Abstract
Plant asparaginyl endopeptidases (AEPs) are expressed as inactive zymogens that perform seed storage protein maturation upon cleavage dependent auto-activation in the low pH environment of storage vacuoles. AEPs 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 AEPs to produce either cyclic or acyclic products can be altered by mutations to the active site region, and that several AEPs 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 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 AEPs, suggesting the many differences in its peptide binding region are the source of its efficient macrocyclization. All considered, it seems either butelase 1 has not fully specialized or a requirement for auto-catalytic cleavage is an evolutionary constraint upon macrocyclizing AEPs. This article is protected by copyright. All rights reserved.
| Original language | English |
|---|---|
| Pages (from-to) | 988-999 |
| Number of pages | 12 |
| Journal | The Plant Journal |
| Volume | 98 |
| Issue number | 6 |
| DOIs | |
| Publication status | Published - Jun 2019 |
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The macrocyclizing protease butelase 1 remains auto-catalytic and reveals the structural basis for ligase activity. / James, Amy M; Haywood, Joel; Leroux, Julie; Ignasiak, Katarzyna; Elliott, Alysha G; Schmidberger, Jason W; Fisher, Mark F; Nonis, Samuel G; Fenske, Ricarda; Bond, Charles S; Mylne, Joshua S.
In: The Plant Journal, Vol. 98, No. 6, 06.2019, p. 988-999.Research output: Contribution to journal › Article
TY - JOUR
T1 - The macrocyclizing protease butelase 1 remains auto-catalytic and reveals the structural basis for ligase activity
AU - James, Amy M
AU - Haywood, Joel
AU - Leroux, Julie
AU - Ignasiak, Katarzyna
AU - Elliott, Alysha G
AU - Schmidberger, Jason W
AU - Fisher, Mark F
AU - Nonis, Samuel G
AU - Fenske, Ricarda
AU - Bond, Charles S
AU - Mylne, Joshua S
PY - 2019/6
Y1 - 2019/6
N2 - Plant asparaginyl endopeptidases (AEPs) are expressed as inactive zymogens that perform seed storage protein maturation upon cleavage dependent auto-activation in the low pH environment of storage vacuoles. AEPs 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 AEPs to produce either cyclic or acyclic products can be altered by mutations to the active site region, and that several AEPs 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 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 AEPs, suggesting the many differences in its peptide binding region are the source of its efficient macrocyclization. All considered, it seems either butelase 1 has not fully specialized or a requirement for auto-catalytic cleavage is an evolutionary constraint upon macrocyclizing AEPs. This article is protected by copyright. All rights reserved.
AB - Plant asparaginyl endopeptidases (AEPs) are expressed as inactive zymogens that perform seed storage protein maturation upon cleavage dependent auto-activation in the low pH environment of storage vacuoles. AEPs 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 AEPs to produce either cyclic or acyclic products can be altered by mutations to the active site region, and that several AEPs 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 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 AEPs, suggesting the many differences in its peptide binding region are the source of its efficient macrocyclization. All considered, it seems either butelase 1 has not fully specialized or a requirement for auto-catalytic cleavage is an evolutionary constraint upon macrocyclizing AEPs. This article is protected by copyright. All rights reserved.
U2 - 10.1101/380295
DO - 10.1101/380295
M3 - Article
VL - 98
SP - 988
EP - 999
JO - The Plant Journal
JF - The Plant Journal
SN - 0960-7412
IS - 6
ER -