title: Engineering and kinetic stabilization of the therapeutic enzyme Anabeana variabilis phenylalanine ammonia lyase.
abstract: Anabeana variabilis phenylalanine ammonia lyase has just recently been discovered and introduced in clinical trials of phenylketonuria enzyme replacement therapy for its outstanding kinetic properties. In the present study, kinetic stabilization of this therapeutically important enzyme has been explored by introduction of a disulfide bond into the structure. Site-directed mutagenesis was performed with quick-change PCR method. Recombinant wild-type and mutated enzymes were expressed in Escherichia coli, and his-tagged proteins were affinity purified. Formation of disulfide bond was confirmed by Ellman's method, and then chemical unfolding, kinetic behavior, and thermal inactivation of mutated enzyme were compared with the wild type. Based on our results, the Q292C mutation resulted in a significant improvement in kinetic stability and resistance against chemical unfolding of the enzyme while kinetic parameters and pH profile of enzyme activity were remained unaffected. The results of the present study provided an insight towards designing phenylalanine ammonia lyases with higher stability.
authors.affiliation: Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran, zarei.j@gmail.com.
authors.forename: Hossein Zarei
authors.initials: HZ
authors.lastname: Jaliani
authors.forename: Safar
authors.initials: S
authors.lastname: Farajnia
authors.forename: Seyyed Abolghasem
authors.initials: SA
authors.lastname: Mohammadi
authors.forename: Abolfazl
authors.initials: A
authors.lastname: Barzegar
authors.forename: Saeed
authors.initials: S
authors.lastname: Talebi
mesh_terms: D017033:Anabaena / Q000201:enzymology* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010650:Phenylalanine Ammonia-Lyase / Q000737:chemistry* / Q000235:genetics*; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D058767:Protein Unfolding / Q000187:drug effects
chemical_list: D004220:Disulfides; D010650:Phenylalanine Ammonia-Lyase; D019791:Guanidine