Search

The human NQO1 (hNQO1) is a flavin adenine nucleotide (FAD)-dependentoxidoreductase that catalyzes the two-electron reduction of quinones to hydro-quinones, being essential for the antioxidant defense system, stabilization oftumor suppressors, and activation of quinone-based chemotherapeutics. More-over, it is overexpressed in several tumors, which makes it an attractive cancerdrug target. To decipher new structural insights into the flavin reductive half-reaction of the catalytic mechanism of hNQO1, we have carried serial crystal-lography experiments at new ID29 beamline of the ESRF to determine, to thebest of our knowledge, the first structure of the hNQO1 in complex withNADH. We have also performed molecular dynamics simulations of freehNQO1 and in complex with NADH. This is the first structural evidence thatthe hNQO1 functional cooperativity is driven by structural communicationbetween the active sites through long-range propagation of cooperative effectsacross the hNQO1 structure. Both structural results and MD simulations havesupported that the binding of NADH significantly decreases protein dynamics

Enzymes catalysing sequential reactions have developed different mechanisms to control the transport and flux of reactants and intermediates along metabolic pathways, which usually involve direct transfer of metabolites from an enzyme to the next one in a cascade reaction. Despite the fact that metabolite or substrate channelling has been widely studied for reactant molecules, such information is seldom available for cofactors in general, and for flavins in particular. Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) act as cofactors in flavoproteins and flavoenzymes involved in a wide range of physiologically relevant processes in all type of organisms. Homo sapiens riboflavin kinase (RFK) catalyses the biosynthesis of the flavin mononucleotide cofactor, and might directly interplay with its flavin client apo-proteins prior to the cofactor transfer. Non-etheless, none of such complexes has been characterized at molecular or atomic level so far. Here, we particularly evaluate the interaction of riboflavin kinase with one of its potential FMN clients, pyridoxine-5'-phosphate oxidase (PNPOx). The interaction capacity of both proteins is assessed by using isothermal titration calorimetry, a methodology that allows to determine dissociation constants for interaction in the micromolar range (in agreement with the expected transient nature of the interaction). Moreover, we show that; i) both proteins become thermally stabilized upon mutual interaction, ii) the tightly bound FMN product can be transferred from RFK to the apo-form of PNPOx producing an efficient enzyme, and iii) the presence of the apo-form of PNPOx slightly enhances RFK catalytic efficiency. Finally, we also show a computational study to predict likely RFK-PNPOx binding modes that can envisage coupling between the FMN binding cavities of both proteins for the potential transfer of FMN.

The Apoptosis-Inducing Factor (AIF) is a moonlighting flavoenzyme involved in the assembly of mitochondrial respiratory complexes in healthy cells, but also able to trigger DNA cleavage and parthanatos. Upon apoptotic-stimuli, AIF redistributes from the mitochondria to the nucleus, where upon association with other proteins such as endonuclease CypA and histone H2AX, it is proposed to organize a DNA–degradosome complex. In this work, we provide evidence for the molecular assembly of this complex as well as for the cooperative effects among its protein components to degrade genomic DNA into large fragments. We have also uncovered that AIF has nuclease activity that is stimulated in the presence of either Mg2+ or Ca2+. Such activity allows AIF by itself and in cooperation with CypA to efficiently degrade genomic DNA. Finally, we have identified TopIB and DEK motifs in AIF as responsible for its nuclease activity. These new findings point, for the first time, to AIF as a nuclease able to digest nuclear dsDNA in dying cells, improving our understanding of its role in promoting apoptosis and opening paths for the development of new therapeutic strategies.

Brucella ovis encodes a bacterial subclass 1 ferredoxin-NADP(H) reductase (BoFPR) that, by similarity with other FPRs, is expected either to deliver electrons from NADPH to the redox-based metabolism and/or to oxidize NADPH to regulate the soxRS regulon that protects bacteria against oxidative damage. Such potential roles for the pathogen survival under infection conditions make of interest to understand and to act on the BoFPR mechanism. Here, we investigate the NADP/H interaction and NADPH oxidation by hydride transfer (HT) to BoFPR. Crystal structures of BoFPR in free and in complex with NADP hardly differ. The latter shows binding of the NADP adenosine moiety, while its redox-reactive nicotinamide protrudes towards the solvent. Nonetheless, pre-steady-state kinetics show formation of a charge-transfer complex (CTC-1) prior to the hydride transfer, as well as conversion of CTC-1 into a second charge-transfer complex (CTC-2) concomitantly with the HT event. Thus, during catalysis nicotinamide and flavin reacting rings stack. Kinetic data also identify the HT itself as the rate limiting step in the reduction of BoFPR by NADPH, as well as product release limiting the overall reaction. Using all-atom molecular dynamics simulations with a thermal effect approach we are able to visualise a potential transient catalytically competent interaction of the reacting rings. Simulations indicate that the architecture of the FAD folded conformation in BoFPR might be key in catalysis, pointing to its adenine as an element to orient the reactive atoms in conformations competent for HT.