title: Kinetic and spectroscopic characterization of the putative monooxygenase domain of human MICAL-1.
abstract: MICALs form a conserved multidomain protein family essential for cytoskeletal rearrangements. To complement structural information available, we produced the FAD-containing monooxygenase-like domain of human MICAL-1 (MICAL-MO) in forms differing for the presence and location of a His-tag, which only influences the protein yields. The K(m) for NADPH of the NADPH oxidase reaction is sensitive to ionic strength and type of ions. The apparent k(cat) (pH 7) is limited by enzyme reduction by NADPH, which occurs without detectable intermediates, as established by anaerobic rapid reaction experiments. The sensitivity to ionic strength and type of ions and the pH dependence of the steady-state kinetic parameters extend MICAL-MO similarity with enzymes of the p-hydroxybenzoate hydroxylase class at the functional level. The reaction is also sensitive to solvent viscosity, providing a tool to monitor the conformational changes predicted to occur during turnover. Finally, it was confirmed that MICAL-MO promotes actin depolymerization, and it was shown that F-actin, but not G-actin, stimulates NADPH oxidation by increasing k(cat) and k(cat)/K(NADPH) (≈5 and ≈200-fold, respectively) with an apparent K(m) for actin of 4.7μM, under conditions that stabilize F-actin. The time-course of NADPH oxidation shows substrate recycling, indicating the possible reversibility of MICAL effect.
authors.affiliation: Dipartimento di Scienze Biomolecolari e Biotecnologie, Universitá degli Studi di Milano, Via Celoria 26, Milan, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Zucchini
authors.forename: Gianluca
authors.initials: G
authors.lastname: Caprini
authors.forename: R Jeroen
authors.initials: RJ
authors.lastname: Pasterkamp
authors.forename: Gabriella
authors.initials: G
authors.lastname: Tedeschi
authors.forename: Maria A
authors.initials: MA
authors.lastname: Vanoni
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D060588:LIM Domain Proteins / Q000737:chemistry / Q000378:metabolism*; D008840:Microfilament Proteins; D006899:Mixed Function Oxygenases; D019255:NADPH Oxidases / Q000378:metabolism*; D000096985:Calponins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D060588:LIM Domain Proteins; D008840:Microfilament Proteins; D006899:Mixed Function Oxygenases; D019255:NADPH Oxidases; C455272:MICAL1 protein, human; D000096985:Calponins