title: Engineering a Catalytically Efficient Recombinant Protein Ligase.
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.
authors.affiliation: School of Biological Sciences, Nanyang Technological University , 60 Nanyang Drive, Singapore 636921.|NTU Institute of Structural Biology, Nanyang Technological University , EMB 06-01, 59 Nanyang Drive, Singapore 636921.
authors.forename: Renliang
authors.initials: R
authors.lastname: Yang
authors.affiliation: School of Biological Sciences, Nanyang Technological University , 60 Nanyang Drive, Singapore 636921.|NTU Institute of Structural Biology, Nanyang Technological University , EMB 06-01, 59 Nanyang Drive, Singapore 636921.
authors.forename: Yee Hwa
authors.initials: YH
authors.lastname: Wong
authors.affiliation: School of Biological Sciences, Nanyang Technological University , 60 Nanyang Drive, Singapore 636921.
authors.forename: Giang K T
authors.initials: GKT
authors.lastname: Nguyen
authors.affiliation: School of Biological Sciences, Nanyang Technological University , 60 Nanyang Drive, Singapore 636921.
authors.forename: James P
authors.identifier: 0000-0003-4433-198X
authors.initials: JP
authors.lastname: Tam
authors.affiliation: School of Biological Sciences, Nanyang Technological University , 60 Nanyang Drive, Singapore 636921.|NTU Institute of Structural Biology, Nanyang Technological University , EMB 06-01, 59 Nanyang Drive, Singapore 636921.
authors.forename: Julien
authors.initials: J
authors.lastname: Lescar
authors.affiliation: School of Biological Sciences, Nanyang Technological University , 60 Nanyang Drive, Singapore 636921.|NTU Institute of Structural Biology, Nanyang Technological University , EMB 06-01, 59 Nanyang Drive, Singapore 636921.
authors.forename: Bin
authors.identifier: 0000-0002-0883-8006
authors.initials: B
authors.lastname: Wu
mesh_terms: D055162:Biocatalysis; D008025:Ligases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D032067:Oldenlandia / Q000201:enzymology; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D008025:Ligases