title: Directed evolution of a thermostable l-aminoacylase biocatalyst.
abstract: Enzymes from extreme environments possess highly desirable traits of activity and stability for application under process conditions. One such example is l-aminoacylase (E.C. 3.5.1.14) from Thermococcus litoralis (TliACY), which catalyzes the enantioselective amide hydrolysis of N-protected l-amino acids, useful for resolving racemic mixtures in the preparation of chiral intermediates. Variants of this enzyme with improved activity and altered substrate preference are highly desirable. We have created a structural homology model of the enzyme and applied various two different directed evolution strategies to identify improved variants. Mutants P237S and F251Y were 2.4-fold more active towards N-benzoyl valine relative to the wild type at 65°C. F251 mutations to basic residues resulted in 4.5-11-fold shifts in the substrate preference towards N-benzoyl phenylalanine relative to N-benzoyl valine. The substrate preference of wild type decreases with increasingly branched and sterically hindered substrates. However, the mutant S100T/M106K disrupted this simple trend by selectively improving the substrate preference for N-benzoyl valine, with a >30-fold shift in the ratio of k(cat) values for N-benzoyl valine and N-benzoyl phenylalanine. Mutations that favoured N-benzoyl-phenylalanine appeared at the active site entrance, whereas those improving activity towards N-benzoyl-valine occurred in the hinge region loops linking the dimerization and zinc-binding domains in each monomer. These observations support a previously proposed substrate induced conformational transition between open and closed forms of aminoacylases.
authors.affiliation: The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, UK.
authors.forename: Brenda M
authors.initials: BM
authors.lastname: Parker
authors.forename: Ian N
authors.initials: IN
authors.lastname: Taylor
authors.forename: John M
authors.initials: JM
authors.lastname: Woodley
authors.forename: John M
authors.initials: JM
authors.lastname: Ward
authors.forename: Paul A
authors.initials: PA
authors.lastname: Dalby
mesh_terms: D000577:Amides / Q000737:chemistry; D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D019143:Evolution, Molecular; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D011487:Protein Conformation; D013379:Substrate Specificity; D019712:Thermococcus / Q000201:enzymology*; D014633:Valine / Q000737:chemistry
chemical_list: D000577:Amides; D050505:Mutant Proteins; D010649:Phenylalanine; C053764:benzoylphenylalanine; D000581:Amidohydrolases; C019672:aminoacylase I; D014633:Valine