Data di Pubblicazione:
2010
Citazione:
STRUCTURE-FUNCTION STUDIES OF NOVEL MEDICALLY RELEVANT FLAVOENZYMES / D. Zucchini ; tutor: Maria Antonietta Vanoni; coordinatore: Francesco Bonomi. Universita' degli Studi di Milano, 2010 Dec 09. 23. ciclo, Anno Accademico 2009/2010. [10.13130/zucchini-daniela_phd2010-12-09].
Abstract:
Flavoproteins are involved in a wide range of biological processes, with a variety of catalytic reactions performed, which range from typical redox catalyses such as the dehydrogenation of an amino acid, or activation of dioxygen, to photochemistry; from DNA damage repair to light emission. Recently, genomics trascriptomic and proteomic approaches led to the identification of new flavoproteins playing a key role in the metabolism of many organisms. Several of these new enzymes are involved in fundamental processes, such as cellular differentiation, apoptosis, protein folding and pathologies. The production and biochemical characterization of two medically-relevant flavoenzymes will be discussed. In a last section we will address a combined computational and experimental study on flavocytochrome b2, a well-characterised flavoenzyme, whose catalytic mechanism is stillat the center of a lively debate.
Seladin-1/DHCR24 is a novel antiapoptotic factor, whose expression levels vary in neurons susceptible of degeneration typical of Alzheimer disease, in certain cancer cell lines and during differentiation. Seladin-1 was shown to be identical to the putative human 3--hydroxysterol 24 reductase (DHCR24), the mutations of which are associated with desmosterolosis, a severe recessive disease that causes multiple congenital anomalies and mental development delays. Sequence analyses and activity assays in whole cell extracts suggested that Seladin-1/DHCR24 may be a FAD-containing NADPH-dependent enzyme that catalyzes the last step of cholesterol biosynthesis from desmosterol. To provide key information for the interpretation of the biological role of the protein, a project aiming to overproduce, purify Seladin-1/DHCR24 was initiated as a prelude to its biochemical characterization. However, in spite of efforts, none of the constructs for protein production in E. coli or S. cerevisiae cells we generated so far led to protein forms suitable for purification. On the contrary, several forms of the N-terminal putative FAD-dependent monooxygenase domain of MICAL (from the Macromolecule Interacting with CasL) have been produced in E.coli and were purified to homogeneity in a stable form and in quantities sufficient to initiate its biochemical characterization. MICAL indicates a family of multidomain proteins involved in the transduction of signals initiated by semaphorins that 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 signaling 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, but its catalytic activity has not been defined yet. At variance with PHBH, MICAL-MO exhibits a detectable NADPH oxidase activity. The rate of the overall reaction is fully determined by that of enzyme reduction by NADPH, which takes place at 25°C and pH 7.0 without detection of spectroscopically distinguishable intermediates. However, MICAL shares with enzymes of the PHBH family NADPH binding the high sensitivity to the ionic strength of the medium and to specific anions suggesting that NADPH binding is governed by electrostatics. Solvent viscosity effects revealed the presence of conformational changes taking place during the catalytic cycle, another property shared with enzymes of the PHBH family that avoid waste of reducing power and release of reactive oxygen species through conformational changes triggered by the redox state of the flavin cofactor and binding of the substrate to be hydroxylated. The search of the physiological activ
Tipologia IRIS:
Tesi di dottorato
Keywords:
Flavoenzymes ; Seladin-1 ; MICAL ; Flvocytochrome b2
Elenco autori:
D. Zucchini
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