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Protein-protein interactions of the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

ABSTRACT: BACKGROUND: Although 2,061 proteins of Pyrococcus horikoshii OT3, a hyperthermophilic archaeon, have been predicted from the recently completed genome sequence, the majority of proteins show no similarity to those from other organisms and are thus hypothetical proteins of unknown function. Because most proteins operate as parts of complexes to regulate biological processes, we systematically analyzed protein-protein interactions in Pyrococcus using the mammalian two-hybrid system to determine the function of the hypothetical proteins. RESULTS: We examined 960 soluble proteins from Pyrococcus and selected 107 interactions based on luciferase reporter activity, which was then evaluated using a computational approach to assess the reliability of the interactions. We also analyzed the expression of the assay samples by western blot, and a few interactions by in vitro pull-down assays. We identified 11 hetero-interactions that we considered to be located at the same operon, as observed in Helicobacter pylori. We annotated and classified proteins in the selected interactions according to their orthologous proteins. Many enzyme proteins showed self-interactions, similar to those seen in other organisms. CONCLUSION: We found 13 unannotated proteins that interacted with annotated proteins; this information is useful for predicting the functions of the hypothetical Pyrococcus proteins from the annotations of their interacting partners. Among the heterogeneous interactions, proteins were more likely to interact with proteins within the same ortholog class than with proteins of different classes. The analysis described here can provide global insights into the biological features of the protein-protein interactions in P. horikoshii.

SUBMITTER: Usui K

PROVIDER: S-EPMC1414084 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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Protein-protein interactions of the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

Usui Kengo K Katayama Shintaro S Kanamori-Katayama Mutsumi M Ogawa Chihiro C Kai Chikatoshi C Okada Makiko M Kawai Jun J Arakawa Takahiro T Carninci Piero P Itoh Masayoshi M Takio Koji K Miyano Masashi M Kidoaki Satoru S Matsuda Takehisa T Hayashizaki Yoshihide Y Suzuki Harukazu H

Genome biology 20051118 12

<h4>Background</h4>Although 2,061 proteins of Pyrococcus horikoshii OT3, a hyperthermophilic archaeon, have been predicted from the recently completed genome sequence, the majority of proteins show no similarity to those from other organisms and are thus hypothetical proteins of unknown function. Because most proteins operate as parts of complexes to regulate biological processes, we systematically analyzed protein-protein interactions in Pyrococcus using the mammalian two-hybrid system to deter ...[more]

PMID: 16356270

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Project description:The gene (PH1074) encoding the NAD kinase of the hyperthermophilic archaeon Pyrococcus horikoshii was identified in the genome database, cloned, and functionally expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment at 90 degrees C for 20 min and one successive HiTrap affinity chromatography step. The purified enzyme was easily precipitated by dialysis against phosphate buffer without NaCl and imidazole and was usually stored in buffer containing 0.5 M NaCl and 0.5 M imidazole to avoid precipitation. The molecular mass of the active enzyme was determined to be 145 kDa by a gel filtration method, and the enzyme was composed of a tetramer of 37-kDa subunits. The archaeal enzyme utilized several nucleoside triphosphates, such as GTP, CTP, UTP, and ITP, as well as ATP and inorganic polyphosphates [poly(P)] as phosphoryl donors for NAD phosphorylation. The enzyme utilized poly(P)27 (the average length of the phosphoryl chain was 27) as the most active inorganic polyphosphate for NAD phosphorylation. Thus, this enzyme is categorized as an inorganic polyphosphate/ATP-dependent NAD kinase. The enzyme was the most thermostable NAD kinase found to date: its activity was not lost by incubation at 95 degrees C for 10 min. The enzyme showed classical Michaelis-Menten-type kinetics for NAD and ATP, but not for poly(P)27. The Km values for NAD were determined to be 0.30 and 0.40 mM when poly(P)27 and ATP, respectively, were used as the phosphoryl donors. The Km value for ATP was 0.29 mM, and the concentration of poly(P)27 which gave half of the maximum enzyme activity was 0.59 mM. The enzyme required several metal cations, such as Mg2+, Mn2+, or Ni2+, for its activity. The deduced amino acid sequence showed a low level of identity to those of E. coli ATP-dependent NAD kinase (31%) and the inorganic polyphosphate/ATP-dependent NAD kinase of Mycobacterium tuberculosis (29%). This is the first description of the characteristics of a poly(P)/ATP-dependent NAD kinase from a hyperthermophilic archaeon.

Dataset Information

Protein-protein interactions of the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

Publications

Protein-protein interactions of the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

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