The newly isolated Clostridium beijerinckii FeFe-hydrogenase CbA5H was characterized by Fourier transform infrared spectroscopy coupled to enzymatic activity assays. This showed for the first time ...that in this enzyme the oxygen-sensitive active state Hox can be simply and reversibly converted to the oxygen-stable inactive Hinact state. This suggests that oxygen sensitivity is not an intrinsic feature of the catalytic center of FeFe-hydrogenases (H-cluster), opening new challenging perspectives on the oxygen sensitivity mechanism as well as new possibilities for exploitation in industrial applications.
FeFe-hydrogenases are efficient H
-catalysts, yet upon contact with dioxygen their catalytic cofactor (H-cluster) is irreversibly inactivated. Here, we combine X-ray crystallography, rational protein ...design, direct electrochemistry, and Fourier-transform infrared spectroscopy to describe a protein morphing mechanism that controls the reversible transition between the catalytic H
-state and the inactive but oxygen-resistant H
-state in FeFe-hydrogenase CbA5H of Clostridium beijerinckii. The X-ray structure of air-exposed CbA5H reveals that a conserved cysteine residue in the local environment of the active site (H-cluster) directly coordinates the substrate-binding site, providing a safety cap that prevents O
-binding and consequently, cofactor degradation. This protection mechanism depends on three non-conserved amino acids situated approximately 13 Å away from the H-cluster, demonstrating that the 1st coordination sphere chemistry of the H-cluster can be remote-controlled by distant residues.
FeFe-hydrogenases reversibly catalyse molecular hydrogen evolution by reduction of two protons. Proton supply to the catalytic site (H-cluster) is essential for enzymatic activity. Cysteine 298 is a ...highly conserved residue in all FeFe-hydrogenases; moreover C298 is structurally very close to the H-cluster and it is important for hydrogenase activity. Here, the function of C298 in catalysis was investigated in detail by means of site saturation mutagenesis, simultaneously studying the effect of C298 replacement with all other 19 amino acids and selecting for mutants with high retained activity. We demonstrated that efficient enzymatic turnover was maintained only when C298 was replaced by aspartic acid, despite the structural diversity between the two residues. Purified CaHydA C298D does not show any significant structural difference in terms of secondary structure and iron incorporation, demonstrating that the mutation does not affect the overall protein fold. C298D retains the hydrogen evolution activity with a decrease of k(cat) only by 2-fold at pH 8.0 and it caused a shift of the optimum pH from 8.0 to 7.0. Moreover, the oxygen inactivation rate was not affected demonstrating that the mutation does not influence O(2) diffusion to the active site or its reactivity with the H-cluster. Our results clearly demonstrate that, in order to maintain the catalytic efficiency and the high turnover number typical of FeFe hydrogenases, the highly conserved C298 can be replaced only by another ionisable residue with similar steric hindrance, giving evidence of its involvement in the catalytic function of FeFe-hydrogenases in agreement with an essential role in proton transfer to the active site.
The FeFe-hydrogenase CpHydA from Clostridium perfringens was immobilized by adsorption on anatase TiO2 electrodes for clean hydrogen production. The immobilized enzyme proved to perform direct ...electron transfer to and from the electrode surface and catalyses both H2 oxidation (H2 uptake) and H2 production (H2 evolution) with a current density for H2 evolution of about 2mAcm−1. The TiO2/CpHydA bioelectrode remained active for several days upon storage and when a reducing potential was set, H2 evolution occurred with a mean Faradaic efficiency of 98%. The high turnover frequency of H2 production and the tight coupling of electron transfer, resulting in a Faradaic efficiency close to 100%, support the exploitation of the novel TiO2/CpHydA stationary electrode as a powerful device for H2 production.
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•A TiO2–FeFe hydrogenase bio-electrode is produced by simple adsorption.•The enzyme retains structure/function and the bio-electrode is stable for several days.•Hydrogen evolution occurs at 98% Faradaic efficiency at −741mV vs SHE.•High turnover frequencies (≥4s−1) compare well with more expensive hybrid systems.
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Genetic variation of phase I drug metabolising enzymes has been shown to greatly influence inter-individual reaction to pharmacological treatments. Among these enzymes, human ...flavin-containing monooxygenase 3 (hFMO3) plays a crucial role and understanding its pharmacogenetics is fundamental for the prediction of individual drug response and the efficacy of therapy. In this work the altered drug metabolism of two common polymorphic variants of hFMO3 (E158K and E308G) are studied by using an electrochemical platform modified with graphene oxide (GO). Electrochemistry was used to characterise the properties of these two engineered and purified hFMO3 variants followed by electrocatalysis experiments in the presence of three different hFMO3 substrates benzydamine, tamoxifen and sulindac sulfide. HPLC quantification of the electrochemically produced metabolites showed that E158K mutation leads to an impairment of N-oxygenation activity while E308G mutation enhances the same activity.
Results demonstrate that electrocatalysis on GO modified glassy carbon electrodes provides a fast and reliable method for measuring kinetic parameters of hFMO3 polymorphic variants. This method can be considered suitable for deciphering metabolic implications of polymorphisms that might lead to adjustment of drug dosages depending on the individual’s genetic makeup, a step closer to the development of personalised medicine.
FeFe-hydrogenases are efficient natural catalysts that can be exploited for hydrogen production. Immobilization of the recombinant FeFe-hydrogenase CaHydA was achieved for the first time on an ...anatase TiO(2) electrode. The enzyme is able to interact and exchange electrons with the electrode and to catalyze hydrogen production with an efficiency of 70%.
Laboratory evolution techniques are becoming increasingly widespread among protein engineers for the development of novel and designed biocatalysts. The palette of different approaches ranges from ...complete randomized strategies to rational and structure-guided mutagenesis, with a wide variety of costs, impacts, drawbacks and relevance to biotechnology. A technique that convincingly compromises the extremes of fully randomized vs. rational mutagenesis, with a high benefit/cost ratio, is saturation mutagenesis. Here we will present and discuss this approach in its many facets, also tackling the issue of randomization, statistical evaluation of library completeness and throughput efficiency of screening methods. Successful recent applications covering different classes of enzymes will be presented referring to the literature and to research lines pursued in our group. The focus is put on saturation mutagenesis as a tool for designing novel biocatalysts specifically relevant to production of fine chemicals for improving bulk enzymes for industry and engineering technical enzymes involved in treatment of waste, detoxification and production of clean energy from renewable sources.
Catechol 1,2-dioxygenases are iron containing enzymes able to convert catechol into cis,cis-muconate, a precursor of the industrially important compound adipic acid. Catechol 1,2-dioxygenase from ...Acinetobacter radioresistens S13 was immobilized on beta-cyclodextrins cross-linked with carbonate groups (nanosponges) with a yield of 29 mg of enzyme per gram of support. This support was chosen for its low cost and its ability to offer different types of interactions with the enzyme. The activity profiles at different pH and temperatures showed a shift of the optimal pH from 8.5, for the free protein, to 9.5, for the immobilized protein and, similarly, a shift in optimal temperature from 30 degrees C to 50 degrees C. The Michaelis-Menten constant, KM, increased from 2.0 +/- 0.3 microM, for the free form, to 16.6 +/- 4.8 microM for the immobilized enzyme, whereas the rate constant, k(cat), values were found to be 32 +/- 2 s(-1) and 27 +/- 3 s(-1) for the free and immobilized forms respectively. The immobilization process also increased the thermostability of the enzyme with 60% residual activity after 90 min at 40 degrees C for the immobilized protein versus 20% for the free enzyme. A residual activity of 75% was found after 15 min at 60 degrees C for the immobilized enzyme while the free form showed a total loss of activity under the same conditions. The activity toward other substrates, such as 3- and 4-methylcatechol and 4-chlorocatechol, was retained by the immobilized enzyme. A small scale bioreactor was constructed and was able to convert catechol into cis,cis-muconic acid with high efficiency for 70 days.
Biological production of hydrogen has a tremendous potential as an environmentally sustainable technology to generate a clean fuel. Among the different available methods to produce biohydrogen, dark ...fermentation features the highest productivity and can be used as a means to dispose of organic waste biomass. Within this approach, Clostridia have the highest theoretical H2 production yield. Nonetheless, most strains show actual yields far lower than the theoretical maximum: improving their efficiency becomes necessary for achieving cost-effective fermentation processes. This review aims at providing a survey of the metabolic network involved in H2 generation in Clostridia and strategies used to improve it through metabolic engineering. Together with current achievements, a number of future perspectives to implement these results will be illustrated.
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