5H0I

Structure of OaAEP1 asparaginyl peptide ligase in its proenzyme form

Summary for 5H0I

• Entry DOI: 10.2210/pdb5h0i/pdb
• Descriptor: Asparaginyl endopeptidase (2 entities in total)
• Functional Keywords: hydrolase
• Biological source: Oldenlandia affinis
• Total number of polymer chains: 2
• Total formula weight: 98787.74

Authors

Yang, R.,Wong, Y.H.,Lescar, J.,Wu, B. (deposition date: 2016-10-04, release date: 2017-03-29, Last modification date: 2024-10-09)

Primary citation

Yang, R.,Wong, Y.H.,Nguyen, G.K.T.,Tam, J.P.,Lescar, J.,Wu, B.
Engineering a Catalytically Efficient Recombinant Protein Ligase
J. Am. Chem. Soc., 139:5351-5358, 2017

PubMed Abstract: Breaking and forming peptidyl bonds are fundamental biochemical reactions in protein chemistry. Unlike proteases that are abundantly available, fast-acting ligases are rare. OaAEP1 is an enzyme isolated from the cyclotide-producing plant oldenlandia affinis that displayed weak peptide cyclase activity, despite having a similar structural fold with other asparaginyl endopeptidases (AEP). Here we report the first atomic structure of OaAEP1, at a resolution of 2.56 Å, in its preactivation form. Our structure and biochemical analysis of this enzyme reveals its activation mechanism as well as structural features important for its ligation activity. Importantly, through structure-based mutagenesis of OaAEP1, we obtained an ultrafast variant having hundreds of times faster catalytic kinetics, capable of ligating well-folded protein substrates using only a submicromolar concentration of enzyme. In contrast, the protein-protein ligation activity in the original wild-type OaAEP1 enzyme described previously is extremely weak. Thus, the structure-based engineering of OaAEP1 described here provides a unique and novel recombinant tool that can now be used to conduct various protein labeling and modifications that were extremely challenging before.

PubMed: 28199119
DOI: 10.1021/jacs.6b12637

Experimental method

X-RAY DIFFRACTION (2.56 Å)