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 α-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 °C. Using calorimetry, a melting point of 79.6 °C was observed at pH 5.3. Cleavage profiles of insulin β-chain indicated that the enzyme exhibited a higher specificity as compared to other plant APs, with a high preference for the Leu₁₅–Tyr₁₆ 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.     

Expression and characterization of the recombinant aspartic proteinase A1 from Arabidopsis thaliana

The present study reports the recombinant expression, purification, and partial characterization of a typical aspartic proteinase from Arabidopsis thaliana (AtAP A1). The cDNA encoding the precursor of AtAP A1 was expressed as a functional protein using the yeast Pichia pastoris. The mature form of the rAtAP A1 was found to be a heterodimeric glycosylated protein with a molecular mass of 47 kDa consisting of heavy and light chain components, approx. 32 and 16 kDa, respectively, linked by disulfide bonds. Glycosylation occurred via the plant specific insert in the light chain. The catalytic properties of the rAtAP A1 were similar to other plant aspartic proteinases with activity in acid pH range, maximal activity at pH 4.0, K_m of 44 μM, and k_cat of 55 s⁻¹ using a synthetic substrate. The enzyme was inhibited by pepstatin A.     

Protective Effect of Lacticaseibacillus casei CRL 431 Postbiotics on Mitochondrial Function and Oxidative Status in Rats with Aflatoxin B1–Induced Oxidative Stress

Probiotics and Antimicrobial Proteins - Studies have shown that the intracellular content of probiotic (postbiotics) has antioxidant properties, which can improve the antioxidant status in vivo....