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The macrocyclizing protease butelase 1 remains autocatalytic and reveals the structural basis for ligase activity
Author(s) -
James Amy M.,
Haywood Joel,
Leroux Julie,
Ignasiak Katarzyna,
Elliott Alysha G.,
Schmidberger Jason W.,
Fisher Mark F.,
is Samuel G.,
Fenske Ricarda,
Bond Charles S.,
Mylne Joshua S.
Publication year - 2019
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.14293
Subject(s) - cleavage (geology) , chemistry , dna ligase , protease , mutant , biochemistry , microbiology and biotechnology , ubiquitin ligase , autocatalysis , in vitro , in vivo , protein engineering , proteolysis , biophysics , stereochemistry , biology , enzyme , genetics , gene , ubiquitin , paleontology , fracture (geology) , catalysis
Summary Plant asparaginyl endopeptidases ( AEP s) are expressed as inactive zymogens that perform maturation of seed storage protein upon cleavage‐dependent autoactivation in the low‐ pH environment of storage vacuoles. The AEP s have attracted attention for their macrocyclization reactions, and have been classified as cleavage or ligation specialists. However, we have recently shown that the ability of AEP s to produce either cyclic or acyclic products can be altered by mutations to the active site region, and that several AEP s are capable of macrocyclization given favorable pH conditions. One AEP extracted from Clitoria ternatea seeds (butelase 1) is classified as a ligase rather than a protease, presenting an opportunity to test for loss of cleavage activity. Here, making recombinant butelase 1 and rescuing an Arabidopsis thaliana mutant lacking AEP , we show that butelase 1 retains cleavage functions in vitro and in vivo . The in vivo rescue was incomplete, consistent with some trade‐off for butelase 1 specialization toward macrocyclization. Its crystal structure showed an active site with only subtle differences from cleaving AEP s, suggesting the many differences in its peptide‐binding region are the source of its efficient macrocyclization. All considered, it seems that either butelase 1 has not fully specialized or a requirement for autocatalytic cleavage is an evolutionary constraint upon macrocyclizing AEP s.