Molecular characterization of the glucose isomerase from the thermophilic bacterium Fervidobacterium gondwanense.

The gene coding for xylose isomerase from the thermophilic bacterium Fervidobacterium gondwanense was cloned and overexpressed in Escherichia coli. The produced xylose isomerase (XylA), which closely resembles counterparts from Thermotoga maritima and T. neapolitana, was purified and characterized. It is optimally active at 70 degrees C, pH 7.3, with a specific activity of 15.0 U/mg for the interconversion of glucose to fructose. When compared with T. maritima XylA at 85 degrees C, a higher catalytic efficiency was observed. Divalent metal ions Co2+ and Mg2+ were found to enhance the thermostability.

Genetic and biochemical characterization of a short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

The gene encoding a short-chain alcohol dehydrogenase, AdhA, has been identified in the hyperthermophilic archaeon Pyrococcus furiosus, as part of an operon that encodes two glycosyl hydrolases, the beta-glucosidase CelB and the endoglucanase LamA. The adhA gene was functionally expressed in Escherichia coli, and AdhA was subsequently purified to homogeneity. The quaternary structure of AdhA is a dimer of identical 26-kDa subunits. AdhA is an NADPH-dependent oxidoreductase that converts alcohols to the corresponding aldehydes/ketones and vice versa, with a rather broad substrate specificity. Maximal specific activities were observed with 2-pentanol (46 U x mg(-1)) and pyruvaldehyde (32 U x mg(-1)) in the oxidative and reductive reaction, respectively. AdhA has an optimal activity at 90 degrees C, at which temperature it has a half life of 22.5 h. The expression of the adhA gene in P. furiosus was demonstrated by activity measurements and immunoblot analysis of cell extracts. A role of this novel type of archaeal alcohol dehydrogenase in carbohydrate fermentation is discussed.

Transcriptional regulation in the hyperthermophilic archaeon Pyrococcus furiosus: coordinated expression of divergently oriented genes in response to beta-linked glucose polymers.

The genetic organization, expression, and regulation of the celB locus of the hyperthermophilic archaeon Pyrococcus furiosus were analyzed. This locus includes the celB gene, which codes for an intracellular beta-glucosidase, and a divergently orientated gene cluster, adhA-adhB-lamA, which codes for two alcohol dehydrogenases and an extracellular beta-1,3-endoglucanase that is transcribed as a polycistronic messenger (the lamA operon). During growth of P. furiosus on either the beta-1,4-linked glucose dimer cellobiose or the beta-1,3-linked glucose polymer laminarin, the activities of both beta-glucosidase and endoglucanase were increased at least fivefold compared with levels during growth on maltose or pyruvate. Northern blot analysis revealed an enhanced transcription of both the celB gene and the lamA operon in the presence of these glucose-containing substrates. The in vivo and in vitro transcription initiation sites of both the celB gene and the lamA operon were identified 25 nucleotides downstream of conserved TATA box motifs. A number of repeating sequences have been recognized in the celB-adhA intergenic region, some of which might be part of a transcriptional regulator-binding site.

Isolation and characterization of the hyperthermostable serine protease, pyrolysin, and its gene from the hyperthermophilic archaeon Pyrococcus furiosus.

The hyperthermostable serine protease pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus was purified from membrane fractions. Two proteolytically active fractions were obtained, designated high (HMW) and low (LMW) molecular weight pyrolysin, that showed immunological cross-reaction and identical NH2-terminal sequences in which the third residue could be glycosylated. The HMW pyrolysin showed a subunit mass of 150 kDa after acid denaturation. Incubation of HMW pyrolysin at 95 degrees C resulted in the formation of LMW pyrolysin, probably as a consequence of COOH-terminal autoproteolysis. The 4194-base pair pls gene encoding pyrolysin was isolated and characterized, and its transcription initiation site was identified. The deduced pyrolysin sequence indicated a prepro-enzyme organization, with a 1249-residue mature protein composed of an NH2-terminal catalytic domain with considerable homology to subtilisin-like serine proteases and a COOH-terminal domain that contained most of the 32 possible N-glycosylation sites. The archaeal pyrolysin showed highest homology with eucaryal tripeptidyl peptidases II on the amino acid level but a different cleavage specificity as shown by its endopeptidase activity toward caseins, casein fragments including alphaS1-casein and synthetic peptides.

Carbon monoxide dehydrogenase from Methanosarcina frisia Gö1. Characterization of the enzyme and the regulated expression of two operon-like cdh gene clusters.

Carbon monoxide dehydrogenase (Cdh) has been anaerobically purified from Methanosarcina frisia Gö1. The enzyme is a Ni2+-, Fe2+-, and S2--containing alpha2beta2 heterotetramer of 214 kDa with a pI of 5.2 and subunits of 94 and 19 kDa. It has a Vmax of 0.3 mmol of CO min-1 mg-1 and Km values for CO and methyl viologen of approximately 0.9 mM and 0.12 mM, respectively. EPR spectroscopy on the reduced enzyme showed two overlapping signals: one indicative for 2 (4Fe-4S)+ clusters and a second signal that is atypical for standard Fe/S clusters. The latter was, together with high-spin EPR signals of the oxidized enzyme tentatively assigned to an Fe/S cluster of high nuclearity.

A cell-free transcription system for the hyperthermophilic archaeon Pyrococcus furiosus.

We describe here the establishment of a cell-free transcription system for the hyperthermophilic Archaeon Pyrococcus furiosus using the cloned glutamate dehydrogenase (gdh) gene as template. The in vitro system that operated up to a temperature of 85 degrees C initiated transcription 23 bp downstream of a TATA box located 45 bp upstream of the translational start codon of gdh mRNA, at the same site as in Pyrococcus cells. Mutational analyses revealed that this TATA box is essential for in vitro initiation of transcription. Pyrococcus transcriptional components were separated into at least two distinct transcription factor activities and RNA polymerase. One of these transcription factors could be functionally replaced by Methanococcus aTFB and Thermococcus TATA bind- ing protein (TBP). Immunochemical analyses demonstrated a structural relationship between Pyrococcus aTFB and Thermococcus TBP. These findings indicate that a TATA box and a TBP are essential components of the Pyrococcus transcriptional machinery.

Exchange of domains of glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus and the mesophilic bacterium Clostridium difficile: effects on catalysis, thermoactivity and stability.

The glutamate dehydrogenase gene from the hyperthermophilic archaeon Pyrococcus furiosus has been functionally expressed in Escherichia coli under the control of the lambda PL promoter. The P. furiosus glutamate dehydrogenase amounted to 20% of the total E. coli cell protein, and the vast majority consisted of hexamers. Following activation by heat treatment, an enzyme could be purified from E. coli that was indistinguishable from the glutamate dehydrogenase purified from P. furiosus. Hybrid genes, that consisted of the coding regions for the homologous glutamate dehydrogenases from P. furiosus and the mesophilic bacterium Clostridium difficile, were constructed and successfully expressed in E. coli. One of the resulting hybrid proteins, containing the glutamate binding domain of the C. difficile enzyme and the cofactor binding domain of the P. furiosus enzyme, did not show a detectable activity. In contrast, the complementary hybrid containing the P. furiosus glutamate and the C. difficile cofactor binding domain was a catalytically active hexamer that showed a reduced substrate affinity but maintained efficient cofactor binding with the specificity found in the Clostridium symbiosum enzyme. Compared with the C. difficile glutamate dehydrogenase, the archaeal-bacterial hybrid is slightly more thermoactive, less thermostable but much more stable towards guanidinium chloride-induced inactivation and denaturation.

The glutamate dehydrogenase-encoding gene of the hyperthermophilic archaeon Pyrococcus furiosus: sequence, transcription and analysis of the deduced amino acid sequence.

Glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon, Pyrococcus woesei, has been isolated, characterized and found to be very similar if not identical to the recently purified GDH from P. furiosus. Using a polymerase chain reaction, based on the N-terminal amino acid sequences of GDH, the P. furiosus gdh gene was identified, cloned into Escherichia coli and sequenced. The transcription start point of gdh has been mapped 1 nucleotide upstream from the ribosome-binding site. Using antiserum raised against purified GDH, expression of gdh was observed in E. coli. The deduced primary sequence of the P. furiosus GDH has been compared to various bacterial, archaeal and eukaryal GDHs and showed a high degree of similarity (32-52%).

Cloning, sequence analysis, and functional expression of the acetyl coenzyme A synthetase gene from Methanothrix soehngenii in Escherichia coli.

In the acetoclastic methanogen Methanothrix soehngenii, acetate is activated to acetyl coenzyme A by acetyl coenzyme A synthetase (Acs). The acs gene, coding for the single Acs subunit, was isolated from a genomic library of M. soehngenii DNA in Escherichia coli by using antiserum raised against the purified Acs. After introduction in E. coli, the acs gene was expressed, resulting in the production of an immunoreactive protein of 68 kDa, which is approximately 5 kDa smaller than the known size of purified Acs. In spite of this difference in size, the Acs enzymes are produced in similar quantities in E. coli and M. soehngenii and show comparable specific activities. Upstream from the acs gene, consensus archaeal expression signals were identified. Immediately downstream from the acs gene there was a putative transcriptional stop signal. The amino acid sequence deduced from the nucleotide sequence of the acs gene showed homology with those of functionally related proteins, i.e., proteins involved in the binding of coenzyme A, ATP, or both.

Cloning, expression, and sequence analysis of the genes for carbon monoxide dehydrogenase of Methanothrix soehngenii.

The cdhA and cdhB genes that code for the large and the small subunits of carbon monoxide dehydrogenase (CDH), respectively, were isolated from a genomic library of Methanothrix soehngenii DNA in Escherichia coli, using polyclonal antibodies raised against purified CDH. After introduction in E. coli or Desulfovibrio vulgaris, the cdh genes appeared to be expressed irrespective of their orientation, yielding immunoreactive proteins of 79 and 19 kDa, corresponding in size to the known subunits of purified CDH. However, no CDH activity could be detected in these heterologous hosts. The cdh genes are preceded by consensus ribosome-binding sites and are arranged in an operon-like structure, with cdhA preceding cdhB. Upstream from this operon, sequences similar to archaeal promoters were identified. The amino acid sequence, deduced from the primary sequence of cdhA, showed homology with ferredoxins and with acyl-CoA oxidase. This is compatible with the proposed functions of CDH.