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Refolding of FAD-monooxygenase domain of MICAL from inclusion bodiesтезисы доклада
- Авторы: Zakharyants A.A., Poloznikov A.A., Smirnova N.A., Chubar T.A., Gazaryan I.G., Tishkov V.I.
- Сборник: Abstracts of International Conference "Biocatalysis-2013: Fundamentals & Applications"
- Тезисы
- Год издания: 2013
- Место издания: Moscow
- Первая страница: 98
- Последняя страница: 99
- Аннотация: MICAL (Molecule Interacting with CasL) is a >1 MDa protein that contains a N-terminal FAD-monooxygenase domain, followed by a calponin homology domain (CH), a double Zn-finger domain (LIM), a proline-rich domain, and coiled-coil ERM motif. The CH and Lim domains have been implicated in signal transduction and cytoskeletal organization. MICALs play role in neural development and plasticity. Several lines of evidence support a role for MICAL in semaphoring/plexin mediated axonal repulsion. MICAL expression is elevated in oligodendrocytes and in meningeal fibroblasts at sites of spinal cord injury, implicating MICALs in neuronal regeneration. It has been recently shown that filamentous (F)-actin is a natural substrate of MICAL, however, there is no robust modification of the actin depolymerization assay that permits easy monitoring of MICAL physiological activity. Expression and refolding conditions of the recombinant FAD-dependent domain of mouse and Drosophila MICAL from inclusion bodies have been optimized. The recombinant enzyme has been tested with potential low molecular substrates (m-hydroxybenzoate and p-hydroxybenzoate). The enzyme also catalyzes the reduction of ferricyanide with NADPH (the so called diaphorase reaction), typical for all FAD-containing and NAD(P)-dependent enzymes. This reaction was used to follow the enzyme activity during the refolding procedure. Diaphorase activity (ferricyanide reduction) of the mouse MICAL was characterized by rate constantans k(FeCN) = 7.1*103 M-1sec-1 and k(NADPH) = 8.2*103 M-1sec-1. MICAL catalytic properties with respect to low molecular weight substrates have been compared to the well-characterized representative of this class of enzymes, p-hydroxybenzoate hydroxylase (PHBH).
- Добавил в систему: Чубарь Татьяна Анатольевна