STRUCTURE-FUNCTION STUDIES OF NOVEL MEDICALLY RELEVANT FLAVOENZYMES

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 activity of MICAL-MO has been initiated by studying its reactivity with actin. We here confirm the proposal that MICAL promotes depolymerization of actin filaments and present preliminary data that support the hypothesis that MICAL may use actin as its second substrate. Flavocytochrome b2 (Fcb2) is the prototype of a family of a-hydroxyacid dehydrogenases, composed by enzymes supposed to share a common mechanism for the oxidation of the substrate in the enzyme reductive half reaction. Computational studies were carried out to contribute to the understanding of the mechanism of substrate dehydrogenation, which is still debated. These studies demonstrated that lactate oxidation takes place with direct transfer of L-lactate αH to the FMN N(5) position as a hydride anion, as opposed to a two step mechanism that implies α-OH hydrogen abstraction (as a proton) by the active site H373 followed by two-electron transfer to the flavin. The same studies revealed the presence of a water molecule (Wat609), conserved in all enzymes of the Fcb2 family and belonging to third shell residues, which may modulate the acid-base properties of the catalytic residue H373, through a Ser371-Wat609-Asp282-His373 hydrogen bond network.. To experimentally test this hypothesis we produced S371A variants of the isolated flavo-dehydrogenase domain as well as of the full-length enzyme. As previously observed for other Fcb2 active site mutants, the S/A substitution clearly impaired protein folding preventing FMN insertion into the isolated flavoprotein domain and allowing us to obtain partially flavinylated full-length forms. Kinetic experiments on full length enzymes demonstrated that the S/A substitution led to a 20-fold decrease of kcat completely attributable to a decrease of the rate of enzyme reductive half reaction, a 10-fold increase of the value of the dissociation constant of the enzyme-sulfite complex but the pH profile and the Km(Kd) for L-lactate were unchanged. Although we cannot rule out that the observed effects of the S/A substitution are due to a change of the overall geometry of the active site, the results are fully consistent with the prediction made through computational studies that removal of Wat609 through the S/A substitution lowers the H373 proton affinity in the reduced enzyme impairing lactate oxidation and concomitant flavin reduction.