MICAL indicates a family of multidomain proteins involved in the transduction of signals initiated by semaphorins, which result in cytoskeletal rearrangements linked to axon steering, cell-cell junctions formation, cell migration and vesicular trafficking, by interacting with a number of proteins critical for signalling events to the cytoskeleton. The N-terminal monoxygenase-like domain (MICAL-MO), structurally similar to p-hydroxybenzoate hydroxylase (PHBH), the prototype of FAD-containing monooxygenases, has been shown to be essential for MICAL function. Its catalytic activity has not been identified yet. It has been proposed that MICAL-MO may act by oxidizing a small molecule or the side chain of a cytoskeletal protein with actin being one of the candidate substrates. The purified enzyme contains stoichiometric amounts of non-covalently bound FAD and the spectral properties are consistent with its structure. The enzyme acts as a poor oxidase, with production of stoichiometric amounts of H2O2, preferring NADPH over NADH as the reductant. At atmospheric oxygen concentrations the rate of the overall reaction is fully determined by that of enzyme reduction by NADPH without detectable formation of intermediates. The effects of the type of ions and the ionic strength of the medium on V/KNADPH, and the pH dependence of V and V/KNADPH highlight the similarity of the NADPH binding mode with that of PHBH, which is mainly electrostatic in nature. The search of the enzyme substrate among small molecules, peptides or proteins has been initiated. We have recently confirmed and extended the hypothesis that MICAL-MO may act on actin by demonstrating that that F-actin enhances the rate of NADPH oxidation (kcat, +actin, 12.3 s-1, versus kcat,-actin, 3.4 s-1; Km actin, 4.7 µM at 300 µM NADPH; KNADPH, 11 µM at 2.4 µM actin). The details of MICAL-MO reaction with F-actin are being studied.
Kinetic and spectroscopic characterization of the putative monooxygenase domain of human MICAL-1 / D. Zucchini, G. Caprini, T. Vitali, R.J. Pasterkamp, G. Tedeschi, M.A. Vanoni. ((Intervento presentato al 17. convegno International Symposium on Flavins and Flavoproteins tenutosi a Berkeley nel 2011.
Kinetic and spectroscopic characterization of the putative monooxygenase domain of human MICAL-1
D. ZucchiniPrimo
;G. CapriniSecondo
;G. TedeschiPenultimo
;M.A. VanoniUltimo
2011
Abstract
MICAL indicates a family of multidomain proteins involved in the transduction of signals initiated by semaphorins, which result in cytoskeletal rearrangements linked to axon steering, cell-cell junctions formation, cell migration and vesicular trafficking, by interacting with a number of proteins critical for signalling events to the cytoskeleton. The N-terminal monoxygenase-like domain (MICAL-MO), structurally similar to p-hydroxybenzoate hydroxylase (PHBH), the prototype of FAD-containing monooxygenases, has been shown to be essential for MICAL function. Its catalytic activity has not been identified yet. It has been proposed that MICAL-MO may act by oxidizing a small molecule or the side chain of a cytoskeletal protein with actin being one of the candidate substrates. The purified enzyme contains stoichiometric amounts of non-covalently bound FAD and the spectral properties are consistent with its structure. The enzyme acts as a poor oxidase, with production of stoichiometric amounts of H2O2, preferring NADPH over NADH as the reductant. At atmospheric oxygen concentrations the rate of the overall reaction is fully determined by that of enzyme reduction by NADPH without detectable formation of intermediates. The effects of the type of ions and the ionic strength of the medium on V/KNADPH, and the pH dependence of V and V/KNADPH highlight the similarity of the NADPH binding mode with that of PHBH, which is mainly electrostatic in nature. The search of the enzyme substrate among small molecules, peptides or proteins has been initiated. We have recently confirmed and extended the hypothesis that MICAL-MO may act on actin by demonstrating that that F-actin enhances the rate of NADPH oxidation (kcat, +actin, 12.3 s-1, versus kcat,-actin, 3.4 s-1; Km actin, 4.7 µM at 300 µM NADPH; KNADPH, 11 µM at 2.4 µM actin). The details of MICAL-MO reaction with F-actin are being studied.Pubblicazioni consigliate
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