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Paolo Ascenzi

Publications and source records attributed to Paolo Ascenzi.

At least 19 recordsLinked to original sources
PubMed2007

Reductive nitrosylation and peroxynitrite-mediated oxidation of heme-hemopexin.

Hemopexin (HPX), which serves as a scavenger and transporter of toxic plasma heme, has been postulated to play a key role in the homeostasis of NO. In fact, HPX-heme(II) reversibly binds NO and facilitates NO scavenging by O(2). HPX-heme is formed by two four-bladed beta-propeller domains. The heme is bound between the two beta-propeller domains, residues His213 and His266 coordinate the heme iron atom. HPX-heme displays structural features of heme-proteins endowed with (pseudo-)enzymatic activities. In this study, the kinetics of rabbit HPX-heme(III) reductive nitrosylation and peroxynitrite-mediated oxidation of HPX-heme(II)-NO are reported. In the presence of excess NO, HPX-heme(III) is converted to HPX-heme(II)-NO by reductive nitrosylation. The second-order rate constant for HPX-heme(III) reductive nitrosylation is (1.3 +/- 0.1) x 10(1) m(-1).s(-1), at pH 7.0 and 10.0 degrees C. NO binding to HPX-heme(III) is rate limiting. In the absence and presence of CO2 (1.2 x 10(-3) m), excess peroxynitrite reacts with HPX-heme(II)-NO (2.6 x 10(-6) m) leading to HPX-heme(III) and NO, via the transient HPX-heme(III)-NO species. Values of the second-order rate constant for HPX-heme(III)-NO formation are (8.6 +/- 0.8) x 10(4) and (1.2 +/- 0.2) x 10(6) m(-1).s(-1) in the absence and presence of CO2, respectively, at pH 7.0 and 10.0 degrees C. The CO2-independent value of the first-order rate constant for HPX-heme(III)-NO denitrosylation is (4.3 +/- 0.4) x 10(-1) s(-1), at pH 7.0 and 10.0 degrees C. HPX-heme(III)-NO denitrosylation is rate limiting. HPX-heme(II)-NO appears to act as an efficient scavenger of peroxynitrite and of strong oxidants and nitrating species following the reaction of peroxynitrite with CO2 (e.g. ONOOC(O)O-, CO3-, and NO2).

Animals↗
PubMed2006

Abacavir modulates peroxynitrite-mediated oxidation of ferrous nitrosylated human serum heme-albumin.

Human serum albumin (SA) is best known for its extraordinary ligand-binding capacity. Here, kinetics of peroxynitrite-mediated oxidation of SA-heme(II)-NO is reported. Peroxynitrite reacts with SA-heme(II)-NO leading to SA-heme(III) and ()NO by way of the transient SA-heme(III)-NO species. Abacavir facilitates peroxynitrite-mediated oxidation of SA-heme(II)-NO, in the absence and presence of CO2. Values of the second order rate constant for peroxynitrite-mediated oxidation of SA-heme(II)-NO are (6.5+/-0.9) x 10(3) M(-1) s(-1) in the absence of CO2 and abacavir, (1.3+/-0.2) x 10(5) M(-1) s(-1) in the presence of CO2, (2.2+/-0.2) x 10(4) M(-1) s(-1) in the presence of abacavir, and (3.6+/-0.3) x 10(5) M(-1) s(-1) in the presence of both CO2 and abacavir. The value of the first-order rate constant for *NO dissociation from the SA-heme(III)-NO complex (=(1.8+/-0.3) x 10(-1) s(-1)) is CO2- and abacavir-independent, representing the rate-limiting step. Present data represent the first evidence for the allosteric modulation of SA-heme reactivity by heterotropic interaction(s).

Dideoxynucleosides↗
PubMed2006

Involvement of Pseudomonas aeruginosa rhodanese in protection from cyanide toxicity.

Cyanide is a serious environmental pollutant and a biocontrol metabolite in plant growth-promoting Pseudomonas species. Here we report on the presence of multiple sulfurtransferases in the cyanogenic bacterium Pseudomonas aeruginosa PAO1 and investigate in detail RhdA, a thiosulfate:cyanide sulfurtransferase (rhodanese) which converts cyanide to less toxic thiocyanate. RhdA is a cytoplasmic enzyme acting as the principal rhodanese in P. aeruginosa. The rhdA gene forms a transcriptional unit with the PA4955 and psd genes and is controlled by two promoters located upstream of PA4955 and rhdA. Both promoters direct constitutive RhdA expression and show similar patterns of activity, involving moderate down-regulation at the stationary phase or in the presence of exogenous cyanide. We previously observed that RhdA overproduction protects Escherichia coli against cyanide toxicity, and here we show that physiological RhdA levels contribute to P. aeruginosa survival under cyanogenic conditions. The growth of a DeltarhdA mutant is impaired under cyanogenic conditions and fully restored upon complementation with rhdA. Wild-type P. aeruginosa outcompetes the DeltarhdA mutant in cyanogenic coculture assays. Hence, RhdA could be regarded as an effector of P. aeruginosa intrinsic resistance to cyanide, insofar as it provides the bacterium with a defense mechanism against endogenous cyanide toxicity, in addition to cyanide-resistant respiration.

Amino Acid Sequence↗
PubMed2006

Peroxynitrite scavenging by ferrous truncated hemoglobin GlbO from Mycobacterium leprae.

Mycobacterium leprae GlbO has been proposed to represent merging of both O(2) uptake/transport and scavenging of nitrogen reactive species. Peroxynitrite reacts with M. leprae GlbO(II)-NO leading to GlbO(III) via the GlbO(III)-NO species. The value of the second order rate constant for GlbO(III)-NO formation is >1x10(8)M(-1)s(-1) in the absence and presence of CO(2) (1.2x10(-3)M). The CO(2)-independent value of the first order rate constant for GlbO(III)-NO denitrosylation is (2.5+/-0.4)x10(1)s(-1). Furthermore, peroxynitrite reacts with GlbO(II)-O(2) leading to GlbO(III) via the GlbO(IV)O species. Values of the second order rate constant for GlbO(IV)O formation are (4.8+/-0.5)x10(4) and (6.3+/-0.7)x10(5)M(-1)s(-1) in the absence and presence of CO(2) (=1.2x10(-3)M), respectively. The value of the second order rate constant for the peroxynitrite-mediated GlbO(IV)O reduction (= (1.5+/-0.2)x10(4)M(-1)s(-1)) is CO(2)-independent. These data argue for a role of GlbO in the defense of M. leprae against nitrosative stress.

Bacterial Proteins↗
PubMed2006

Structural determinants in the group III truncated hemoglobin from Campylobacter jejuni.

Truncated hemoglobins (trHbs) constitute a distinct lineage in the globin superfamily, distantly related in size and fold to myoglobin and monomeric hemoglobins. Their phylogenetic analyses revealed that three groups (I, II, and III) compose the trHb family. Group I and II trHbs adopt a simplified globin fold, essentially composed of a 2-on-2 alpha-helical sandwich, wrapped around the heme group. So far no structural data have been reported for group III trHbs. Here we report the three-dimensional structure of the group III trHbP from the eubacterium Campylobacter jejuni. The 2.15-A resolution crystal structure of C. jejuni trHbP (cyano-met form) shows that the 2-on-2 trHb fold is substantially conserved in the trHb group III, despite the absence of the Gly-based sequence motifs that were considered necessary for the attainment of the trHb specific fold. The heme crevice presents important structural modifications in the C-E region and in the FG helical hinge, with novel surface clefts at the proximal heme site. Contrary to what has been observed for group I and II trHbs, no protein matrix tunnel/cavity system is evident in C. jejuni trHbP. A gating movement of His(E7) side chain (found in two alternate conformations in the crystal structure) may be instrumental for ligand entry to the heme distal site. Sequence conservation allows extrapolating part of the structural results here reported to the whole trHb group III.

Amino Acid Sequence↗
PubMed2006

Cellulose acetate butyrate-pH/thermosensitive polymer microcapsules containing aminated poly(vinyl alcohol) microspheres for oral administration of DNA.

The aim of this work is to safely transport bioadhesive microspheres loaded with DNA to intestine and to test their bioadhesive properties. Poly(vinyl alcohol) (PVA) microspheres were prepared by dispersion reticulation with glutaraldehyde and further aminated. These microspheres were firstly loaded with plasmid DNA by electrostatic interactions and then entrapped in cellulose acetate butyrate (CAB) microcapsules for gastric protection. The entrapped PVA microspheres do not have enough force by swelling to produce the rupture of CAB shell, therefore the resistance of microcapsules was weakened by incorporating different amount of the pH/thermosensitive polymer (SP) based on poly(N-isopropylacrylamide-co-methyl methacrylate-co-methacrylic acid) (NIPAAm-co-MM-co-MA). This polymer is insoluble in gastric juice at pH 1.2 and 37 degrees C, but quickly solubilized in intestinal fluids (pH 6.8 and pH 7.4). Therefore, DNA loaded PVA microspheres were not expelled in acidic media but were almost entirely discharged in small intestine or colon. The integrity of DNA after entrapment was tested by agarose gel electrophoresis indicating that no DNA degradation occurs during encapsulation. The percentage of adhered microspheres on the mucus surface of everted intestinal tissue was 65+/-18% for aminated PVA microspheres without DNA and almost 50+/-15% for those loaded with DNA. Non-aminated PVA microspheres display the lowest adhesive properties (33+/-12%). In conclusion DNA loaded microspheres were progressively discharged in intestine. The integrity of DNA was not modified after entrapment and release, as proved by agarose gel electrophoresis. Both loaded and un-loaded aminated microspheres display good bioadhesive properties.

Administration, Oral↗
PubMed2006

Cyanide binding and heme cavity conformational transitions in Drosophila melanogaster hexacoordinate hemoglobin.

The reason for the presence of hemoglobin-like molecules in insects, such as Drosophila melanogaster, that live in fully aerobic environments has yet to be determined. Heme endogenous hexacoordination (where HisE7 and HisF8 axial ligands to the heme Fe atom are both provided by the protein) is a recently discovered mechanism proposed to modulate O(2) affinity in hemoglobins from different species. Previous results have shown that D. melanogaster hemoglobin 1 (product of the glob1 gene) displays heme endogenous hexacoordination in both the ferrous and ferric states. Here we present kinetic data characterizing the exogenous cyanide ligand binding process, and the three-dimensional structure (at 1.4 A resolution) of the ensuing cyano-met D. melanogaster hemoglobin. Comparison with the crystal structure of the endogenously hexacoordinated D. melanogaster hemoglobin shows that the transition to the cyano-met form is supported by conformational readjustment in the CD-D-E region of the protein, which removes HisE7 from the heme. The structural and functional features of D. melanogaster hemoglobin are examined in light of previous results achieved for human and mouse neuroglobins and for human cytoglobin, which display heme endogenous hexacoordination. The study shows that, despite the rather constant value for cyanide association rate constants for the ferric hemoproteins, different distal site conformational readjustments and/or heme sliding mechanisms are displayed by the known hexacoordinate hemoglobins as a result of exogenous ligand binding.

Animals↗
PubMed2006

CO2 impairs peroxynitrite-mediated inhibition of human caspase-3.

Peroxynitrite (ONOO-) is a transient powerful oxidant produced in vivo as the reaction of nitrogen monoxide (.NO) with superoxide (O2.-). The peroxynitrite reactivity is modulated by carbon dioxide (CO2) which enhances the peroxynitrite-mediated nitration of aromatics and partially impairs the oxidation of thiols. Here, the effect of CO2 on the peroxynitrite-mediated inhibition of human caspase-3, the execution enzyme of the apoptotic cascade, is reported. Peroxynitrite inhibits the catalytic activity of human caspase-3 by oxidizing the Sgamma atom of the Cys catalytic residue. In the absence of CO2, 1.0 equivalent of peroxynitrite inactivates 1.0 equivalent of human caspase-3. In the presence of the physiological concentration of CO2 (=1.3x10(-3) M), 1.0 equivalent of peroxynitrite inactivates only 0.38 equivalents of human caspase-3. Peroxynitrite affects the kcat value of the human caspase-3 catalyzed hydrolysis of N-acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin, without altering Km. Both in the absence and presence of CO2, the reducing agent dithiothreitol does not prevent human caspase-3 inhibition by peroxynitrite and does not reverse the peroxynitrite-induced inactivation of human caspase-3. These results represent the first evidence for modulation of peroxynitrite-mediated inhibition of cysteine proteinase action by CO2, supporting the role of CO2 in fine tuning of cell processes (e.g., apoptosis).

Apoptosis↗
PubMed2006

Structure-function relationship of estrogen receptor alpha and beta: impact on human health.

17Beta-estradiol (E2) controls many aspects of human physiology, including development, reproduction and homeostasis, through regulation of the transcriptional activity of its cognate receptors (ERs). The crystal structures of ERs with agonists and antagonists and the use of transgenic animals have revealed much about how hormone binding influences ER conformation(s) and how this conformation(s), in turn, influences the interaction of ERs with co-activators or co-repressors and hence determines ER binding to DNA and cellular outcomes. This information has helped to shed light on the connection between E2 and the development or progression of numerous diseases. Current therapeutic strategy in the treatment of E2-related pathologies relies on the modulation of ER trancriptional activity by anti-estrogens; however, data accumulated during the last five years reveal that ER activities are not only restricted to the nucleus. ERs are very mobile proteins continuously shuttling between protein targets located within various cellular compartments (e.g., membrane, nucleus). This allows E2 to generate different and synergic signal transduction pathways (i.e., non-genomic and genomic) which provide plasticity for cell response to E2. Understanding the structural basis and the molecular mechanisms by which ER transduce E2 signals in target cells will allow to create new pharmacologic therapies aimed at the treatment of a variety of human diseases affecting the cardiovascular system, the reproductive system, the skeletal system, the nervous system, the mammary gland, and many others.

Amino Acid Sequence↗
PubMed2006

Molecular dynamics simulation of Leishmania major surface metalloprotease GP63 (leishmanolysin).

One of the molecular factors contributing to Leishmania sp. virulence and pathogenesis is the major surface metalloprotease GP63, alternatively called leishmanolysin, MSP, and PSP (EC 3.4.24.36). Here, the molecular dynamics simulation of Leishmania major GP63 in water at pH 7 is reported. Upon solvation, GP63 undergoes a sharp structural relaxation with respect to the crystal structure. The fluctuation pattern occurs essentially in solvent-exposed nonstructured regions. By contrast, the active site turns out to be rigid. Essential dynamics and dynamic-domain analyses, both carried out on the equilibrated portion of GP63, show that the fingerprint fluctuations of GP63 are practically characterized by the motion of a large part of the N-terminal domain. These results appear to be in line with substrate recognition and (pro)enzyme activation played by the N-terminal domain of GP63. A systematic analysis among a series of 10 homologs of GP63 also shows that the residues involved in the interdomain bending result highly conserved. This finding also suggests possible relationship between the maintainance of proteolytic activity and the similarity of the dynamical properties of the related enzymes.

Animals↗
PubMed2006

O2-mediated oxidation of hemopexin-heme(II)-NO.

Hemopexin (HPX), serving as scavenger and transporter of toxic plasma heme, has been postulated to play a key role in the homeostasis of NO. Here, kinetics of HPX-heme(II) nitrosylation and O2-mediated oxidation of HPX-heme(II)-NO are reported. NO reacts reversibly with HPX-heme(II) yielding HPX-heme(II)-NO, according to the minimum reaction scheme: HPX-heme(II)+NO kon<-->koff HPX-heme(II)-NO values of kon, koff, and K (=kon/koff) are (6.3+/-0.3)x10(3)M-1s-1, (9.1+/-0.4)x10(-4)s-1, and (6.9+/-0.6)x10(6)M-1, respectively, at pH 7.0 and 10.0 degrees C. O2 reacts with HPX-heme(II)-NO yielding HPX-heme(III) and NO3-, by means of the ferric heme-bound peroxynitrite intermediate (HPX-heme(III)-N(O)OO), according to the minimum reaction scheme: HPX-heme(II)-NO+O2 hon<--> HPX-heme(III)-N(O)OO l-->HPX-heme(III)+NO3- the backward reaction rate is negligible. Values of hon and l are (2.4+/-0.3)x10(1)M-1s-1 and (1.4+/-0.2)x10(-3)s-1, respectively, at pH 7.0 and 10.0 degrees C. The decay of HPX-heme(III)-N(O)OO (i.e., l) is rate limiting. The HPX-heme(III)-N(O)OO intermediate has been characterized by optical absorption spectroscopy in the Soret region (lambdamax=409 nm and epsilon409=1.51x10(5)M-1cm-1). These results, representing the first kinetic evidence for HPX-heme(II) nitrosylation and O2-mediated oxidation of HPX-heme(II)-NO, might be predictive of transient (pseudo-enzymatic) function(s) of heme carriers.

Heme↗
PubMed2006

Truncated hemoglobin GlbO from Mycobacterium leprae alleviates nitric oxide toxicity.

As a consequence of reductive genome evolution, the obligate intracellular pathogen Mycobacterium leprae has minimized the repertoire of genes implicated in defense against reactive oxygen and nitrogen species. Genes for multiple hemoglobin types coexist in mycobacterial genomes, but M. leprae has retained only glbO, encoding a group-II truncated hemoglobin. Mycobacterium tuberculosis GlbO has been involved in oxygen transfer and respiration during hypoxia, but a role in protection from nitric oxide (NO) has not been documented yet. Here, we report that the in vitro reaction of oxygenated recombinant M. leprae GlbO with NO results in an immediate stoichiometric formation of nitrate, concomitant with heme-protein oxidation. Overexpression of GlbO alleviates the growth inhibition of Escherichia colihmp (flavohemoglobin gene) mutants in the presence of NO-donors, partly complementing the defect in Hmp synthesis. A promoter element upstream of glbO was predicted in silico, and confirmed by using a glbO::lacZ transcriptional fusion in the heterologous Mycobacterium smegmatis system. The glbO::lacZ fusion was expressed through the whole growth cycle of M. smegmatis, and moderately induced by NO. We propose that M. leprae, by retaining the unique truncated hemoglobin GlbO, may have coupled O2 delivery to the terminal oxidase with a defensive mechanism to scavenge NO from respiratory enzymes. These activities would help to sustain the obligate aerobic metabolism required for intracellular survival of leprosy bacilli.

Base Sequence↗
PubMed2006

Determination of anti-HIV drug concentration in human plasma by MALDI-TOF/TOF.

The antiretroviral therapeutic drug monitoring is becoming increased in clinical care to determine the best dosage regimen adapted to each patient. Here, the determination of the anti-HIV drugs lamivudine, lopinavir, and ritonavir concentration in the plasma of HIV-infected patients by MALDI-TOF/TOF is reported. The volume of the plasma sample was 600 microL. Plasma samples were extracted by solid-phase (divinylbenzene and N-vinylpyrrolidone) and evaporated in a water bath under a nitrogen stream. The extracted samples were reconstituted with methanol (100 microL), mixed (1:1) with a saturated matrix solution (4-hydroxybenzoic acid in 50% acetonitrile-0.1% trifluoracetic acid), and spotted onto the MALDI-TOF/TOF sample target plate. The lamivudine, lopinavir and ritonavir concentration was determined by standard additions analysis. Regression of standard additions was linear over the anti-HIV drug concentration ranges explored (lamivudine, 0.010-1.0 pmol/microL; lopinavir and ritonavir, 0.0025-0.50 pmol/microL). Moreover, emtricitabine (i.e., the fluorinated analog of lamivudine) was used as the internal standard to determine the lamivudine concentration. The calibration curve was linear on the emtricitabine concentration ranging between 0.050 and 5.0 pmol/microL. The absolute recovery ranged between 80 and 110%. Values of the lamivudine, lopinavir and ritonavir concentration determined by MALDI-TOF/TOF are in excellent agreement with those obtained by HPLC-UV and HPLC-MS/MS. MALDI-TOF/TOF experiments allowed also the detection of the ritonavir metabolite R5. Zidovudine was undetectable by MALDI-TOF/TOF analysis because also the minimal laser intensity may induce the anti-HIV drug photolysis. The MALDI-TOF/TOF technique is useful to determine very low concentrations of anti-HIV drugs (0.0025-0.010 pmol/microL).

Anti-HIV Agents↗
PubMed2006

Simultaneous determination of 16 anti-HIV drugs in human plasma by high-performance liquid chromatography.

Therapeutic drug monitoring (TDM) is pivotal to improve the management of HIV infection. Here, a HPLC-UV method has been developed to quantify simultaneously seven HIV protease inhibitors (amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir; PIs), seven nucleoside reverse transcriptase inhibitors (abacavir, didanosine, emtricitabine, lamivudine, stavudine, zalcitabine, and zidovudine; NRTIs), and two non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine; NNRTIs) in human plasma. The volume of the plasma sample was 600 microL. This method involved automated solid-phase extraction with Oasis HLB Cartridge 1 cc (divinylbenzene and N-vinylpyrrolidone) and evaporation in a water bath under nitrogen stream. The extracted samples were reconstituted with 100 microL methanol. Twenty microliters of these samples were injected into a HPLC-UV system, the analytes were eluted on an analytical C(18) Symmetry column (250 mm x 4.6mm I.D.) with a particle size of 5 microm. The mobile phase (0.01 M KH(2)PO(4) and acetonitrile) was delivered at 1.0 mL/min with linear gradient elution. The total run time for a single analysis was 35 min, the anti-HIV drugs were detected by UV at 240 and 260 nm. The calibration curves were linear up to 10 microg/mL. The absolute recovery ranged between 88 and 120%. The in vitro stability of anti-HIV drugs (0.005-10 microg/mL) in plasma has been studied at 24.0 degrees C. On these bases, a two to four analyte method has been tailored to the individual needs of the HIV-infected patient. The HPLC-UV method here reported has been validated and is currently applied to monitor PIs, NRTIs, and NNRTIs in plasma of HIV-infected patients. It allows to monitor the largest number of anti-HIV drugs simultaneously, appearing useful in a routine laboratory, and represents an essential step to elucidate the utility of a formal therapeutic drug monitoring for the optimal follow-up of HIV-infected patients.

Anti-HIV Agents↗
PubMed2006

Inhibition of polyamine and spermine oxidases by polyamine analogues.

Polyamine oxidase (PAO) and spermine oxidase (SMO) are involved in the catabolism of polyamines--basic regulators of cell growth and proliferation. The discovery of selective inhibitors of PAO and SMO represents an important tool in studying the involvement of these enzymes in polyamine homeostasis and a starting point for the development of novel antineoplastic drugs. Here, a comparative study on murine PAO (mPAO) and SMO (mSMO) inhibition by the polyamine analogues 1,8-diaminooctane, 1,12-diaminododecane, N-prenylagmatine (G3), guazatine and N,N1-bis(2,3-butadienyl)-1,4-butanediamine (MDL72527) is reported. Interestingly, 1,12-Diaminododecane and G3 behave as specific inhibitors of mPAO, values of K(i) for mPAO inhibition being lower than those for mSMO inactivation by several orders of magnitude. The analysis of molecular models of mPAO and mSMO indicates a significant reduction of the hydrophobic pocket located in maize PAO (MPAO) at the wider catalytic tunnel opening. This observation provides a rationale to explain the lower affinity displayed by G3, guazatine and MDL72527 for mPAO and mSMO as compared to MPAO. The different behaviour displayed by 1,12-diaminododecane towards mPAO and mSMO reveals the occurrence of basic differences in the ligand binding mode of the two enzymes, the first enzyme interacting mainly with substrate secondary amino groups and the second one with substrate primary amino groups. Thus, the data reported here provide the basis for the development of novel and selective inhibitors able to discriminate between mammalian SMO and PAO activities.

Amino Acid Sequence↗
PubMed2006

Allosteric modulation of drug binding to human serum albumin.

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its extraordinary ligand binding capacity. The three homologous domains of HSA (labeled I, II, and III), each in turn composed of two subdomains (named A and B), give rise to the three-dimensional structure of HSA. This flexible structural organization allows the protein structure to adapt to a variety of ligands. As conformational adaptability of HSA extends well beyond the immediate vicinity of the binding site(s), cooperativity and allosteric modulation arise among binding sites; this makes HSA similar to a multimeric protein. Although kinetic and thermodynamic parameters for ligand binding to HSA calculated by quantitative structure-activity relationship models are in excellent agreement with those obtained in vitro, cooperative and allosteric equilibria between different binding sites and competition between drugs or between drugs and endogenous ligands make difficult the interpretation of HSA binding properties in vivo. Binding of exogenous and endogenous ligands to HSA appears to be relevant in drug therapy and management. Here, the allosteric modulation of drug binding to HSA is briefly reviewed.

Allosteric Regulation↗
PubMed2005

Cyanide detoxification by recombinant bacterial rhodanese.

Cyanide is a major environmental pollutant of the chemical and metallurgical industries. Although extremely toxic, cyanide can enzymatically be converted to the less toxic thiocyanate by rhodaneses (thiosulfate:cyanide sulfurtransferases, EC 2.8.1.1). We engineered a genetic system to express high levels of recombinant Pseudomonas aeruginosa rhodanese (r-RhdA) in Escherichia coli, and used this organism to test the role of r-RhdA in cyanide detoxification. Inducible expression of the rhdA gene under the control of the hybrid T7-lacO promoter yielded active r-RhdA over a 4-h period, though r-RhdA-expressing E. coli showed decreased viability starting from 1 h post-induction. At this time, Western blot analysis and enzymatic assay showed r-RhdA partition between the cytoplasm (95%) and the periplasm (5%). The accessibility of thiosulfate to r-RhdA was a limiting step for the sulfur transfer reaction in the cellular system, but cyanide conversion to thiocyanate could be increased upon permeabilization of the bacterial membrane. Specific r-RhdA activity was higher in the whole-cell assay than in the in vitro assay with pure enzyme (2154 vs. 816 micromol min-1 mg-1 r-RhdA, respectively), likely reflecting enzyme stability. The r-RhdA-dependent cyanide detoxification resulted in increased resistance of r-RhdA overexpressing E. coli to 5 mM cyanide. Bacterial survival was paralleled by release of thiocyanate into the medium. Our results indicate that cyanide detoxification by engineered E. coli cells is feasible under laboratory conditions, and suggest that microbial rhodaneses may contribute to cyanide transformation in natural environments.

Biodegradation, Environmental↗