title: Exon grafting yields a "two active-site" lysozyme.
abstract: The design of enzymes with enhanced stability and activity has long been a goal in protein engineering. We report a strategy to engineer an additional active site for human lysozyme, grafted the entire human lysozyme exon 2, which encodes the catalytically competent domain, into the gene at a position corresponding to an exposed loop region in the translated protein. Exon 2 grafting created a novel lysozyme with twice the activity of the wild type enzyme, equal activity came from each of the two active sites. We dissected the contributions of each active site using site-directed mutagenesis of the catalytic doublets of (E35A/D53A), circular dichroism, fluorescence spectra, and molecular modeling. Temperature and pH stability of the "two active-site" enzyme were similar to those of wild-type lysozyme. Thus, we provide a novel strategy for engineering the active site of enzymes.
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, PR China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Li
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.forename: Yanqun
authors.initials: Y
authors.lastname: Fei
authors.forename: Dazhi
authors.initials: D
authors.lastname: Jiang
authors.forename: Yongjie
authors.initials: Y
authors.lastname: Sheng
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Sun
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
mesh_terms: D001665:Binding Sites; D003198:Computer Simulation; D004789:Enzyme Activation; D004795:Enzyme Stability; D005091:Exons / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods; D006801:Humans; D008835:Micrococcaceae / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D013329:Structure-Activity Relationship
chemical_list: D009113:Muramidase