Oligomerization of Sulfolobus solfataricus signature amidase is promoted by acidic pH and high temperature.
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 degrees 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.
Scotto D'Abusco
Anna
A
Dipartimento di Scienze Biochimiche A. Rossi-Fanelli, Università La Sapienza, P.le A. Moro 5, 00185 Roma, Italy.
Casadio
Rita
R
Tasco
Gianluca
G
Giangiacomo
Laura
L
Giartosio
Anna
A
Calamia
Valentina
V
Di Marco
Stefania
S
Chiaraluce
Roberta
R
Consalvi
Valerio
V
Scandurra
Roberto
R
Politi
Laura
L
D000577:Amides / Q000378:metabolism; D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
D000577:Amides; D019843:Archaeal Proteins; D011994:Recombinant Proteins; D000581:Amidohydrolases; C019665:amidase