tailieunhanh - Báo cáo khoa học: Optimization of D-amino acid oxidase for low substrate concentrations – towards a cancer enzyme therapy

d-Amino acid oxidase (DAAO) has recently become of interest as a biocat-alyst 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 Km for this substrate. | ỊFEBS Journal Optimization of D-amino acid oxidase for low substrate concentrations - towards a cancer enzyme therapy Elena Rosini Loredano Pollegioni Sandro Ghisla Roberto Orru and Gianluca Molla Dipartimento di Biotecnologie e Scienze Molecolari Universita degli studi dell Insubria and The Protein Factory Centro Interuniversitario di Biotecnologie Proteiche Politecnico di Milano and Universita degli studi dell Insubria Varese Italy Keywords cancer therapy cell death hydrogen peroxide kinetics oxygen reactivity Correspondence L. Pollegioni Dipartimento di Biotecnologie e Scienze Molecolari Universita degli studi dell Insubria J. H. Dunant 3 21100 Varese Italy Fax 39 0332 421500 Tel 39 0332 421506 E-mail Present address Dipartimento di Genetica e Microbiologia Universita degli studi di Pavia Via Ferrata 1 27100 Pavia Italy Received 8 May 2009 revised 24 June 2009 accepted 1 July 2009 doi 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 Km 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 Km 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. Introduction Chemotherapy together with surgery and radiotherapy is widely used for the .

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