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We report here the characterization of the first agmatine/cadaverine aminopropyl transferase (ACAPT), the enzyme responsible for polyamine biosynthesis from an archaeon. The gene PF0127 encoding ACAPT in the hyperthermophile Pyrococcus furiosus was cloned and expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. P. furiosus ACAPT is a homodimer of 65 kDa. The broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diaminopropane, putrescine, cadaverine, and sym-nor-spermidine all serve as substrates. While maximal catalytic activity was observed with cadaverine, agmatine was the preferred substrate on the basis of the [k.sub.cat]/[K.sub.m] value. P. furiosus ACAPT is thermoactive and thermostable with an apparent melting temperature of 108[degrees]C that increases to 112[degrees]C in the presence of cadaverine. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. The crystal structure of the enzyme determined to 1.8-[angstrom] resolution confirmed its dimeric nature and provided insight into the proteolytic analyses as well as into mechanisms of thermal stability. Analysis of the polyamine content of P.furiosus showed that spermidine, cadaverine, and sym.nor-spermidine are the major components, with small amounts of sym-nor-spermine and N-(3-aminopropyl)cadaverine (APC). This is the first report in Archaea of an unusual polyamine APC that is proposed to play a role in stress adaptation.

Pyrococcus furiosus utilizes starch and its degradation products, such as maltose, as primary carbon sources, but the pathways by which these [alpha]-glucans are processed have yet to be defined. For example, its genome contains genes proposed to encode five amylolytic enzymes (including a cyclodextrin glucanotransferase [CGTase] and amylopullulanase), as well as two transporters for maltose and maltodextrins (Mal-I and Mal-II), and a range of intracellular enzymes have been purified that reportedly metabolize altodextrins and maltose. However, precisely which of these enzymes are involved in starch processing is not clear. In this study, starch metabolism in P. furiosus was examined by biochemical analyses in conjunction with global transcriptional response data for cells grown on a variety of glucans. In addition, DNA sequencing led to the correction of two key errors in the genome sequence, and these change the predicted properties of amylopullulanase (now designated PF1935*) and CGTase (PF0478*). Based on all of these data, a pathway is proposed that is specific for starch utilization that involves one transporter (Mal-II [PF1933 to PF1939]) and only three enzymes, amylopullulanase (PF1935*), 4-[alpha]-glucanotransferase (PF0272), and maltodextrin phosphorylase (PF1535). Their expression is upregulated on starch, and together they generate glucose and glucose-1-phosphate, which then feed into the novel glycolytic pathway of this organism. In addition, the results indicate that several hypothetical proteins encoded by three gene clusters are also involved in the transport and processing of 1-glucan substrates by P. furiosus.

The original genome annotation of the hyperthermophilic archaeon Pyrococcus furiosus contained 2,065 open reading frames (ORFs). The genome was subsequently automatically annotated in two public databases by the Institute for Genomic Research (TIGR) and the National Center for Biotechnology Information (NCBI). Remarkably, more than 500 of the originally annotated ORFs differ in size in the two databases, many very significantly. For example, more than 170 of the predicted proteins differ at their N termini by more than 25 amino acids. Similar discrepancies were observed in the TIGR and NCBI databases with the other archaeal and bacterial genomes examined. In addition, the two databases contain 60 (NCBI) and 221 (TIGR) ORFs not present in the original annotation of P. furiosus. In the present study we have experimentally assessed the validity of 88 previously unannotated ORFs. Transcriptional analyses showed that 11 of 61 ORFs examined were expressed in P. furiosus when grown at either 95 or 72[degrees]C. In addition, 7 of 54 ORFs examined yielded heat-stable recombinant proteins when they were expressed in Escherichia coli, although only one of the seven ORFs was expressed in P. furiosus under the growth conditions tested. It is concluded that the P. furiosus genome contains at least 17 ORFs not previously recognized in the original annotation. This study serves to highlight the discrepancies in the public databases and the problems of accurately defining the number and sizes of ORFs within any microbial genome.

The main aim of the study is to obtain the recombinant form of Pyrococcus furiosus NAD(P)H:rubredoxin oxidoreductase (NROR), to reconstitute an in vitro recombinant superoxide reduction pathway and to show that NADPH can serve as an electron source for superoxide reduction to peroxide. It is demonstrated that the complete pathway from NAD(P)H to reduction of superoxide reductase (SOR) via NROR and rubredoxin can be reconstituted in an in vitro recombinant system.

Cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus contain high specific activity (11 U/mg) of lysine aminopeptidase (KAP), as measured by the hydrolysis of L-lysyl-p-nitroanilide (Lys-pNA). The enzyme was purified by multistep chromatography. KAP is a homotetramer (38.2 kDa per subunit) and, as purified, contains 2.0 [+ or -] 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of [Co.sup.2+] ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100[degrees]C. The enzyme had a narrow substrate specificity with di-, tri-, and tetrapeptides, and it hydrolyzed only basic N-terminal residues at high rates. Mass spectroscopy analysis of the purified enzyme was used to identify, in the P. furiosus genome database, a gene (PF1861) that encodes a product corresponding to 346 amino acids. The recombinant protein containing a polyhistidine tag at the N terminus was produced in Escherichia coli and purified using affinity chromatography. Its properties, including molecular mass, metal ion dependence, and pH and temperature optima for catalysis, were indistinguishable from those of the native form, although the thermostability of the recombinant form was dramatically lower than that of the native enzyme (half-life of approximately 6 h at 100[degrees]C). Based on its amino acid sequence, KAP is part of the M18 family of peptidases and represents the first prokaryotic member of this family. KAP is also the first lysine-specific aminopeptidase to be purified from an archaeon.

Rubrerythrin was purified by multistep chromatography under anaerobic, reducing conditions from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimer with a molecular mass of 39.2 kDa and contains 2.9 [+ or -] 0.2 iron atoms per subunit. The purified protein had peroxidase activity at 85[degrees]C using hydrogen peroxide with reduced P. furiosus rubredoxin as the electron donor. The specific activity was 36 [micro]mol of rubredoxin oxidized/min/mg with apparent [K.sub.m] values of 35 and 70 [micro]M for hydrogen peroxide and rubredoxin, respectively. When rubrerythrin was combined with rubredoxin and P. furiosus NADH:rubredoxin oxidoreductase, the complete system used NADH as the electron donor to reduce hydrogen peroxide with a specific activity of 7.0 [micro]mol of [H.sub.2][O.sub.2] reduced/min/mg of rubrerythrin at 85[degrees]C. Strangely, as-purified (reduced) rubrerythrin precipitated when oxidized by either hydrogen peroxide, air, or ferricyanide. The gene (PF1283) encoding rubrerythrin was expressed in Escherichia coli grown in medium with various metal contents. The purified recombinant proteins each contained approximately three metal atoms/subunit, ranging from 0.4 Fe plus 2.2 Zn to 1.9 Fe plus 1.2 Zn, where the metal content of the protein depended on the metal content of the E. coli growth medium. The peroxidase activities of the recombinant forms were proportional to the iron content. P. furiosus rubrerythrin is the first to be characterized from a hyperthermophile or from an archaeon, and the results are the first demonstration that this protein functions in an NADH-dependent, hydrogen peroxide: rubredoxin oxidoreductase system. Rubrerythrin is proposed to play a role in the recently defined anaerobic detoxification pathway for reactive oxygen species.

Results show that casein hydrolyzed peptides and elemental sulfur significantly enhance growth of Pyrococcus furiosus and that sulfur is needed for the utilization of peptides. Data also show that sulfur decreases activities of hydrogenase I and II and a membrane-bound hydrogenase suggesting to its metabolic regulatory role.

The biologically active, C-terminally epitope-tagged derivative (FLAGed cyt cy) of Rhodobacter capsulatus cytochrome (cyt) cy was purified and analyzed to characterize the binding properties of the cyt to the cytoplasmic membrane. The purified Rhodobacter capsulatus FLAGed cyt cy exhibited optical spectral properties and redox midpoint potentials that were similar to c-type cytochromes from bacterial and plant mitochondrial sources. Furthermore, the purified mature cyt cy contained an N-terminal signal sequence-like extension that can anchor to the periplasmic membrane.

Photosynthetic-negative (Ps-) mutants of Rhodobacter capsulatus without CycH lack c-type chromosomes due to their inability to undergo cytochrome c biogenesis. The Rhodobacter capsulatus cycH mutants possess complex Ps- Nadi- growth phenotypes. The Ps+ phenotype cannot be complemented by the incomplete cytochromes of the mutants and the absence of cytochrome co and cp have made them cytochrome cd negative-bacteria.

Isolation of a group of Rhodobacter capsulatus mutants, in which the water-soluble cytochrome c2 (cyt c2) or the membrane-bound c-type cytochrome (cyt cy) is absent, helps study the involvement of cyt cy in respiratory electron transport, with or without the alternate respiratory pathway that is dependent on quinol oxidase. Assessment of the mutants' growth and inhibitor responsiveness distribution, their rates of respiration in a particular inhibitor environment and c-type cytochrome oxidation-reduction kinetics reveals that the bc1 complex and cyt c oxidase are linked by cyt cy during respiratory electron transfer.

Presents a study which concluded that cytochrome cy of Rhodobacter capsulatus is attached to the cytoplasmis membrane by an uncleaved signal sequence-like anchor. Research methodology; Statistical information gathered from the study; Analysis of results obtained.