Structural insights into the substrate specificity and activity of ervatamins, the papain‐like cysteine proteases from a tropical plant, Ervatamia coronaria

Abstract

Multiple proteases of the same family are quite often present in the same species in biological systems. These multiple proteases, despite having high homology in their primary and tertiary structures, show deviations in properties such as stability, activity, and specificity. It is of interest, therefore, to compare the structures of these multiple proteases in a single species to identify the structural changes, if any, that may be responsible for such deviations. Ervatamin‐A, ervatamin‐B and ervatamin‐C are three such papain‐like cysteine proteases found in the latex of the tropical plant Ervatamia coronaria, and are known not only for their high stability over a wide range of temperature and pH, but also for variations in activity and specificity among themselves and among other members of the family. Here we report the crystal structures of ervatamin‐A and ervatamin‐C, complexed with an irreversible inhibitor 1‐[l‐N‐(trans‐epoxysuccinyl)leucyl]amino‐4‐guanidinobutane (E‐64), together with enzyme kinetics and molecular dynamic simulation studies. A comparison of these results with the earlier structures helps in a correlation of the structural features with the corresponding functional properties. The specificity constants (kcat/Km) for the ervatamins indicate that all of these enzymes have specificity for a branched hydrophobic residue at the P2 position of the peptide substrates, with different degrees of efficiency. A single amino acid change, as compared to ervatamin‐C, in the S2 pocket of ervatamin‐A (Ala67→Tyr) results in a 57‐fold increase in its kcat/Km value for a substrate having a Val at the P2 position. Our studies indicate a higher enzymatic activity of ervatamin‐A, which has been subsequently explained at the molecular level from the three‐dimensional structure of the enzyme and in the context of its helix polarizibility and active site plasticity.

Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid

“…In regard to the observed cleavages for substrates with Gly in P 1 and concomitant preference for Lys/Arg in P 3 (Fig. 5D), dionain adequately carries a negatively charged interface by Asn 62 and Asp 63 that could comprise a stabilizing S 3 subsite. In fact, this interface results from a dionain-1-specific 2-residue insertion (Asp 63 -Arg 64 ) that gives rise to a main-chain protrusion toward subsites S 3 -S 4 (Fig.…”

Subclassification and Biochemical Analysis of Plant Papain-Like Cysteine Proteases Displays Subfamily-Specific Characteristics

“…1A, marked with asterisks). These studies concern papain, chymopapain, and caricain from papaya (Carica papaya; Drenth et al, 1968;Katerelos et al, 1996;Maes et al, 1996), CysEP of castor bean (Ricinus communis; Than et al, 2004), actinidin of kiwi (Actinidia deliciosa; Varughese et al, 1992), ervatamin-A of crape jasmine (Ervatamia coronaria; Ghosh et al, 2008), and EP-B2 of barley (Hordeum vulgare; Bethune et al, 2006).…”

Ultrahigh and High Resolution Structures and Mutational Analysis of Monomeric Streptococcus pyogenes SpeB Reveal a Functional Role for the Glycine-rich C-terminal Loop

“…deviation of 2.04 Å for 68 conserved core C␣ atoms) and ervatamin-C (Protein Data Bank entry 2PRE) (r.m.s. deviation of 2.03 Å for 68 conserved core C␣ atoms) (37), and Staphylococcus aureus staphopain (1CV8) (r.m.s. deviation of 1.57 Å for 62 conserved core C␣ atoms) (38).…”