title: Optimization of D-amino acid oxidase for low substrate concentrations--towards a cancer enzyme therapy.
abstract: D-amino acid oxidase (DAAO) has recently become of interest as a biocatalyst for industrial applications and for therapeutic treatments. It has been used in gene-directed enzyme prodrug therapies, in which its production of H2O2 in tumor cells can be regulated by administration of substrate. This approach is limited by the locally low O2 concentration and the high K(m) for this substrate. Using the directed evolution approach, one DAAO mutant was identified that has increased activity at low O2 and D-Ala concentrations and a 10-fold lower K(m) for O2. We report on the mechanism of this DAAO variant and on its cytotoxicity towards various mammalian cancer cell lines. The higher activity observed at low O2 and D-Ala concentrations results from a combination of modifications of specific kinetic steps, each being of small magnitude. These results highlight the potential in vivo applicability of this evolved mutant DAAO for tumor therapy.
authors.affiliation: Dipartimento di Biotecnologie e Scienze Molecolari, Università degli studi dell'Insubria, Varese, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Rosini
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
authors.forename: Sandro
authors.initials: S
authors.lastname: Ghisla
authors.forename: Roberto
authors.initials: R
authors.lastname: Orru
authors.forename: Gianluca
authors.initials: G
authors.lastname: Molla
mesh_terms: D000409:Alanine / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D045744:Cell Line, Tumor; D003605:D-Amino-Acid Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D010100:Oxygen / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000970:Antineoplastic Agents; D005656:Fungal Proteins; D006861:Hydrogen Peroxide; D003605:D-Amino-Acid Oxidase; D000409:Alanine; D010100:Oxygen