Structure-function characterization of the recombinant aspartic proteinase A1 from Arabidopsis thaliana.
Aspartic proteinases (APs) are involved in several physiological processes in plants, including protein processing, senescence, and stress response and share many structural and functional features with mammalian and microbial APs. The heterodimeric aspartic proteinase A1 from Arabidopsis thaliana (AtAP A1) was the first acid protease identified in this model plant, however, little information exists regarding its structure function characteristics. Circular dichroism analysis indicated that recombinant AtAP A1 contained an higher alpha-helical content than most APs which was attributed to the presence of a sequence known as the plant specific insert in the mature enzyme. rAtAP A1 was stable over a broad pH range (pH 3-8) with the highest stability at pH 5-6, where 70-80% of the activity was retained after 1 month at 37 degrees C. Using calorimetry, a melting point of 79.6 degrees C was observed at pH 5.3. Cleavage profiles of insulin beta-chain indicated that the enzyme exhibited a higher specificity as compared to other plant APs, with a high preference for the Leu(15)-Tyr(16) peptide bond. Molecular modeling of AtAP A1 indicated that exposed histidine residues and their interaction with nearby charged groups may explain the pH stability of rAtAP A1.
Mazorra-Manzano
Miguel A
MA
Centro de Investigación en Alimentación y Desarrollo AC (CIAD), Hermosillo, Sonora, Mexico. mazorra@ciad.mx
Tanaka
Takuji
T
Dee
Derek R
DR
Yada
Rickey Y
RY
D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000493:pharmacokinetics; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000493:pharmacokinetics; D057055:Aspartic Acid Proteases / Q000737:chemistry; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D029681:Arabidopsis Proteins; D011994:Recombinant Proteins; D057055:Aspartic Acid Proteases; C022325:aspartic proteinase A; C552247:AT1G11910 protein, Arabidopsis; D016282:Aspartic Acid Endopeptidases