BACTEROMETRICS · Bioremediation of toxic metals and radionuclides using naturally evolved bacteria capable of intra-cellular reduction without oxidative stress

Bioremediation of toxic metals and radionuclides using naturally evolved bacteria capable of intra-cellular reduction without oxidative stress

The fact that toxic metals and radionuclides can naturally be precipitated by bacterial reduction has been regarded as a promising approach for inexpensive bioremediation in the surroundings of old nuclear waste repository sites. This reaction can be catalysed in the envelope-located electron transport chain of iron- or sulphate-respiring bacteria. In this case, however, the process can be inhibited by nitrate and oxygen, and reduced species can be back-oxidized after release in the extracellular environment. In parallel, metal reduction by cytosolic enzymes often involves 1-electron transfer mechanisms that generate reactive oxygen species, which poison the cells and hamper remediation. Our efforts were focused on strengthening an alternative 'safe' pathway based on the widely distributed ChrR enzyme family of NAD(P)H-dependant FMN-reductases, which catalyse obligatory two-electron transfers that lessen metal toxicity and increase bacterial remediation capacity with minimum oxidative stress. These reductases contain a non-covalently bound flavin mononucleotide (FMN) as prosthetic group, through which electrons are transferred from NAD(P)H to a broad spectrum of substrates, including metal ions, like chromate and uranyl, and nitroaromatic anticancer prodrugs. Escherichia coli chromate reductase ChrR has been extensively studied for its remediation properties, and improved for metal reduction in variants obtained by random mutagenesis or predictions inferred from statistical models. Our initial goal consisted in developing a new biological tool for remediation of chromate and uranyl, based on whole bacterial cells expressing improved ChrR enzymes. This approach was unsuccessful, essentially because the membrane of living bacteria is not sufficiently permeable to metals to allow whole cells benefiting from the strengthened reduction power of improved enzymes. Therefore, to investigate the molecular basis of ChrR activity modulation, we solved the crystal structure of the wild-type protein, determined the kinetic parameters of ChrR variants, and carried out an extended structure-function analysis, involving Fourier-transform infrared (FTIR) spectroscopy, electrochemical titration of oxidation-reduction potentials associated to FMN in ChrR enzymes, and size-exclusion chromatography experiments. For the first time, our results show that while the enzyme smallest functional unit is dimeric, ChrR proteins mainly form tetramers, under different experimental conditions including substrate concentrations historically applied to characterise the reduction kinetics of ChrR variants. In random improved variants, mutations G150S and N154T are distant from the active site (FMN) and are thus likely influencing activity by long-distance structural effects. The most effective amino acid substitution is Y128N, which is centrally localised in a complex hydrogen-bonding network established at the tetramer interface between FMN centres of two distinct dimers. The central role of this network on ChrR activity modulation was confirmed by determining the kinetic properties of new mutants Y85N, R125T and E146T, specially tailored in that goal. The structure-function analysis combined to thorough determination of kinetic parameters suggests that some of ChrR mutations specifically modify the affinity and possibly docking sites for chromate and NADH, while other directly influence the electron transfer mechanism and associated protonation reactions. As ChrR improvement consisted of minor mutations, we have been seeking for similarly improved enzymes that may have naturally evolved in environments where microorganisms have been living in contact with toxic metals for several decades. Thus, this study provides new fundamental knowledge on properties of NAD(P)H-dependent flavoproteins, as well as new insights in the development of a novel biological tool for ex-situ bioremediation of metals and radionuclides.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз