Flavoenzymes: action mechanisms and biotechnology



A large number of key metabolic processes rely on oxido-reduction reactions mediated by proteins, enzymes and coenzymes. Flavoproteins and flavoenzymes are common components of these routes due to their unique ability to connect processes of two electrons with those of a single one. These proteins can participate in redox processes because the isoalloxazine moiety of their FMN or FAD cofactors is a redox agent that can exist in three different states: fully oxidised (quinone), one-electron reduced (semiquinone), and two-electron reduced (hydroquinone). The large versatility of FMN and FAD in vivo can only be understood when the flavoprotein is considered as a whole, since they only acts as successful cofactors when their reactive potentiality is tuned by the protein. This makes each flavoprotein highly specific with respect to electron partners and reaction. Therefore, flavoproteins can be considered like efficient and sophisticated molecular instruments that use molecular recognition to control redox processes. Our goal is to increase the use of flavoproteins in biotechnological and therapeutic applications. This needs understanding of the factors that provide them versatility, as well as of the mechanisms in which they are involved. We are applying a combination of biochemical and biophysical tools with theoretical methods to investigate and control the chemistry of several flavoenzymes. 


Biosynthesis of flavin cofactors: Prokaryotic bifunctional enzymes as drug targets

Riboflavin (RF) can be de novo synthesized by plants, yeast and most prokaryotes, but RF uptake from the environment is essential for human nutrition and animal feeding. All organisms are able to transform RF, first into FMN, and then into FAD, by the sequential action of two activities, an ATP:riboflavin kinase (RFK) and an ATP:FMN adenylyltransferase (FMNAT). However, whereas eukaryotes and archaea use two different enzymes for FMN and FAD production, most prokaryotes depend on a single bifunctional enzyme, FAD synthetase (FADS). Deficiency of RFK or FMNAT activities prevents the assembly of flavoproteins essential for cell survival, causing accumulation of the corresponding apoproteins that are unable to carry out their functions. Moreover, changes in the RFK and FMNAT expression levels cause several cellular stresses, suggesting they are also involved in diverse cellular functions. As examples; amyotrophic lateral sclerosis is related with decrease in the expression of both proteins; RFK overexpression up-regulates FMN, FAD and glutathione levels, increasing activity of the cellular antioxidant system and promoting prostate cancer growth and survival; and, RFK enhances signal transductions related to cellular defence depending on the tumor necrosis factor. Differential molecular characteristics to carry out the same chemistry between the enzymatic components for FMN and FAD production in prokaryotes and eukaryotes have been revealed, making selective inhibition of prokaryotic FADS a feasible treatment for diseases produced by pathogens, and, therefore, worth to be explored. The first stage in this process is to increase the structural-functional knowledge of all these enzymes, but particularly prokaryotic FADS. In this line, we use as model the FADS from Corynebacterium ammoniagenes. We have determined the binding and catalytic parameters for the substrates at the two catalytic sites, and localized them in each one of the modules of this enzyme. No homology is found among the FADS module responsible for the FMNAT activity and the monofunctional FMNAT enzymes in mammals. Moreover, despite the homology at the RFK module of FADS with regard to the corresponding monofunctional enzymes in eukarya, a much more complex structural reorganization is envisaged for the prokaryotic protein. Finally, the quaternary organization identified for FADS from Corynebacterium ammoniagenes suggests a possible role of this protein in the flavin homeostasis in prokaryotes. Studies are also under progress with FADS from additional different pathogens.


The apoptosis inducing factor (AIF)

Multicellular organisms have developed complex mechanisms to eliminate potentially dangerous body cells by a mechanism called apoptosis. Deregulation of this mechanism is involved in a number of carcinogenic processes. A cisteine proteases family (caspases) was identified as responsible for apoptosis, but the existence of an alternative mechanism involving the apoptosis inducing factor (AIF) was evidenced. AIF is a phylogenetically preserved mitochondrial flavoenzyme sharing homology with different families of reductases. In intact mitochondria, AIF presents a NADH-oxido-reductase activity through its flavin cofactor, FAD, that has been linked to the stability of the oxidative phosphorylation complexes I and III. However, neither its role as reductase, nor how it generates stability of mitochondrial complexes are clear. Additionally, when mitochondria receive an apoptotic stimulus AIF is translocated to the nucleus, where it induces chromatinolysis. Since the AIF redox state influences its conformation, and, by extension, its pro-apoptotic function, there is some controversy about the relationship between these two AIF functions. The interest in the design of new therapies to modulate caspase-independent apoptosis pathways has increased in the last years, making AIF a potential target to treat pathological disorders (cancer or degenerative diseases) in which this protein causes a defect or excess of apoptosis. Since one of the possibilities for modulating the AIF proapoptotic function means regulating its redox activity, it is imperative to answer several questions related with such activity. Our research on human AIF focus on providing with some of these answers by understanding the molecular mechanisms linked the AIF oxido-reduction processes. This work has been initiated in collaboration with the Programmed Cell Death and Physiopathology of Tumor Cells group leaded by Dr. S. Susin at the Centre de Recherche des Cordeliers, Paris.


Photosynthesis: a key energy transformation electron transfer chain dependent on flavoproteins

During photosynthesis an electron transfer chain produced through the formation of transitory short life complexes that involve the flavoenzyme ferredoxin-NADP+ reductase (FNR) convert light energy into chemical energy (as NADPH) usable by the cell. Under iron deficiency, some algae and cyanobacteria, like Anabaena, incorporate another flavoprotein to this chain, flavodoxin (Fld). Since the growth of such organisms is limited by the iron availability in many parts of the oceans, this chain supports a central role in global photosynthetic productivity. Despite the main biological function of this system is the production of reduction powder as NADPH, but the reverse electron transfer processes also take place in vivo. Moreover, although the photosynthetic function was the first related to FNR, flavoproteins with FNR activity have been described in chloroplasts, phototropic and heterotrophic bacteria, apicoplasts and, animals and yeast mitochondria. Therefore, the electron flow in the photosynthetic and non-photosynthetic directions in Anabaena was chosen as a model for the study of the parameters involved in determining the catalytic mechanism and efficiency of enzymes of the FNR family. Our work has contributed to describe the structural features that determine key aspects of the interaction and electron transfer between the components of this system to efficiently perform their physiological functions. Simulation algorithms using QM/MM methods and experimental data have recently allowed producing a structural model of the catalytically competent complex. The knowledge acquired in this system also allowed us glimpsing the possibility to re-design functions of these flavoproteins and to produce heterologous enzymatic systems. Understanding how the protein environment modulates the flavin reduction potential has allowed producing a battery of FNR and Fld variants with different redox properties. On the other hand, one of the most interesting changes in protein function is that involving coenzyme specificity alteration in oxido-reduction dependent pyridine nucleotide enzymes, NADP+/H by NAD+/H and viceversa, for which Anabaena FNR has produced interesting results.


Collaborations in other flavoenzyme dependent systems

NADPH-flavodoxin (ferredoxin) reductases (FPR) from bacteria: In collaboration with the groups of Dr. E. Ceccarelli and N. Cortez from the Universidad Nacional de Rosario, Argentina, we are comparatively studying bacterial and plastidic enzymes. Since in many bacteria these enzymes are either essential or involved in the response against oxidative stress, they might be interesting drug targets for the treatment of infections caused by pathogens.

Oxidases: The Aryl-alcohol oxidase: In collaboration with Dr. Martinez’s, at the Centro de Investigaciones Biológicas, CSIC, Madrid, we are describing the chemical and structural mechanism of the extracellular flavoenzyme aryl-alcohol oxidase. This enzyme, involved in lignin degradation, catalyzes the oxidation of aromatic alcohols and aldehydes through a synchronous concerted mechanism with the concomitant reduction of O2 to H2O2, and different active site residues are involved in activating the substrate during catalysis.

A flavoprotein dependent electron transfer chain regulates tetralin biodegradation: We are collaborating with F. Reyes, Universidad Pablo Olavide, Sevilla, to describe the mechanism of action of the electron transfer chain in which the flavoenzyme activator ThnY is involved in tetralin biodegradation.


Methodologies used in our research:

  • Production of native and mutant proteins through the use of protein engineering techniques
  • Homologous and heterologous protein expression in different microorganisms
  • Purification of proteins (electrophoresis, chromatographic methods, HPLC, FPLC...)
  • Work under anaerobic conditions
  • Absorption spectrometry: kinetic studies in steady-state, differential spectroscopy, midpoint reduction potential determination.
  • Use of transient kinetic techniques such as stopped-flow and laser flash photolysis.
  • Fluorescence spectroscopy and circular dichroism.
  • Protein crystallization and X-Ray diffraction for the determination of 3D protein structures.
  • Electron Paramagnetic Resonance related techniques (ESEEM, HYSCORE, ENDOR).
  • Isothermal Titration and Differential Scanning Calorimetries
  • Docking, Molecular Dynamics and QM/MM simulations.