The macrocyclizing protease butelase 1 remains auto-catalytic and reveals the structural basis for ligase activity

Amy M James, Joel Haywood, Julie Leroux, Katarzyna Ignasiak, Alysha G Elliott, Jason W Schmidberger, Mark F Fisher, Samuel G Nonis, Ricarda Fenske, Charles S Bond, Joshua S Mylne

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3 Citations (Scopus)

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 languageEnglish
Pages (from-to)988-999
Number of pages12
JournalThe Plant Journal
Volume98
Issue number6
DOIs
Publication statusPublished - Jun 2019

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asparaginylendopeptidase
Ligases
ligases
automobiles
Peptide Hydrolases
proteinases
Clitoria
active sites
Catalytic Domain
Seed Storage Proteins
Clitoria ternatea
Enzyme Precursors
zymogens
seed storage proteins
Vacuoles
Arabidopsis
crystal structure

Cite this

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title = "The macrocyclizing protease butelase 1 remains auto-catalytic and reveals the structural basis for ligase activity",
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.",
author = "James, {Amy M} and Joel Haywood and Julie Leroux and Katarzyna Ignasiak and Elliott, {Alysha G} and Schmidberger, {Jason W} and Fisher, {Mark F} and Nonis, {Samuel G} and Ricarda Fenske and Bond, {Charles S} and Mylne, {Joshua S}",
year = "2019",
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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

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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.

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