Characterization of the 20S proteasome from the actinomycete Frankia.
Frankia is an actinomycete that fixes atmospheric nitrogen in symbiotic association with the root systems of a variety of non-leguminous plants, denominated actinorhizal plants. Information on the biology of proteolysis in Frankia is almost non-existent as it is extremely difficult to grow this organism. We have purified 20S proteasomes from Frankia strain ACN14a/ts-r. It is composed of one alpha-subunit and one beta-subunit, which assemble into the canonical structure of four rings of seven subunits each. The enzyme displayed a chymotrypsin-like activity against synthetic substrates and was sensitive to lactacystin, a specific proteasome inhibitor. Analysis of the structural genes and the flanking regions revealed a similar organization to Rhodococcus erythropolis, Mycobacterium tuberculosis and Streptomyces coelicolor and showed that the beta-subunit is encoded with a 52-amino-acid propeptide that is cleaved off in the course of the assembly. We report also for the first time the in vitro assembly of chimeric proteasomes composed of Frankia and Rhodococcus erythropolis subunits, which are correctly assembled and proteolytically active.
Pouch
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Max-Planck-Institut für Biochemie, Abteilung Molekulare Strukturbiologie, Am Klopferspitz 18a, D-82152 Martinsried, Germany.
Cournoyer
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Baumeister
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D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004269:DNA, Bacterial / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D004269:DNA, Bacterial; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex