 to +50 nt around the predicted translation starts codons revealed a clear upstream pattern matching the consensus sequence of the archaeal TATA box located unusually close to the translation starts. For genes with the TATA boxes that best matched the consensus sequence, the distance between the TATA box and the translation start codon appears to be shorter than 30 nt. Two other promoter elements distinguished were also found unusually close to the translation start codons: a transcription initiator element with significant elevation of C and T frequencies at the −1 position and a BRE element with more frequent A bases at position −29 to −32 (counting from the translation start site). We also show that one of the mapped genes is transcribed as the first gene of an operon. For a set of genes likely to be internal in operons the upstream signal extracted by computer analysis was a Shine-Dalgarno pattern matching the complementary sequence of P. aerophilum 16 S rRNA. Together these results suggest that the translation of proteins encoded by single genes or genes that are first in operons in the hyperthermophilic crenarchaeon P. aerophilum proceeds mostly, if not exclusively, through leaderless transcripts. Internal genes in operons are likely to undergo translation via a mechanism that is facilitated by ribosome binding to the Shine-Dalgarno sequence.
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a thermostable endonuclease iii homolog from the archaeon Pyrobaculum aerophilum
Nucleic Acids Research, 2001Co-Authors: Hanjing Yang, Sorel Fitzgibbon, Isabella T Phan, Mahmud K K Shivji, Richard D Wood, Wendy M Clendenin, Elizabeth C Hyman, Jeffrey H MillerAbstract:: Pyrimidine adducts in cellular DNA arise from modification of the pyrimidine 5,6-double bond by oxidation, reduction or hydration. The biological outcome includes increased mutation rate and potential lethality. A major DNA N:-glycosylase responsible for the excision of modified pyrimidine bases is the base excision repair (BER) glycosylase endonuclease III, for which functional homologs have been identified and characterized in Escherichia coli, yeast and humans. So far, little is known about how hyperthermophilic Archaea cope with such pyrimidine damage. Here we report characterization of an endonuclease III homolog, PaNth, from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100 degrees C. The predicted product of 223 amino acids shares significant sequence homology with several [4Fe-4S]-containing DNA N:-glycosylases including E.coli endonuclease III (EcNth). The histidine-tagged recombinant protein was expressed in E.coli and purified. Under optimal conditions of 80-160 mM NaCl and 70 degrees C, PaNth displays DNA glycosylase/ss-lyase activity with the modified pyrimidine base 5,6-dihydrothymine (DHT). This activity is enhanced when DHT is paired with G. Our data, showing the structural and functional similarity between PaNth and EcNth, suggests that BER of modified pyrimidines may be a conserved repair mechanism in Archaea. Conserved amino acid residues are identified for five subfamilies of endonuclease III/UV endonuclease homologs clustered by phylogenetic analysis.
Sorel Fitzgibbon - One of the best experts on this subject based on the ideXlab platform.
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genome sequence of the hyperthermophilic crenarchaeon Pyrobaculum aerophilum
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Sorel Fitzgibbon, Heidi Ladner, Melvin I Simon, Jeffrey H MillerAbstract:We determined and annotated the complete 2.2-megabase genome sequence of Pyrobaculum aerophilum, a facultatively aerobic nitrate-reducing hyperthermophilic (T-opt = 100 degrees C) crenarchaeon. Clues were found suggesting explanations of the organism's surprising intolerance to sulfur, which may aid in the development of methods for genetic studies of the organism. Many interesting features worthy of further genetic studies were revealed. Whole genome computational analysis confirmed experiments showing that P. aerophilum (and perhaps all crenarchaea) lack 5' untranslated regions in their mRNAs and thus appear not to use a ribosome-binding site (Shine-Dalgarno)-based mechanism for translation initiation at the 5' end of transcripts. Inspection of the lengths and distribution of mononucleotide repeat-tracts revealed some interesting features. For instance, it was seen that mononucleotide repeat-tracts of Gs (or Cs) are highly unstable, a pattern expected for an organism deficient in mismatch repair. This result, together with an independent study on mutation rates, suggests a "mutator" phenotype.
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biochemical characterization of uracil processing activities in the hyperthermophilic archaeon Pyrobaculum aerophilum
Journal of Biological Chemistry, 2001Co-Authors: Alessandro A. Sartori, Sorel Fitzgibbon, Jeffrey H Miller, Primo Schar, Josef JiricnyAbstract:Abstract Deamination of cytosine to uracil and 5-methylcytosine to thymine represents a major mutagenic threat particularly at high temperatures. In double-stranded DNA, these spontaneous hydrolytic reactions give rise to G·U and G·T mispairs, respectively, that must be restored to G·C pairs prior to the next round of DNA replication; if left unrepaired, 50% of progeny DNA would acquire G·C → A·T transition mutations. The genome of the hyperthermophilic archaeon Pyrobaculum aerophilum has been recently shown to encode a protein, Pa-MIG, a member of the endonuclease III family, capable of processing both G·U and G·T mispairs. We now show that this latter activity is undetectable in crude extracts of P. aerophilum. However, uracil residues in G·U mispairs, in A·U pairs, and in single-stranded DNA were efficiently removed in these extracts. These activities were assigned to a ∼22-kDa polypeptide named Pa-UDG (P. aerophilum uracil-DNA glycosylase). The recombinantPa-UDG protein is highly thermostable and displays a considerable degree of homology to the recently described uracil-DNA glycosylases from Archaeoglobus fulgidus andThermotoga maritima. Interestingly, neitherPa-MIG nor Pa-UDG was inhibited by UGI, a generic inhibitor of the UNG family of uracil glycosylases. Yet a small fraction of the total uracil processing activity present in crude extracts of P. aerophilum was inhibited by this peptide. This implies that the hyperthermophilic archaeon possesses at least a three-pronged defense against the mutagenic threat of hydrolytic deamination of cytosines in its genomic DNA.
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biochemical characterization of uracil processing activities in the hyperthermophilic archaeon Pyrobaculum aerophilum
Journal of Biological Chemistry, 2001Co-Authors: Alessandro A. Sartori, Sorel Fitzgibbon, Jeffrey H Miller, Primo Schar, Josef JiricnyAbstract:Deamination of cytosine to uracil and 5-methylcytosine to thymine represents a major mutagenic threat particularly at high temperatures. In double-stranded DNA, these spontaneous hydrolytic reactions give rise to G.U and G.T mispairs, respectively, that must be restored to G.C pairs prior to the next round of DNA replication; if left unrepaired, 50% of progeny DNA would acquire G.C --> A.T transition mutations. The genome of the hyperthermophilic archaeon Pyrobaculum aerophilum has been recently shown to encode a protein, Pa-MIG, a member of the endonuclease III family, capable of processing both G.U and G.T mispairs. We now show that this latter activity is undetectable in crude extracts of P. aerophilum. However, uracil residues in G.U mispairs, in A.U pairs, and in single-stranded DNA were efficiently removed in these extracts. These activities were assigned to a approximately 22-kDa polypeptide named Pa-UDG (P. aerophilum uracil-DNA glycosylase). The recombinant Pa-UDG protein is highly thermostable and displays a considerable degree of homology to the recently described uracil-DNA glycosylases from Archaeoglobus fulgidus and Thermotoga maritima. Interestingly, neither Pa-MIG nor Pa-UDG was inhibited by UGI, a generic inhibitor of the UNG family of uracil glycosylases. Yet a small fraction of the total uracil processing activity present in crude extracts of P. aerophilum was inhibited by this peptide. This implies that the hyperthermophilic archaeon possesses at least a three-pronged defense against the mutagenic threat of hydrolytic deamination of cytosines in its genomic DNA.
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leaderless transcripts of the crenarchaeal hyperthermophile Pyrobaculum aerophilum
Journal of Molecular Biology, 2001Co-Authors: Malgorzata M Slupska, Sorel Fitzgibbon, John Besemer, Angela G. King, Mark Borodovsky, Jeffrey H MillerAbstract:Abstract We mapped transcription start sites for ten unrelated protein-encoding Pyrobaculum aerophilum genes by primer extension and S 1 nuclease mapping. All of the mapped transcripts start at the computationally predicted translation start codons, two of which were supported by N-terminal protein sequencing. A whole genome computational analysis of the regions from −50 to +50 nt around the predicted translation starts codons revealed a clear upstream pattern matching the consensus sequence of the archaeal TATA box located unusually close to the translation starts. For genes with the TATA boxes that best matched the consensus sequence, the distance between the TATA box and the translation start codon appears to be shorter than 30 nt. Two other promoter elements distinguished were also found unusually close to the translation start codons: a transcription initiator element with significant elevation of C and T frequencies at the −1 position and a BRE element with more frequent A bases at position −29 to −32 (counting from the translation start site). We also show that one of the mapped genes is transcribed as the first gene of an operon. For a set of genes likely to be internal in operons the upstream signal extracted by computer analysis was a Shine-Dalgarno pattern matching the complementary sequence of P. aerophilum 16 S rRNA. Together these results suggest that the translation of proteins encoded by single genes or genes that are first in operons in the hyperthermophilic crenarchaeon P. aerophilum proceeds mostly, if not exclusively, through leaderless transcripts. Internal genes in operons are likely to undergo translation via a mechanism that is facilitated by ribosome binding to the Shine-Dalgarno sequence.
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a thermostable endonuclease iii homolog from the archaeon Pyrobaculum aerophilum
Nucleic Acids Research, 2001Co-Authors: Hanjing Yang, Sorel Fitzgibbon, Isabella T Phan, Mahmud K K Shivji, Richard D Wood, Wendy M Clendenin, Elizabeth C Hyman, Jeffrey H MillerAbstract:: Pyrimidine adducts in cellular DNA arise from modification of the pyrimidine 5,6-double bond by oxidation, reduction or hydration. The biological outcome includes increased mutation rate and potential lethality. A major DNA N:-glycosylase responsible for the excision of modified pyrimidine bases is the base excision repair (BER) glycosylase endonuclease III, for which functional homologs have been identified and characterized in Escherichia coli, yeast and humans. So far, little is known about how hyperthermophilic Archaea cope with such pyrimidine damage. Here we report characterization of an endonuclease III homolog, PaNth, from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100 degrees C. The predicted product of 223 amino acids shares significant sequence homology with several [4Fe-4S]-containing DNA N:-glycosylases including E.coli endonuclease III (EcNth). The histidine-tagged recombinant protein was expressed in E.coli and purified. Under optimal conditions of 80-160 mM NaCl and 70 degrees C, PaNth displays DNA glycosylase/ss-lyase activity with the modified pyrimidine base 5,6-dihydrothymine (DHT). This activity is enhanced when DHT is paired with G. Our data, showing the structural and functional similarity between PaNth and EcNth, suggests that BER of modified pyrimidines may be a conserved repair mechanism in Archaea. Conserved amino acid residues are identified for five subfamilies of endonuclease III/UV endonuclease homologs clustered by phylogenetic analysis.
Todd M Lowe - One of the best experts on this subject based on the ideXlab platform.
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methylation guide rna evolution in archaea structure function and genomic organization of 110 c d box srna families across six Pyrobaculum species
Nucleic Acids Research, 2018Co-Authors: Andrew V Uzilov, David L Bernick, Todd M Lowe, Andrea Corredor, Patrick P DennisAbstract:Author(s): Lui, Lauren M; Uzilov, Andrew V; Bernick, David L; Corredor, Andrea; Lowe, Todd M; Dennis, Patrick P | Abstract: Archaeal homologs of eukaryotic C/D box small nucleolar RNAs (C/D box sRNAs) guide precise 2'-O-methyl modification of ribosomal and transfer RNAs. Although C/D box sRNA genes constitute one of the largest RNA gene families in archaeal thermophiles, most genomes have incomplete sRNA gene annotation because reliable, fully automated detection methods are not available. We expanded and curated a comprehensive gene set across six species of the crenarchaeal genus Pyrobaculum, particularly rich in C/D box sRNA genes. Using high-throughput small RNA sequencing, specialized computational searches and comparative genomics, we analyzed 526 Pyrobaculum C/D box sRNAs, organizing them into 110 families based on synteny and conservation of guide sequences which determine methylation targets. We examined gene duplications and rearrangements, including one family that has expanded in a pattern similar to retrotransposed repetitive elements in eukaryotes. New training data and inclusion of kink-turn secondary structural features enabled creation of an improved search model. Our analyses provide the most comprehensive, dynamic view of C/D box sRNA evolutionary history within a genus, in terms of modification function, feature plasticity, and gene mobility.
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methylation guide rna evolution in archaea structure function and genomic organization of 110 c d box srna families across six Pyrobaculum species
bioRxiv, 2017Co-Authors: Lauren M. Lui, David L Bernick, Todd M Lowe, Andrew V Uzilov, Andrea Corredor, Patrick P DennisAbstract:Archaeal homologs of eukaryotic C/D box small nucleolar RNAs (C/D box sRNAs) guide precise 2′-O-methyl modification of ribosomal and transfer RNAs. Although C/D box sRNA genes constitute one of the largest RNA gene families in archaeal thermophiles, most genomes have incomplete sRNA gene annotation because reliable, fully automated detection methods are not available. We expanded and curated a comprehensive gene set across six species of the crenarchaeal genus Pyrobaculum, particularly rich in C/D box sRNA genes. Using high-throughput small RNA sequencing, specialized computational searches, and comparative genomics, we analyzed 526 Pyrobaculum C/D box sRNAs, organizing them into 110 families based on synteny and conservation of guide sequences which determine methylation targets. We examined gene duplications and rearrangements, including one family that has expanded in a pattern similar to retrotransposed repetitive elements in eukaryotes. New training data and inclusion of kink-turn secondary structural features enabled creation of an improved search model. Our analyses provide the most comprehensive, dynamic view of C/D box sRNA evolutionary history within a genus, in terms of modification function, feature plasticity, and gene mobility.
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Standards in Genomic Sciences (2012) 6:336-345 DOI:10.4056/sigs.2645906 The Genomic Standards Consoritum
2015Co-Authors: David L Bernick, Patricia P. Chan, Aaron E Cozen, Kevin Karplus, Joanna K. C. Coker, Julie N. Murphy, Lauren M. Lui, Todd M LoweAbstract:Pyrobaculum oguniense TE7 is an aerobic hyperthermophilic crenarchaeon isolated from a hot spring in Japan. Here we describe its main chromosome of 2,436,033 bp, with three large-scale inversions and an extra-chromosomal element of 16,887 bp. We have annotated 2,800 protein-coding genes and 145 RNA genes in this genome, including nine H/ACA-like small RNA, 83 predicted C/D box small RNA, and 47 transfer RNA genes. Comparative anal-yses with the closest known relative, the anaerobe Pyrobaculum arsenaticum from Italy, re-veals unexpectedly high synteny and nucleotide identity between these two geographically distant species. Deep sequencing of a mixture of genomic DNA from multiple cells has illu-minated some of the genome dynamics potentially shared with other species in this genus
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Reclassification of Thermoproteus neutrophilus Stetter and Zillig 1989 as Pyrobaculum neutrophilum comb. nov. based on phylogenetic analysis.
International Journal of Systematic and Evolutionary Microbiology, 2013Co-Authors: Patricia P. Chan, Aaron E Cozen, Todd M LoweAbstract:The hyperthermophilic crenarchaeon Thermoproteus neutrophilus V24StaT was originally classified before sequence-based phylogenetic analysis became standard for bacterial taxonomy. Subsequent phylogenetic analyses by various groups have shown that strain V24StaT groups more closely with strains of the genus Pyrobaculum than with those in the genus Thermoproteus . Based on phylogenetic comparison of rRNA gene sequences and ribosomal proteins, we propose that strain V24StaT be reclassified as Pyrobaculum neutrophilum comb. nov., with the type strain V24StaT ( = DSM 2338T = JCM 9278T). An emended description of the genus Pyrobaculum is also presented.
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Complete genome sequence of Pyrobaculum oguniense
Standards in Genomic Sciences, 2012Co-Authors: David L Bernick, Patricia P. Chan, Aaron E Cozen, Kevin Karplus, Joanna K. C. Coker, Julie N. Murphy, Todd M LoweAbstract:Pyrobaculum oguniense TE7 is an aerobic hyperthermophilic crenarchaeon isolated from a hot spring in Japan. Here we describe its main chromosome of 2,436,033 bp, with three large-scale inversions and an extra-chromosomal element of 16,887 bp. We have annotated 2,800 protein-coding genes and 145 RNA genes in this genome, including nine H/ACA-like small RNA, 83 predicted C/D box small RNA, and 47 transfer RNA genes. Comparative analyses with the closest known relative, the anaerobe Pyrobaculum arsenaticum from Italy, reveals unexpectedly high synteny and nucleotide identity between these two geographically distant species. Deep sequencing of a mixture of genomic DNA from multiple cells has illuminated some of the genome dynamics potentially shared with other species in this genus.
Imke Schroder - One of the best experts on this subject based on the ideXlab platform.
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adaptation to a high tungsten environment Pyrobaculum aerophilum contains an active tungsten nitrate reductase
Biochemistry, 2010Co-Authors: Simon De Vries, Milica Momcilovic, Marc J F Strampraad, Julian P Whitelegge, Ashkan Baghai, Imke SchroderAbstract:Nitrate reductases (Nars) belong to the DMSO reductase family of molybdoenzymes. The hyperthermophilic denitrifying archaeon Pyrobaculum aerophilum exhibits nitrate reductase (Nar) activity even at...
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properties of a thermostable nitrate reductase from the hyperthermophilic archaeon Pyrobaculum aerophilum
Journal of Bacteriology, 2001Co-Authors: Sepideh Afshar, Eric E. Johnson, Simon De Vries, Imke SchroderAbstract:The nitrate reductase of the hyperthermophilic archaeon Pyrobaculum aerophilum was purified 137-fold from the cytoplasmic membrane. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the enzyme complex consists of three subunits with apparent molecular weights of 130,000, 52,000, and 32,000. The enzyme contained molybdenum (0.8-mol/mol complex), iron (15.4-mol/mol complex) and cytochrome b (0.49mol/mol complex) as cofactors. The P. aerophilum nitrate reductase distinguishes itself from nitrate reductases of mesophilic bacteria and archaea by its very high specific activity using reduced benzyl viologen as the electron donor (Vmax with nitrate, 1,162 s 21 (326 U/mg); Vmax with chlorate, 1,348 s 21 (378 U/mg) [assayed at 75°C]). The Km values for nitrate and chlorate were 58 and 140 mM, respectively. Azide was a competitive inhibitor and cyanide was a noncompetitive inhibitor of the nitrate reductase activity. The temperature optimum for activity was >95°C. When incubated at 100°C, the purified nitrate reductase had a half-life of 1.5 h. This study constitutes the first description of a nitrate reductase from a hyperthermophilic archaeon. Nitrate serves as electron acceptor to many prokaryotic microbes that thrive under anaerobic conditions. Nitrate respiration occurs via two independent pathways, the denitrification pathway and the ammonification pathway (3). Nitrate is reduced sequentially to dinitrogen gas in the denitrification pathway, while ammonium is the product of the two-step ammonification pathway. The first reaction, in which nitrate is reduced to nitrite via the membrane-bound nitrate reductase, is identical in both pathways (3, 21). In general, the dissimilatory nitrate reductase is conserved among bacteria and archaea that have been investigated thus far. The enzyme has been extensively studied in mesophilic nitrate reducing bacteria such as the ammonifier Escherichia coli and the denitrifiers Paracoccus denitrificans, Pseudomonas stuzeri, Pseudomonas denitrificans, and others (5, 6, 11, 12, 16). The E. coli NarGHI enzyme is one of the best-characterized enzymes (3). The enzyme complex consists of three subunits (11, 16). The a subunit (NarG) has an Mr of 145,000 and contains a molybdopterin cofactor at its active site, where nitrate is reduced to nitrite. The b subunit (NarH) has an Mr of 58,000 and is the location of one [3Fe-4S] center and three [4Fe-4S] centers. Both the a and b subunits are attached to the cytoplasmic membrane by the 25,000-Da g subunit (NarI). This polypeptide contains cytochrome b and functions to oxidize the menaquinol or ubiquinol of the quinone pool. Electrons are transferred from the quinol pool via the b subunit to the a subunit active site (3). Recently, nitrate reductases from several archaeal species have been described. While Haloferax volcanii contains a heterotrimeric enzyme complex similar to the bacterial dissimilatory nitrate reductases, the nitrate reductase from Haloferax denitrificans was purified as a heterodimeric enzyme possibly lacking the
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effect of tungstate on nitrate reduction by the hyperthermophilic archaeon Pyrobaculum aerophilum
Applied and Environmental Microbiology, 1998Co-Authors: Sepideh Afshar, Harold G Monbouquette, Imke SchroderAbstract:metal oxyanion WO4 22 for its anaerobic growth on yeast extract, peptone, and nitrate as carbon and energy sources. The addition of 1 mM MoO4 22 did not replace WO4 22 for the growth of P. aerophilum. However, cell growth was completely inhibited by the addition of 100 mM MoO4 22 to the culture medium. At lower tungstate concentrations (0.3 mM and less), nitrite was accumulated in the culture medium. The accumulation of nitrite was abolished at higher WO4 22 concentrations (<0.7 mM). High-temperature enzyme assays for the nitrate, nitrite, and nitric oxide reductases were performed. The majority of all three denitrification pathway enzyme activities was localized to the cytoplasmic membrane, suggesting their involvement in the energy metabolism of the cell. While nitrite and nitric oxide specific activities were relatively constant at different tungstate concentrations, the activity of nitrate reductase was decreased fourfold at WO4 22 levels of 0.7 mM or higher. The high specific activity of the nitrate reductase enzyme observed at low WO4 22 levels (0.3 mM or less) coincided with the accumulation of nitrite in the culture medium. This study documents the first example of the effect of tungstate on the denitrification process of an extremely thermophilic archaeon. We demonstrate here that nitrate reductase synthesis in P. aerophilum occurs in the presence of high concentrations of tungstate.
Sabato Dauria - One of the best experts on this subject based on the ideXlab platform.
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physicochemical characterization of a thermostable alcohol dehydrogenase from Pyrobaculum aerophilum
PLOS ONE, 2013Co-Authors: Annalisa Vitale, Natasha Thorne, Scott Lovell, Kevin P Battaile, Xin Hu, Sabato Dauria, Min Shen, Douglas S AuldAbstract:In this work we characterize an alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII). We have previously found that PyAeADHII has no activity when standard ADH substrates are used but is active when α-tetralone is used as substrate. Here, to gain insights into enzyme function, we screened several chemical libraries for enzymatic modulators using an assay employing α-tetralone. The results indicate that PyAeADHII activity in the presence of α-tetralone was inhibited by compounds such as flunarizine. We also examined metal coordination of the enzyme in solution by performing metal substitution of the enzyme-bound zinc (Zn2+) with cobalt. The solution-based absorption spectra for cobalt substituted PyAeADHII supports substitution at the structural Zn2+ site. To gain structural insight, we obtained the crystal structure of both wild-type and cobalt-substituted PyAeADHII at 1.75 A and 2.20 A resolution, respectively. The X-ray data confirmed one metal ion per monomer present only at the structural site with otherwise close conservation to other ADH enzymes. We next determined the co-crystal structure of the NADPH-bound form of the enzyme at 2.35 A resolution to help define the active site region of the enzyme and this data shows close structural conservation with horse ADH, despite the lack of a catalytic Zn2+ ion in PyAeADHII. Modeling of α-tetralone into the NADPH bound structure suggests an arginine as a possible catalytic residue. The data presented here can yield a better understanding of alcohol dehydrogenases lacking the catalytic zinc as well as the structural features inherent to thermostable enzymes.
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alcohol dehydrogenase from the hyperthermophilic archaeon Pyrobaculum aerophilum stability at high temperature
Archives of Biochemistry and Biophysics, 2012Co-Authors: Alessio Ausili, Annalisa Vitale, Tullio Labella, Alfonso Barbarisi, Francesco Rosso, Juan C Gomezfernandez, Sabato DauriaAbstract:Abstract The structural and stability properties of a novel zinc-dependent alcohol dehydrogenase from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII) were investigated by Fourier transformed infrared spectroscopy (FTIR). This enzyme is a thermostable homo-tetramer belonging to the GroES-fold motif proteins characterized by an irregular β-barrel conformation. Our results revealed a protein with a secondary structure rich in β-sheet (32% of the total secondary elements) and it showed a three-step thermal unfolding pathway. The complete enzyme denaturation was preceded by the formation of a relaxed tertiary/quaternary structure and previously by an excited native-like conformation. Two-dimensional correlation spectroscopy analysis (2D-COS) and differential scanning calorimetry (DSC) experiments supported these data and allowed us to determine the protein melting temperature at 96.9 °C as well as to suggest the sequence of the events that occurred during the protein denaturation process.
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properties and evolution of an alcohol dehydrogenase from the crenarchaeota Pyrobaculum aerophilum
Gene, 2010Co-Authors: Annalisa Vitale, Tullio Labella, Alfonso Barbarisi, Francesco Rosso, Sabato DauriaAbstract:Abstract The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the medium chain dehydrogenase/reductase (MDR) superfamily was identified in the hyperthermophilic archaeon, Pyrobaculum aerophilum . The P. aerophilum ADH gene ( Pae2687 ) was over-expressed in Escherichia coli , and the protein (PyAeADHII) was purified to homogeneity and characterized. The PyAeADHII belongs to a medium chain class because its monomer size is 330 residues and even if it is structurally similar to other enzymes belonging to MDR superfamily, it lacks key residues involved in the coordination of the catalytic Zn ion and in the binding of alcoholic substrates typical of other ADHs. Consistently, PyAeADHII does not show activity on a large number of alcohols, aldheydes or ketones. It is active only when α-tetralone is used as a substrate. The enzyme has a strict requirement for NADP(H) as the coenzyme and has remarkable thermophilicity, displaying activity at temperatures up to 95 °C. The study of the metabolic pathways of P. aerophilum can provide information on the evolution of genes and enzymes and may be crucial for understanding the evolution of eukaryotic cells.
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properties and evolution of an alcohol dehydrogenase from the crenarchaeota Pyrobaculum aerophilum
Gene, 2010Co-Authors: Annalisa Vitale, Tullio Labella, Alfonso Barbarisi, Francesco Rosso, Sabato DauriaAbstract:Abstract The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the medium chain dehydrogenase/reductase (MDR) superfamily was identified in the hyperthermophilic archaeon, Pyrobaculum aerophilum . The P. aerophilum ADH gene ( Pae2687 ) was over-expressed in Escherichia coli , and the protein (PyAeADHII) was purified to homogeneity and characterized. The PyAeADHII belongs to a medium chain class because its monomer size is 330 residues and even if it is structurally similar to other enzymes belonging to MDR superfamily, it lacks key residues involved in the coordination of the catalytic Zn ion and in the binding of alcoholic substrates typical of other ADHs. Consistently, PyAeADHII does not show activity on a large number of alcohols, aldheydes or ketones. It is active only when α-tetralone is used as a substrate. The enzyme has a strict requirement for NADP(H) as the coenzyme and has remarkable thermophilicity, displaying activity at temperatures up to 95 °C. The study of the metabolic pathways of P. aerophilum can provide information on the evolution of genes and enzymes and may be crucial for understanding the evolution of eukaryotic cells.
