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.

Plant proteases and programmed cell death

“…In a comprehensive analysis of Ftshi mutant phenotypes, Mishra et al (2019) have found that inactive FtsH homologs affect chloroplast function and plant development. Furthermore, in the right conditions, some proteases can ligate peptides, for example in the production of cyclic peptides by asparaginyl endopeptidases such as legumain and butelase (Zauner et al , 2018; James et al , 2019). Also, the presence of smaller molecular weight protein products is not necessarily the result of proteolysis.…”

Plant derived cyclic peptides: from discovery to biotechnological applications

“…However, cyclic peptides that are smaller than nine residues cannot be cyclized . To facilitate the broad utilisation of butelase1, it has been developed to be produced recombinantly in E. coli (James et al, 2019) and yeast (Pi et al, 2019).…”

Contributions to the studies of asparaginyl peptide ligases and to the design of inhibitors targeting bacterial tRNA methyltransferases

“…Enzyme mediated protein/peptide ligation has been exploited for a number of applications in peptide ligation (Proft 2010, Chen et al 2015, peptide cyclization (Saska et al 2007, Nguyen et al 2015b, Nguyen et al 2016a, 2016b, Amy et al 2019, Hemu et al 2016, peptide labelling (Popp et al 2007, John et sl. 2009, Yamamoto and Nagamune 2009, John et al 2008, Antos et al 2016, Craik 2010, Conlan et al 2011, Giang et al 2014, Saska et al 2007), protein-lipid conjugation (John et al 2008, protein thioesters synthesis (Cao et al 2015), living-cell surface labelling (Bi et al 2017, Swee et al 2015 and antibodydrug conjugation (Fang et al 2016).…”