Functional-genomics-based identification and characterization of open reading frames encoding alpha-glucoside-processing enzymes in the hyperthermophilic archaeon Pyrococcus furiosus.
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ABSTRACT:
SUBMITTER: Comfort DA
PROVIDER: S-EPMC2258559 | biostudies-literature | 2008 Feb
REPOSITORIES: biostudies-literature
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Functional-genomics-based identification and characterization of open reading frames encoding alpha-glucoside-processing enzymes in the hyperthermophilic archaeon Pyrococcus furiosus.
Comfort Donald A DA Chou Chung-Jung CJ Conners Shannon B SB VanFossen Amy L AL Kelly Robert M RM
Applied and environmental microbiology 20071221 4
Bioinformatics analysis and transcriptional response information for Pyrococcus furiosus grown on alpha-glucans led to the identification of a novel isomaltase (PF0132) representing a new glycoside hydrolase (GH) family, a novel GH57 beta-amylase (PF0870), and an extracellular starch-binding protein (1,141 amino acids; PF1109-PF1110), in addition to several other putative alpha-glucan-processing enzymes. ...[more]
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Project description:Oxidative Stress Protection and the Repair Response To Hydrogen Peroxide in the Hyperthermophilic Archaeon Pyrococcus furiosus Pyrococcus furiosus is a shallow marine, anaerobic archaeon that grows optimally at 100°C. Addition of H2O2 (0.5 mM) to a growing culture resulted in cessation of growth with a 2 hour lag before normal growth resumed. Whole genome transcriptional profiling revealed that the main response occurs within 30 min of peroxide addition, with the up-regulation of 62 open reading frames (ORFs), 36 of which are part of 10 potential operons. More than half of the up-regulated ORFs are of unknown function while some others encode proteins that are involved potentially in sequestering iron and sulfide, in DNA repair and in generating NADPH. This response is thought to involve primarily damage repair rather than protection, since cultures exposed to sub-toxic levels of H2O2 were not more resistant to the subsequent addition of H2O2 (0.5 – 5.0 mM). Consequently, there is little if any induced protective response to peroxide, rather, the organism maintains a constitutive protective mechanism involving high levels of oxidoreductase-type enzymes such as superoxide reductase, rubrerythrin and alkyl hydroperoxide reductase I. The related hyperthermophiles P. woesei and Thermococcus kodakaraensis were more sensitive to peroxide than P. furiosus, apparently due to the lack of several of its peroxide-responsive ORFs.
2010-02-25 | GSE20470 | GEO
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2010-05-14 | E-GEOD-20470 | biostudies-arrayexpress
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2006-09-28 | GSE5919 | GEO
Project description:The reverse gyrase gene rgy from the hyperthermophilic archaeon Pyrococcus furiosus was cloned and sequenced. The gene is 3,642 bp (1,214 amino acids) in length. The deduced amino acid sequence has relatively high similarity to the sequences of the Methanococcus jannaschii reverse gyrase (48% overall identity), the Sulfolobus acidocaldarius reverse gyrase (41% identity), and the Methanopynrus kandleri reverse gyrase (37% identity). The P. furiosus reverse gyrase is a monomeric protein, containing a helicase-like module and a type I topoisomerase module, which resembles the enzyme from S. acidocaldarius more than that from M. kandleri, a heterodimeric protein encoded by two separate genes. The control region of the P. furiosus rgy gene contains a typical archaeal putative box A promoter element which is located at position -26 from the transcription start identified by primer extension experiments. The initiating ATG codon is preceded by a possible prokaryote-type ribosome-binding site. Purified P. furiosus reverse gyrase has a sedimentation coefficient of 6S, suggesting a monomeric structure for the native protein. The enzyme is a single polypeptide with an apparent molecular mass of 120 kDa, in agreement with the gene structure. The sequence of the N terminus of the protein corresponded to the deduced amino acid sequence. Phylogenetic analysis indicates that all known reverse gyrase topoisomerase modules form a subgroup inside subfamily IA of type I DNA topoisomerases (sensu Wang [J. C. Wang, Annu. Rev. Biochem. 65:635-692, 1996]). Our results suggest that the fusion between the topoisomerase and helicase modules of reverse gyrase occurred before the divergence of the two archaeal phyla, Crenoarchaeota and Euryarchaeota.
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Project description:Insights into the Metabolism of Elemental Sulfur by the Hyperthermophilic Archaeon Pyrococcus furiosus: Characterization of a Coenzyme A-Dependent NAD(P)H Sulfur Oxidoreductase The hyperthermophilic archaeon Pyrococcus furiosus, uses carbohydrates as a carbon source and produces acetate, CO2 and H2 as end products. When S° is added to a growing culture, within 10 min the rate of H2 production rapidly decreases and H2S is detected. After one hour cells contain high NADPH- and coenzyme A-dependent S° reduction activity (0.7 units/mg, 85°C) located in the cytoplasm. The enzyme responsible for this activity was purified to electrophoretic homogeneity (specific activity, 100 units/mg) and is termed NAD(P)H elemental sulfur oxidoreductase (NSR). NSR is a homodimeric flavoprotein (Mr 100 kDa) and is encoded by PF1186. This was previously assigned to an enzyme that reduces coenzyme A disulfide, which is a side-reaction of NSR. Whole genome DNA microarray and quantitative PCR analyses showed that the expression of NSR is up-regulated up to 7-fold within 10 min of S° addition. This primary response to S° also involves the up-regulation (> 16-fold) of a 13 gene cluster encoding a membrane-bound oxidoreductase (MBX). MBX is proposed replace the homologous 14 gene cluster that encodes the ferredoxin-oxidizing, H2-evolving membrane-bound hydrogenase (MBH), which is down-regulated >12-fold within 10 min of S° addition. Although an activity for MBX could not be demonstrated, it is proposed to conserve energy by oxidizing ferredoxin and reducing NADP, which is used by NSR to reduce S°. A secondary response to S° is observed 30 min after S° addition and includes the up-regulation of genes encoding proteins involved in amino acid biosynthesis and iron metabolism, as well as two so-called sulfur-induced proteins, termed SipA and SipB. This novel S°-reducing system involving NSR and MBX is so far unique to the heterotrophic Thermococcales, and is in contrast to the cytochrome- and quinone-based S°-reducing system in autotrophic archaea and bacteria. Keywords: time course, kinetic, sulfur metabolism, archaea, Pyrococcus furiosus, hyperthermophile
2007-05-25 | GSE7022 | GEO