Francesca Valetti - Academia.edu (original) (raw)
Papers by Francesca Valetti
![Research paper thumbnail of Isolation of strains from a bio-hydrogen plant: Novel [FeFe]-hydrogenases for Exploitation in Biotechnology](https://attachments.academia-assets.com/92103125/thumbnails/1.jpg)
](![Research paper thumbnail of Studies of Cysteine 298 Role in [FeFe]-hydrogenases](https://attachments.academia-assets.com/92103126/thumbnails/1.jpg)
](The exclusive functional replacement of cysteine with aspartic acid, an ionisable residue, and th... more The exclusive functional replacement of cysteine with aspartic acid, an ionisable residue, and the shift in the pH activity profile demonstrate experimentally the central role of C298 in the proton transfer pathway to the active site during [FeFe]-hydrogenase catalysis. Since C298 is strongly conserved among all the known functional [FeFe]hydrogenases, it is reasonable that the results obtained here for CaHydA have broader implications regarding its functional significance in the entire class. Future work will aim at a more detailed characterisation of the most relevant variants, application of site saturation mutagenesis to other residues within the enzyme core and study of other [FeFe]-hydrogenases.
![Research paper thumbnail of Oxygen Stability in the New [FeFe]-Hydrogenase from Clostridium beijerinckii SM10 (CbA5H)](https://attachments.academia-assets.com/92103119/thumbnails/1.jpg)
](Biochemistry, 2016
The newly isolated [FeFe]-hydrogenase CbA5H was characterized by FTIR spectroscopy coupled to enz... more The newly isolated [FeFe]-hydrogenase CbA5H was characterized by FTIR 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 the exploitation in industrial applications.
Waste Management, 2016
(AAM) is copyrighted and published by Elsevier. It is posted here by agreement between Elsevier a... more (AAM) is copyrighted and published by Elsevier. It is posted here by agreement between Elsevier and the University of Turin. Changes resulting from the publishing process-such as editing, corrections, structural formatting, and other quality control mechanisms-may not be reflected in this version of the text. The definitive version of the text was subsequently published in WASTE MANAGEMENT, 56, 2016, 10.1016/j.wasman.2016.06.039. You may download, copy and otherwise use the AAM for non-commercial purposes provided that your license is limited by the following restrictions: (1) You may use this AAM for non-commercial purposes only under the terms of the CC-BY-NC-ND license. (2) The integrity of the work and identification of the author, copyright owner, and publisher must be preserved in any copy.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2016
Background: Ar-BVMO is a recently discovered Baeyer-Villiger monooxygenase from the genome of Aci... more Background: Ar-BVMO is a recently discovered Baeyer-Villiger monooxygenase from the genome of Acinetobacter radioresistens S13 closely related to medically relevant ethionamide monooxygenase EtaA (prodrug activator) and capable of inactivating the imipenem antibiotic. Methods: The co-substrate preference as well as steady-state and rapid kinetics studies of the recombinant purified protein were carried out using stopped-flow spectroscopy under anaerobic and aerobic conditions. K d values were measured by isothermal calorimetry. Enzymatic activity was determined by measuring the amount of product formed using high pressure liquid chromatography or gas chromatography. Site-directed mutagenesis experiments were performed to decipher the role of the active site arginine-292. Results: Ar-BVMO was found to oxidize ethionamide as well as linear ketones. Mechanistic studies on the wild type enzyme using stopped-flow spectroscopy allowed for the detection of the characteristic oxygenating C4a-(hydro)peroxyflavin intermediate, which decayed rapidly in the presence of the substrate. Replacement of arginine 292 in Ar-BVMO by glycine or alanine resulted in greatly reduced or no Baeyer-Villiger activity, respectively, demonstrating the crucial role of this residue in catalysis of ketone substrates. However, both the R292A and R292G mutants are capable of carrying out N-and S-oxidation reactions. Conclusions: Substrate profiling of Ar-BVMO confirms its close relationship to EtaA; ethionamide is one of its substrates. The active site Arginine 292 is required for its Baeyer-Villiger activity but not for heteroatom oxidation. General Significance: A single mutation converts Ar-BVMO to a unique S-or Nmonooxygenase, a useful biocatalyst for the production of oxidized metabolites of human drug metabolizing enzymes.
Bioelectrochemistry, 2015
of Canis familiaris cytochrome P450 2D15 with gold nanoparticles: an alternative to 7 animal test... more of Canis familiaris cytochrome P450 2D15 with gold nanoparticles: an alternative to 7 animal testing in drug discovery. Bioelectrochemistry (2015) 105: 110-116.], 8 9 which has been published in final form at 10 [
Fatty acid amide hydrolase (FAAH) is an integral membrane protein that participates in terminatio... more Fatty acid amide hydrolase (FAAH) is an integral membrane protein that participates in termination of cannabinoid signalling mechanisms by catalysing the hydrolysis and thus inactivation of the endogenous cannabinoid anandamide and other natural lipids involved in signalling. Inhibition of FAAH activity, which increases the anandamide levels, is implicated in the treatment of pain,
Yeast, 2003
An amine oxidase from the yeast Kluyveromyces marxianus was induced, purified and completely char... more An amine oxidase from the yeast Kluyveromyces marxianus was induced, purified and completely characterized; it was shown to belong to the class of coppercontaining amine oxidases (E.C. 1.4.3.6). The enzyme was induced by putrescine and, very strongly, by copper(II); structural-functional characterization of the enzyme was performed, including determination of molecular weight, glycosylation, copper and TPQ content, isoelectric point, K M and k CAT (with benzylamine as substrate), pH, temperature and ionic strength effect on catalysis, substrate and inhibitor specificity. A 700 bp clone was isolated containing the cDNA that encodes for the C-terminus of the enzyme; the amino acid sequence deduced (the first available for a benzylamine oxidase from yeast) was compared to that of other copper amine oxidases from microorganisms and higher organisms. From the results obtained, the putrescine/benzylamine oxidase from Kluyveromyces marxianus was found to have a good homology with other enzymes of this class from microorganisms, and particularly with AO I from Aspergillus niger. Nonetheless, some features resulted closer to those of animal amine oxidases and histaminases. Some potential biotechnological applications are proposed. The cDNA Accession No. is AJ320485.
Yeast, 1994
The presence of adenylate cyclase activity was first demonstrated in membrane fractions from the ... more The presence of adenylate cyclase activity was first demonstrated in membrane fractions from the budding yeast Kluyveromyces marxianus. The enzyme showed a Mn2+-and M$+-dependent activity, with optimal pH at around 6 as observed in other yeast species. As in Saccharomyces cerevisiae, where adenylate cyclase is regulated by RASl and RAS2, we detected a guanyl nucleotide-dependent activity. Interestingly Y 13-259 monoclonal antibody, raised against mammalian p21Ha-'=, inhibited M$+ plus GTP-y-S-dependent CAMP production, suggesting that the GTP binding proteins involved in adenylate cyclase regulation could be Ras proteins. The same antibody recognized on Western blot and immunoprecipitated a 40 kDa polypeptide from K. marxianus crude membranes. This polypeptide was not detected by an anti-US2 polyclonal antibody raised against S. cerevisiae RAS2 protein, suggesting that Ras proteins from the two species could be structurally different.
European Journal of Biochemistry, 2003
This paper reports the isolation and characterization of the regulatory moiety of the multicompon... more This paper reports the isolation and characterization of the regulatory moiety of the multicomponent enzyme phenol hydroxylase from Acinetobacter radioresistens S13 grown on phenol as the only carbon and energy source. The whole enzyme comprises an oxygenase moiety (PHO), a reductase moiety (PHR) and a regulatory moiety (PHI). PHR contains one FAD and one iron-sulfur cluster, whose function is electron transfer from NADH to the dinuclear iron centre of the oxygenase. PHI is required for catalysis of the conversion of phenol to catechol in vitro, but is not required for PHR activity towards alternative electron acceptors such as cytochrome c and Nitro Blue Tetrazolium. The molecular mass of PHI was determined to be 10 kDa by SDS/PAGE, 8.8 kDa by MALDI-TOF spectrometry and 18 kDa by gel-permeation. This finding suggests that the protein in its native state is a homodimer. The isoelectric point is 4.1. PHI does not contain any redox cofactor and does not bind ANS, a fluorescent probe for hydrophobic sites. The N-terminal sequence is similar to those of the regulatory proteins of phenol hydroxylase from A. calcoaceticus and Pseudomonas CF 600. In the reconstituted system, optimal reaction rate was achieved when the stoichiometry of the components was 2 PHR monomers: 1 PHI dimer: 1 PHO (alphabetagamma) dimer. PHI interacts specifically with PHR, promoting the enhancement of FAD fluorescence emission. This signal is diagnostic of a conformational change of PHR that might result in a better alignment with respect to PHO.
Dalton Transactions, 2009
Catalytic properties of catechol 1,2-dioxygenase from Acinetobacter radioresistens S13 immobilize... more Catalytic properties of catechol 1,2-dioxygenase from Acinetobacter radioresistens S13 immobilized on nanosponges
Chemical Communications, 2011
[FeFe]-hydrogenases are efficient natural catalysts that can be exploited for hydrogen production... more [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%.
ChemBioChem, 2009
Catechol 1,2-dioxygenases and chlorocatechol dioxygenases are Fe(III)-dependent enzymes that do n... more Catechol 1,2-dioxygenases and chlorocatechol dioxygenases are Fe(III)-dependent enzymes that do not require a reductant to perform the ortho cleavage of the aromatic ring. The reaction mechanism is common to the two enzymes, and active-site residues must play a key role in the fine-tuning of specificity. Protein engineering was applied for the first time to the catalytic pocket of a catechol 1,2-dioxygenase by site-specific and site-saturation mutagenesis with the purpose of redesigning the pocket shape for improved catalysis on bulky derivatives. Mutants were analysed for changes in kinetic parameters: variants for residue 69 show an inversion of specificity with a preference towards 4-chlorocatechol (decrease of K(M) by a factor of 20) and activity on the rarely recognised substrate 4,5-dichlorocatechol, thus creating a novel, engineered chlorocatechol dioxygenase. A L69A substitution conveys gain-of-function activity towards 4-tert-butylcatechol. Mutations of position 72 enhance k(cat) towards chlorinated substrates. The biphasic Arrhenius plot observed in A72S suggests the involvement of a dynamic switch in the fine regulation of the enzyme.
Biotechnology and Applied Biochemistry, 2014
Enzymes entrapped in wet, nanoporous silica gel have great potential as bioreactors for bioremedi... more Enzymes entrapped in wet, nanoporous silica gel have great potential as bioreactors for bioremediation because of their improved thermal, chemical, and mechanical stability with respect to enzymes in solution. The B isozyme of catechol 1,2 dioxygenase from Acinetobacter radioresistens and its mutants of Leu69 and Ala72, designed for an increased reactivity toward the environmental pollutant chlorocatechols, were encapsulated using alkoxysilanes and alkyl alkoxysilanes as precursors in varying proportions. Encapsulation of the mutants in a hydrophobic tetramethoxysilane/ dimethoxydimethylsilane-based matrix yielded a remarkable 10-to 12-fold enhancement in reactivity toward chlorocatechols. These gels also showed a fivefold increase in relative reactivity toward chlorocatechols with respect to the natural substrate catechol, thus compensating for their relatively low activity for these substrates in solution. The encapsulated enzyme, unlike the enzyme in solution, proved resilient in assays carried out in urban wastewater and bacteria-contaminated solutions mimicking environmentally relevant conditions. Overall, the combination of a structure-based rational design of enzyme mutants, and the selection of a suitable encapsulation material, proved to be a powerful approach for the production and optimization of a potential bioremediation device, with increased activity and resistance toward bacterial degradation.
Biosensors and Bioelectronics, 1998
This work demonstrates that non-physiological electron transfer (ET) can occur in solution betwee... more This work demonstrates that non-physiological electron transfer (ET) can occur in solution between wild type D. vulgaris flavodoxin (Fld) and horse heart cytochrome c (cyt-c), D. vulgaris cytochrome c553 (cyt-c553) and the haem domain of B. megaterium cytochrome P450 (cyt-P450 BMP). Second order rate constants of the ET reaction between [Fld] Sq /[cyt-c] Ox , [Fld] Sq /[cyt-c553] Ox and [Fld] Sq /[cyt-P450 BMP] Ox , were found to be 6.16 × 10 5 , 1.80 × 10 4 and in the region of 10 5 respectively. These data are interpreted in terms of complementarity between the surfaces of the two proteins, their surface and redox potentials. Analysis of the ET results obtained from the separate wild type proteins supported the rational design approach in the creation of Fld-based chimeras. The preliminary design of the chimeras reported here is a 3D prototype for an artificial flavo-cytochrome obtained by covalent linkage of a Fld module to cyt-c553 via a disulphide bond. Theoretical ET rates calculated on the modelled flavo-cytochrome are encouraging the construction of these chimeric systems at DNA level. This work is now underway. The relevance of this molecular lego approach is to be seen in the long term goal of producing engineered multi-domain systems to be applied in the field of biosensors and bioelectronics to fulfil specific requirements. Novel catalytic devices can be obtained by using natural redox proteins in different combinations: this process mimics the natural evolution of proteins such as gene shuffling and gene fusion.
Biochemistry, 2002
Cytochrome c-553 from Desulfovibrio vulgaris exhibits a highly exposed heme and an unusually low ... more Cytochrome c-553 from Desulfovibrio vulgaris exhibits a highly exposed heme and an unusually low reduction potential with respect to other c-type cytochromes. Solvent heme exposure has been indicated as one of the most important factors in modulating the midpoint potential of the redox center. To test this hypothesis, a unique surface-exposed cysteine has been substituted for either M23 or G51 to produce the corresponding mutants and allow the formation of homodimers through a specific disulfide bridge. The reduction potentials, determined via spectroelectrochemistry, show an increase from +20 +/- 5 mV for the wt to +88 +/- 5 and +105 +/- 5 mV for the M23C-M23C homodimer and G51C-G51C homodimer, respectively. Chemical denaturation of the homodimers leads to parameters related to the hydrophobicity (m) and the number of buried side chains (n(B)), which suggest a decrease of exposure of the heme as a result of dimerization. These results are consistent with the heme-accessible surface area (ASA) calculated from a computer model of the homodimers. The ASA values show a decrease from 73 A(2) for the wt to 66 and 50 A(2) per heme for the M23C-M23C homodimer and G51C-G51C homodimer, respectively. The trend of the m- and n(B)-values, the degree of solvent accessibility, and the midpoint potential observed upon formation of the homodimers indicate a correlation between the reduction potential values and the exclusion of water from the heme surface.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2011
Intradiol-cleaving catechol 1,2 dioxygenases are Fe(III) dependent enzymes that act on catechol a... more Intradiol-cleaving catechol 1,2 dioxygenases are Fe(III) dependent enzymes that act on catechol and substituted catechols, including chlorocatechols pollutants, by inserting molecular oxygen in the aromatic ring. Members of this class are the object of intense biochemical investigations aimed at the understanding of their catalytic mechanism, particularly for designing mutants with selected catalytic properties. We report here an in depth investigation of catechol 1,2 dioxygenase IsoB from Acinetobacter radioresistens LMG S13 and its A72G and L69A mutants. By applying a multidisciplinary approach that includes high resolution X-rays crystallography, mass spectrometry and single crystal microspectrophotometry, we characterised the phospholipid bound to the enzyme and provided a structural framework to understand the inversion of substrate specificity showed by the mutants. Our results might be of help for the rational design of enzyme mutants showing a biotechnologically relevant substrate specificity, particularly to be used in bioremediation. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
European Journal of Biochemistry, 2003
Phenol hydroxylase (PH) from Acinetobacter radioresistens S13 represents an example of multicompo... more Phenol hydroxylase (PH) from Acinetobacter radioresistens S13 represents an example of multicomponent aromatic ring monooxygenase made up of three moieties: a reductase (PHR), an oxygenase (PHO) and a regulative component (PHI). The function of the oxygenase component (PHO), here characterized for the first time, is to bind molecular oxygen and catalyse the mono-hydroxylation of substrates (phenol, and with less efficiency, chloro- and methyl-phenol and naphthol). PHO was purified from extracts of A. radioresistens S13 cells and shown to be a dimer of 206 kDa. Each monomer is composed by three subunits: alpha (54 kDa), beta (38 kDa) and gamma (11 kDa). The gene encoding PHO alpha (named mopN) was cloned and sequenced and the corresponding amino acid sequence matched with that of functionally related oxygenases. By structural alignment with the catalytic subunits of methane monooxygenase (MMO) and alkene monooxygenase, we propose that PHO alpha contains the enzyme active site, harbouring a dinuclear iron centre Fe-O-Fe, as also suggested by spectral analysis. Conserved hydrophobic amino acids known to define the substrate recognition pocket, are also present in the alpha-subunit. The prevalence of alpha-helices (99.6%) as studied by CD confirmed the hypothized structural homologies between PHO and MMO. Three parameters (optimum ionic strength, temperature and pH) that affect kinetics of the overall phenol hydroxylase reaction were further analyzed with a fixed optimal PHR/PHI/PHO ratio of 2/1/1. The highest level of activity was evaluated between 0.075 and 0.1 m of ionic strength, the temperature dependence showed a maximum of activity at 24 degrees C and finally the pH for optimal activity was determined to be 7.5.
![Research paper thumbnail of Site saturation mutagenesis demonstrates a central role for cysteine 298 as proton donor to the catalytic site in CaHydA [FeFe]-hydrogenase. PLoS One 2012](https://attachments.academia-assets.com/83989931/thumbnails/1.jpg)
](The exclusive functional replacement of cysteine with aspartic acid, an ionisable residue, and th... more The exclusive functional replacement of cysteine with aspartic acid, an ionisable residue, and the shift in the pH activity profile demonstrate experimentally the central role of C298 in the proton transfer pathway to the active site during [FeFe]-hydrogenase catalysis. Since C298 is strongly conserved among all the known functional [FeFe]hydrogenases, it is reasonable that the results obtained here for CaHydA have broader implications regarding its functional significance in the entire class. Future work will aim at a more detailed characterisation of the most relevant variants, application of site saturation mutagenesis to other residues within the enzyme core and study of other [FeFe]-hydrogenases.
Biochemistry, 2016
The newly isolated [FeFe]-hydrogenase CbA5H was characterized by FTIR spectroscopy coupled to enz... more The newly isolated [FeFe]-hydrogenase CbA5H was characterized by FTIR 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 the exploitation in industrial applications.
Waste Management, 2016
(AAM) is copyrighted and published by Elsevier. It is posted here by agreement between Elsevier a... more (AAM) is copyrighted and published by Elsevier. It is posted here by agreement between Elsevier and the University of Turin. Changes resulting from the publishing process-such as editing, corrections, structural formatting, and other quality control mechanisms-may not be reflected in this version of the text. The definitive version of the text was subsequently published in WASTE MANAGEMENT, 56, 2016, 10.1016/j.wasman.2016.06.039. You may download, copy and otherwise use the AAM for non-commercial purposes provided that your license is limited by the following restrictions: (1) You may use this AAM for non-commercial purposes only under the terms of the CC-BY-NC-ND license. (2) The integrity of the work and identification of the author, copyright owner, and publisher must be preserved in any copy.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2016
Background: Ar-BVMO is a recently discovered Baeyer-Villiger monooxygenase from the genome of Aci... more Background: Ar-BVMO is a recently discovered Baeyer-Villiger monooxygenase from the genome of Acinetobacter radioresistens S13 closely related to medically relevant ethionamide monooxygenase EtaA (prodrug activator) and capable of inactivating the imipenem antibiotic. Methods: The co-substrate preference as well as steady-state and rapid kinetics studies of the recombinant purified protein were carried out using stopped-flow spectroscopy under anaerobic and aerobic conditions. K d values were measured by isothermal calorimetry. Enzymatic activity was determined by measuring the amount of product formed using high pressure liquid chromatography or gas chromatography. Site-directed mutagenesis experiments were performed to decipher the role of the active site arginine-292. Results: Ar-BVMO was found to oxidize ethionamide as well as linear ketones. Mechanistic studies on the wild type enzyme using stopped-flow spectroscopy allowed for the detection of the characteristic oxygenating C4a-(hydro)peroxyflavin intermediate, which decayed rapidly in the presence of the substrate. Replacement of arginine 292 in Ar-BVMO by glycine or alanine resulted in greatly reduced or no Baeyer-Villiger activity, respectively, demonstrating the crucial role of this residue in catalysis of ketone substrates. However, both the R292A and R292G mutants are capable of carrying out N-and S-oxidation reactions. Conclusions: Substrate profiling of Ar-BVMO confirms its close relationship to EtaA; ethionamide is one of its substrates. The active site Arginine 292 is required for its Baeyer-Villiger activity but not for heteroatom oxidation. General Significance: A single mutation converts Ar-BVMO to a unique S-or Nmonooxygenase, a useful biocatalyst for the production of oxidized metabolites of human drug metabolizing enzymes.
Bioelectrochemistry, 2015
of Canis familiaris cytochrome P450 2D15 with gold nanoparticles: an alternative to 7 animal test... more of Canis familiaris cytochrome P450 2D15 with gold nanoparticles: an alternative to 7 animal testing in drug discovery. Bioelectrochemistry (2015) 105: 110-116.], 8 9 which has been published in final form at 10 [
Fatty acid amide hydrolase (FAAH) is an integral membrane protein that participates in terminatio... more Fatty acid amide hydrolase (FAAH) is an integral membrane protein that participates in termination of cannabinoid signalling mechanisms by catalysing the hydrolysis and thus inactivation of the endogenous cannabinoid anandamide and other natural lipids involved in signalling. Inhibition of FAAH activity, which increases the anandamide levels, is implicated in the treatment of pain,
Yeast, 2003
An amine oxidase from the yeast Kluyveromyces marxianus was induced, purified and completely char... more An amine oxidase from the yeast Kluyveromyces marxianus was induced, purified and completely characterized; it was shown to belong to the class of coppercontaining amine oxidases (E.C. 1.4.3.6). The enzyme was induced by putrescine and, very strongly, by copper(II); structural-functional characterization of the enzyme was performed, including determination of molecular weight, glycosylation, copper and TPQ content, isoelectric point, K M and k CAT (with benzylamine as substrate), pH, temperature and ionic strength effect on catalysis, substrate and inhibitor specificity. A 700 bp clone was isolated containing the cDNA that encodes for the C-terminus of the enzyme; the amino acid sequence deduced (the first available for a benzylamine oxidase from yeast) was compared to that of other copper amine oxidases from microorganisms and higher organisms. From the results obtained, the putrescine/benzylamine oxidase from Kluyveromyces marxianus was found to have a good homology with other enzymes of this class from microorganisms, and particularly with AO I from Aspergillus niger. Nonetheless, some features resulted closer to those of animal amine oxidases and histaminases. Some potential biotechnological applications are proposed. The cDNA Accession No. is AJ320485.
Yeast, 1994
The presence of adenylate cyclase activity was first demonstrated in membrane fractions from the ... more The presence of adenylate cyclase activity was first demonstrated in membrane fractions from the budding yeast Kluyveromyces marxianus. The enzyme showed a Mn2+-and M$+-dependent activity, with optimal pH at around 6 as observed in other yeast species. As in Saccharomyces cerevisiae, where adenylate cyclase is regulated by RASl and RAS2, we detected a guanyl nucleotide-dependent activity. Interestingly Y 13-259 monoclonal antibody, raised against mammalian p21Ha-'=, inhibited M$+ plus GTP-y-S-dependent CAMP production, suggesting that the GTP binding proteins involved in adenylate cyclase regulation could be Ras proteins. The same antibody recognized on Western blot and immunoprecipitated a 40 kDa polypeptide from K. marxianus crude membranes. This polypeptide was not detected by an anti-US2 polyclonal antibody raised against S. cerevisiae RAS2 protein, suggesting that Ras proteins from the two species could be structurally different.
European Journal of Biochemistry, 2003
This paper reports the isolation and characterization of the regulatory moiety of the multicompon... more This paper reports the isolation and characterization of the regulatory moiety of the multicomponent enzyme phenol hydroxylase from Acinetobacter radioresistens S13 grown on phenol as the only carbon and energy source. The whole enzyme comprises an oxygenase moiety (PHO), a reductase moiety (PHR) and a regulatory moiety (PHI). PHR contains one FAD and one iron-sulfur cluster, whose function is electron transfer from NADH to the dinuclear iron centre of the oxygenase. PHI is required for catalysis of the conversion of phenol to catechol in vitro, but is not required for PHR activity towards alternative electron acceptors such as cytochrome c and Nitro Blue Tetrazolium. The molecular mass of PHI was determined to be 10 kDa by SDS/PAGE, 8.8 kDa by MALDI-TOF spectrometry and 18 kDa by gel-permeation. This finding suggests that the protein in its native state is a homodimer. The isoelectric point is 4.1. PHI does not contain any redox cofactor and does not bind ANS, a fluorescent probe for hydrophobic sites. The N-terminal sequence is similar to those of the regulatory proteins of phenol hydroxylase from A. calcoaceticus and Pseudomonas CF 600. In the reconstituted system, optimal reaction rate was achieved when the stoichiometry of the components was 2 PHR monomers: 1 PHI dimer: 1 PHO (alphabetagamma) dimer. PHI interacts specifically with PHR, promoting the enhancement of FAD fluorescence emission. This signal is diagnostic of a conformational change of PHR that might result in a better alignment with respect to PHO.
Dalton Transactions, 2009
Catalytic properties of catechol 1,2-dioxygenase from Acinetobacter radioresistens S13 immobilize... more Catalytic properties of catechol 1,2-dioxygenase from Acinetobacter radioresistens S13 immobilized on nanosponges
Chemical Communications, 2011
[FeFe]-hydrogenases are efficient natural catalysts that can be exploited for hydrogen production... more [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%.
ChemBioChem, 2009
Catechol 1,2-dioxygenases and chlorocatechol dioxygenases are Fe(III)-dependent enzymes that do n... more Catechol 1,2-dioxygenases and chlorocatechol dioxygenases are Fe(III)-dependent enzymes that do not require a reductant to perform the ortho cleavage of the aromatic ring. The reaction mechanism is common to the two enzymes, and active-site residues must play a key role in the fine-tuning of specificity. Protein engineering was applied for the first time to the catalytic pocket of a catechol 1,2-dioxygenase by site-specific and site-saturation mutagenesis with the purpose of redesigning the pocket shape for improved catalysis on bulky derivatives. Mutants were analysed for changes in kinetic parameters: variants for residue 69 show an inversion of specificity with a preference towards 4-chlorocatechol (decrease of K(M) by a factor of 20) and activity on the rarely recognised substrate 4,5-dichlorocatechol, thus creating a novel, engineered chlorocatechol dioxygenase. A L69A substitution conveys gain-of-function activity towards 4-tert-butylcatechol. Mutations of position 72 enhance k(cat) towards chlorinated substrates. The biphasic Arrhenius plot observed in A72S suggests the involvement of a dynamic switch in the fine regulation of the enzyme.
Biotechnology and Applied Biochemistry, 2014
Enzymes entrapped in wet, nanoporous silica gel have great potential as bioreactors for bioremedi... more Enzymes entrapped in wet, nanoporous silica gel have great potential as bioreactors for bioremediation because of their improved thermal, chemical, and mechanical stability with respect to enzymes in solution. The B isozyme of catechol 1,2 dioxygenase from Acinetobacter radioresistens and its mutants of Leu69 and Ala72, designed for an increased reactivity toward the environmental pollutant chlorocatechols, were encapsulated using alkoxysilanes and alkyl alkoxysilanes as precursors in varying proportions. Encapsulation of the mutants in a hydrophobic tetramethoxysilane/ dimethoxydimethylsilane-based matrix yielded a remarkable 10-to 12-fold enhancement in reactivity toward chlorocatechols. These gels also showed a fivefold increase in relative reactivity toward chlorocatechols with respect to the natural substrate catechol, thus compensating for their relatively low activity for these substrates in solution. The encapsulated enzyme, unlike the enzyme in solution, proved resilient in assays carried out in urban wastewater and bacteria-contaminated solutions mimicking environmentally relevant conditions. Overall, the combination of a structure-based rational design of enzyme mutants, and the selection of a suitable encapsulation material, proved to be a powerful approach for the production and optimization of a potential bioremediation device, with increased activity and resistance toward bacterial degradation.
Biosensors and Bioelectronics, 1998
This work demonstrates that non-physiological electron transfer (ET) can occur in solution betwee... more This work demonstrates that non-physiological electron transfer (ET) can occur in solution between wild type D. vulgaris flavodoxin (Fld) and horse heart cytochrome c (cyt-c), D. vulgaris cytochrome c553 (cyt-c553) and the haem domain of B. megaterium cytochrome P450 (cyt-P450 BMP). Second order rate constants of the ET reaction between [Fld] Sq /[cyt-c] Ox , [Fld] Sq /[cyt-c553] Ox and [Fld] Sq /[cyt-P450 BMP] Ox , were found to be 6.16 × 10 5 , 1.80 × 10 4 and in the region of 10 5 respectively. These data are interpreted in terms of complementarity between the surfaces of the two proteins, their surface and redox potentials. Analysis of the ET results obtained from the separate wild type proteins supported the rational design approach in the creation of Fld-based chimeras. The preliminary design of the chimeras reported here is a 3D prototype for an artificial flavo-cytochrome obtained by covalent linkage of a Fld module to cyt-c553 via a disulphide bond. Theoretical ET rates calculated on the modelled flavo-cytochrome are encouraging the construction of these chimeric systems at DNA level. This work is now underway. The relevance of this molecular lego approach is to be seen in the long term goal of producing engineered multi-domain systems to be applied in the field of biosensors and bioelectronics to fulfil specific requirements. Novel catalytic devices can be obtained by using natural redox proteins in different combinations: this process mimics the natural evolution of proteins such as gene shuffling and gene fusion.
Biochemistry, 2002
Cytochrome c-553 from Desulfovibrio vulgaris exhibits a highly exposed heme and an unusually low ... more Cytochrome c-553 from Desulfovibrio vulgaris exhibits a highly exposed heme and an unusually low reduction potential with respect to other c-type cytochromes. Solvent heme exposure has been indicated as one of the most important factors in modulating the midpoint potential of the redox center. To test this hypothesis, a unique surface-exposed cysteine has been substituted for either M23 or G51 to produce the corresponding mutants and allow the formation of homodimers through a specific disulfide bridge. The reduction potentials, determined via spectroelectrochemistry, show an increase from +20 +/- 5 mV for the wt to +88 +/- 5 and +105 +/- 5 mV for the M23C-M23C homodimer and G51C-G51C homodimer, respectively. Chemical denaturation of the homodimers leads to parameters related to the hydrophobicity (m) and the number of buried side chains (n(B)), which suggest a decrease of exposure of the heme as a result of dimerization. These results are consistent with the heme-accessible surface area (ASA) calculated from a computer model of the homodimers. The ASA values show a decrease from 73 A(2) for the wt to 66 and 50 A(2) per heme for the M23C-M23C homodimer and G51C-G51C homodimer, respectively. The trend of the m- and n(B)-values, the degree of solvent accessibility, and the midpoint potential observed upon formation of the homodimers indicate a correlation between the reduction potential values and the exclusion of water from the heme surface.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2011
Intradiol-cleaving catechol 1,2 dioxygenases are Fe(III) dependent enzymes that act on catechol a... more Intradiol-cleaving catechol 1,2 dioxygenases are Fe(III) dependent enzymes that act on catechol and substituted catechols, including chlorocatechols pollutants, by inserting molecular oxygen in the aromatic ring. Members of this class are the object of intense biochemical investigations aimed at the understanding of their catalytic mechanism, particularly for designing mutants with selected catalytic properties. We report here an in depth investigation of catechol 1,2 dioxygenase IsoB from Acinetobacter radioresistens LMG S13 and its A72G and L69A mutants. By applying a multidisciplinary approach that includes high resolution X-rays crystallography, mass spectrometry and single crystal microspectrophotometry, we characterised the phospholipid bound to the enzyme and provided a structural framework to understand the inversion of substrate specificity showed by the mutants. Our results might be of help for the rational design of enzyme mutants showing a biotechnologically relevant substrate specificity, particularly to be used in bioremediation. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
European Journal of Biochemistry, 2003
Phenol hydroxylase (PH) from Acinetobacter radioresistens S13 represents an example of multicompo... more Phenol hydroxylase (PH) from Acinetobacter radioresistens S13 represents an example of multicomponent aromatic ring monooxygenase made up of three moieties: a reductase (PHR), an oxygenase (PHO) and a regulative component (PHI). The function of the oxygenase component (PHO), here characterized for the first time, is to bind molecular oxygen and catalyse the mono-hydroxylation of substrates (phenol, and with less efficiency, chloro- and methyl-phenol and naphthol). PHO was purified from extracts of A. radioresistens S13 cells and shown to be a dimer of 206 kDa. Each monomer is composed by three subunits: alpha (54 kDa), beta (38 kDa) and gamma (11 kDa). The gene encoding PHO alpha (named mopN) was cloned and sequenced and the corresponding amino acid sequence matched with that of functionally related oxygenases. By structural alignment with the catalytic subunits of methane monooxygenase (MMO) and alkene monooxygenase, we propose that PHO alpha contains the enzyme active site, harbouring a dinuclear iron centre Fe-O-Fe, as also suggested by spectral analysis. Conserved hydrophobic amino acids known to define the substrate recognition pocket, are also present in the alpha-subunit. The prevalence of alpha-helices (99.6%) as studied by CD confirmed the hypothized structural homologies between PHO and MMO. Three parameters (optimum ionic strength, temperature and pH) that affect kinetics of the overall phenol hydroxylase reaction were further analyzed with a fixed optimal PHR/PHI/PHO ratio of 2/1/1. The highest level of activity was evaluated between 0.075 and 0.1 m of ionic strength, the temperature dependence showed a maximum of activity at 24 degrees C and finally the pH for optimal activity was determined to be 7.5.