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Characterization of a novel zinc-containing, lysine-specific aminopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus.

ABSTRACT: 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 +/- 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of Co2+ 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.

SUBMITTER: Story SV

PROVIDER: S-EPMC1064047 | biostudies-literature | 2005 Mar

REPOSITORIES: biostudies-literature

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Characterization of a novel zinc-containing, lysine-specific aminopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus.

Story Sherry V SV Shah Claudia C Jenney Francis E FE Adams Michael W W MW Adams Michael W W MW

Journal of bacteriology 20050301 6

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 +/- 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of Co2+ ions (0.2 mM). Optimal KAP activity with L ...[more]

PMID: 15743956

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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 100Â°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 â 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. Pyrococcus furiosus (DSM 3638) was grown at 95Â°C in a 20-liter fermentor using maltose as the carbon and energy source. An exponential-phase culture of P. furiosus that had undergone three successive transfers in the experimental medium was used to inoculate the 20-liter fermentor. The culture was shocked with 0.5 mM of hydrogen peroxide (H2O2) when cell density was in mid-exponential phase (~ 5.0 Â´ 107 cells/ml, as determined by direct microscopic cell count). To obtain RNA for microarray and for quantitative PCR (QPCR) analyses, samples (2 liter) were rapidly removed from the fermentor and cooled to 4Â°C. Total RNA was extracted using acid-phenol and stored at -80Â°C until needed. A total of 3 biological replicates in triplicate (3 copies on the same slide) was used in the data set.

Dataset Information

Characterization of a novel zinc-containing, lysine-specific aminopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus.

Publications

Characterization of a novel zinc-containing, lysine-specific aminopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus.

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