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Optimization of d ‐amino acid oxidase for low substrate concentrations – towards a cancer enzyme therapy
Author(s) -
Rosini Elena,
Pollegioni Loredano,
Ghisla Sandro,
Orru Roberto,
Molla Gianluca
Publication year - 2009
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2009.07191.x
Subject(s) - enzyme , cancer therapy , substrate (aquarium) , chemistry , biochemistry , cancer , amino acid , medicine , biology , ecology
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 H 2 O 2 in tumor cells can be regulated by administration of substrate. This approach is limited by the locally low O 2 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 O 2 and d ‐Ala concentrations and a 10‐fold lower K m for O 2 . 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 O 2 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.