© 2005 Heron Publishing—Victoria, Canada
Oligomerization of Sulfolobus solfataricus signature amidase is promoted by acidic pH and high temperature
Anna Scotto D'Abusco (1), Rita Casadio (2), Gianluca Tasco (2), Laura Giangiacomo (1), Anna Giartosio (1), Valentina Calamia (1), Stefania Di Marco (3), Roberta Chiaraluce (1), Valerio Consalvi (1), Roberto Scandurra (1, 4) and Laura Politi (1)
1. Dipartimento di Scienze Biochimiche “A. Rossi-Fanelli,” Università “La Sapienza,” P.le A. Moro 5, 00185 Roma, Italy / 2. CIRB Biocomputing Group, University of Bologna, Via Irnerio 42, 40126 Bologna, Italy / 3. IRBM P. Angeletti,Via Pontina Km 30.600, 00040 Pomezia, Roma, Italy / 4. Corresponding author ([email protected]) / Received July 4, 2005; accepted September 13, 2005; published online October 21, 2005
The recombinant amidase from the hyperthermophylic archaeon Sulfolobus solfataricus (SSAM) a signature amidase, was cloned, purified and characterized. The enzyme is active on a large number of aliphatic and aromatic amides over the temperature range 60–95 °C and at pH values between 4.0 and 9.5, with an optimum at pH 5.0. The recombinant enzyme is in the form of a dimer of about 110 kD that reversibly associates into an octamer in a pH-dependent reaction. The pH dependence of the state of association was studied using gel permeation chromatography, analytical ultracentrifugation and dynamic light scattering techniques.
At pH 7.0 all three techniques show the presence of two species, in about equal amounts, which is compatible with the existence of a dimeric and an octameric form. In decreasing pH, the dimers formed the octameric species and in increasing pH, the octameric species was converted to dimers. Above pH 8.0, only dimers were present, below pH 3.0 only octamers were present. The association of dimers into octamers decreased in non-polar solvents and increased with temperature. A mutant (Y41C) was obtained that did not show this behavior.
Keywords: amidase signature, amide metabolism, hyperthermophile.
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