Publications by authors named "Linda Celeste Montemiglio"

18 Publications

  • Page 1 of 1

A novel strategy for molecular interfaces optimization: The case of Ferritin-Transferrin receptor interaction.

Comput Struct Biotechnol J 2020 24;18:2678-2686. Epub 2020 Sep 24.

Center for Life Nanoscience, Istituto Italiano di Tecnologia, Viale Regina Elena 291, 00161 Rome, Italy.

Protein-protein interactions regulate almost all cellular functions and rely on a fine tune of surface amino acids properties involved on both molecular partners. The disruption of a molecular association can be caused even by a single residue mutation, often leading to a pathological modification of a biochemical pathway. Therefore the evaluation of the effects of amino acid substitutions on binding, and the design of protein-protein interfaces, is one of the biggest challenges in computational biology. Here, we present a novel strategy for computational mutation and optimization of protein-protein interfaces. Modeling the interaction surface properties using the Zernike polynomials, we describe the shape and electrostatics of binding sites with an ordered set of descriptors, making possible the evaluation of complementarity between interacting surfaces. With a Monte Carlo approach, we obtain protein mutants with controlled molecular complementarities. Applying this strategy to the relevant case of the interaction between Ferritin and Transferrin Receptor, we obtain a set of Ferritin mutants with increased or decreased complementarity. The extensive molecular dynamics validation of the method results confirms its efficacy, showing that this strategy represents a very promising approach in designing correct molecular interfaces.
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http://dx.doi.org/10.1016/j.csbj.2020.09.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7548301PMC
September 2020

Dissecting the Cytochrome P450 OleP Substrate Specificity: Evidence for a Preferential Substrate.

Biomolecules 2020 10 6;10(10). Epub 2020 Oct 6.

Institute of Molecular Biology and Pathology c/o Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza, University of Rome, National Research Council, P.le Aldo Moro, 5, 00185 Rome, Italy.

The cytochrome P450 OleP catalyzes the epoxidation of aliphatic carbons on both the aglycone 8.8a-deoxyoleandolide (DEO) and the monoglycosylated L-olivosyl-8.8a-deoxyoleandolide (L-O-DEO) intermediates of oleandomycin biosynthesis. We investigated the substrate versatility of the enzyme. X-ray and equilibrium binding data show that the aglycone DEO loosely fits the OleP active site, triggering the closure that prepares it for catalysis only on a minor population of enzyme. The open-to-closed state transition allows solvent molecules to accumulate in a cavity that forms upon closure, mediating protein-substrate interactions. docking of the monoglycosylated L-O-DEO in the closed OleP-DEO structure shows that the L-olivosyl moiety can be hosted in the same cavity, replacing solvent molecules and directly contacting structural elements involved in the transition. X-ray structures of aglycone-bound OleP in the presence of L-rhamnose confirm the cavity as a potential site for sugar binding. All considered, we propose L-O-DEO as the optimal substrate of OleP, the L-olivosyl moiety possibly representing the molecular wedge that triggers a more efficient structural response upon substrate binding, favoring and stabilizing the enzyme closure before catalysis. OleP substrate versatility is supported by structural solvent molecules that compensate for the absence of a glycosyl unit when the aglycone is bound.
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http://dx.doi.org/10.3390/biom10101411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600006PMC
October 2020

Biodistribution PET/CT Study of Hemoglobin-DFO-Zr Complex in Healthy and Lung Tumor-Bearing Mice.

Int J Mol Sci 2020 Jul 15;21(14). Epub 2020 Jul 15.

Department of Cancer Biology, Institute of Biology, Warsaw University of Life Sciences, 02-787 Warsaw, Poland.

Proteins, as a major component of organisms, are considered the preferred biomaterials for drug delivery vehicles. Hemoglobin (Hb) has been recently rediscovered as a potential drug carrier, but its use for biomedical applications still lacks extensive investigation. To further explore the possibility of utilizing Hb as a potential tumor targeting drug carrier, we examined and compared the biodistribution of Hb in healthy and lung tumor-bearing mice, using for the first time Zr labelled Hb in a positron emission tomography (PET) measurement. Hb displays a very high conjugation yield in its fast and selective reaction with the maleimide-deferoxamine (DFO) bifunctional chelator. The high-resolution X-ray structure of the Hb-DFO complex demonstrated that cysteine β93 is the sole attachment moiety to the αβ-protomer of Hb. The Hb-DFO complex shows quantitative uptake of Zr in solution as determined by radiochromatography. Injection of 0.03 mg of Hb-DFO-Zr complex in healthy mice indicates very high radioactivity in liver, followed by spleen and lungs, whereas a threefold increased dosage results in intensification of PET signal in kidneys and decreased signal in liver and spleen. No difference in biodistribution pattern is observed between naïve and tumor-bearing mice. Interestingly, the liver Hb uptake did not decrease upon clodronate-mediated macrophage depletion, indicating that other immune cells contribute to Hb clearance. This finding is of particular interest for rapidly developing clinical immunology and projects aiming to target, label or specifically deliver agents to immune cells.
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http://dx.doi.org/10.3390/ijms21144991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7404105PMC
July 2020

Lack of orientation selectivity of the heme insertion in murine neuroglobin revealed by resonance Raman spectroscopy.

FEBS J 2020 Sep 27;287(18):4082-4097. Epub 2020 Feb 27.

Dipartimento di Chimica "Ugo Schiff", Università di Firenze, Florence, Italy.

Different murine neuroglobin variants showing structural and dynamic alterations that are associated with perturbation of ligand binding have been studied: the CD loop mutants characterized by an enhanced flexibility (Gly-loop and Gly-loop ), the F106A mutant, and the double Gly-loop /F106A mutant. Their ferric resonance Raman spectra in solution and in crystals are almost identical. In the high-frequency region, the identification of a double set of core size marker bands indicates the presence of two 6-coordinate low spin species. The resonance Raman data, together with the corresponding crystal structures, indicate the presence of two neuroglobin conformers with a reversed (A conformer) or a canonical (B conformer) heme insertion orientation. With the identification of the marker bands corresponding to each conformer, the data indicate that the B conformer increases at the expense of the A form, predominantly in the Gly-loop /F106A double mutant, as confirmed by X-ray crystallography. This is the first time that a reversed heme insertion has been identified by resonance Raman in a native 6-coordinate low-spin heme protein. This diagnostic tool could be extended to other heme proteins in order to detect heme orientational disorder, which are likely to be correlated to functionally relevant heme dynamics. DATABASE: Crystallographic structure: structural data are deposited in the Protein Data Bank under the 6RA6 PDB entry.
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http://dx.doi.org/10.1111/febs.15241DOI Listing
September 2020

Ligand pathways in neuroglobin revealed by low-temperature photodissociation and docking experiments.

IUCrJ 2019 Sep 10;6(Pt 5):832-842. Epub 2019 Jul 10.

Department of Biochemical Sciences 'A. Rossi Fanelli', University of Rome Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy.

A combined biophysical approach was applied to map gas-docking sites within murine neuroglobin (Ngb), revealing snapshots of events that might govern activity and dynamics in this unique hexacoordinate globin, which is most likely to be involved in gas-sensing in the central nervous system and for which a precise mechanism of action remains to be elucidated. The application of UV-visible microspectroscopy , solution X-ray absorption near-edge spectroscopy and X-ray diffraction experiments at 15-40 K provided the structural characterization of an Ngb photolytic intermediate by cryo-trapping and allowed direct observation of the relocation of carbon monoxide within the distal heme pocket after photodissociation. Moreover, X-ray diffraction at 100 K under a high pressure of dioxygen, a physiological ligand of Ngb, unravelled the existence of a storage site for O in Ngb which coincides with Xe-III, a previously described docking site for xenon or krypton. Notably, no other secondary sites were observed under our experimental conditions.
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http://dx.doi.org/10.1107/S2052252519008157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760443PMC
September 2019

Structural analysis of the transferrin receptor multifaceted ligand(s) interface.

Biophys Chem 2019 11 1;254:106242. Epub 2019 Aug 1.

Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza, University of Rome, Rome, Italy; Institute of Molecular Biology and Pathology, National Research Council, Rome, Italy. Electronic address:

The transferrin receptor 1 (TfR1) is one of the key regulators of iron homeostasis for most higher organisms. It mediates cellular iron import through a constitutive clathrin-dependent endocytosis mechanism and by recruiting iron- regulator proteins as transferrin, Hereditary Hemochromatosis factor (HFE) and serum ferritin in response to cellular demand. The receptor is also opportunistically exploited by several viruses and the malaria parasite as a preferential door for cell invasion. In this review, we analyze the structural information available for TfR1 and all its functional complexes to figure out how structural signals in a single receptor can guide the recognition of multiple ligands and how the conservation of key residues in TfR1 might have a role in iron uptake and cell infection.
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http://dx.doi.org/10.1016/j.bpc.2019.106242DOI Listing
November 2019

Proximal and distal control for ligand binding in neuroglobin: role of the CD loop and evidence for His64 gating.

Sci Rep 2019 03 29;9(1):5326. Epub 2019 Mar 29.

Dip. di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, P.le A. Moro 5, 00185, Rome, Italy.

Neuroglobin (Ngb) is predominantly expressed in neurons of the central and peripheral nervous systems and it clearly seems to be involved in neuroprotection. Engineering Ngb to observe structural and dynamic alterations associated with perturbation in ligand binding might reveal important structural determinants, and could shed light on key features related to its mechanism of action. Our results highlight the relevance of the CD loop and of Phe106 as distal and proximal controls involved in ligand binding in murine neuroglobin. We observed the effects of individual and combined mutations of the CD loop and Phe106 that conferred to Ngb higher CO binding velocities, which we correlate with the following structural observations: the mutant F106A shows, upon CO binding, a reduced heme sliding hindrance, with the heme present in a peculiar double conformation, whereas in the CD loop mutant "Gly-loop", the original network of interactions between the loop and the heme was abolished, enhancing binding via facilitated gating out of the distal His64. Finally, the double mutant, combining both mutations, showed a synergistic effect on CO binding rates. Resonance Raman spectroscopy and MD simulations support our findings on structural dynamics and heme interactions in wild type and mutated Ngbs.
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http://dx.doi.org/10.1038/s41598-019-41780-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441039PMC
March 2019

Cryo-EM structure of the human ferritin-transferrin receptor 1 complex.

Nat Commun 2019 03 8;10(1):1121. Epub 2019 Mar 8.

Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza University of Rome, P.le A. Moro 5, 00185, Rome, Italy.

Human transferrin receptor 1 (CD71) guarantees iron supply by endocytosis upon binding of iron-loaded transferrin and ferritin. Arenaviruses and the malaria parasite exploit CD71 for cell invasion and epitopes on CD71 for interaction with transferrin and pathogenic hosts were identified. Here, we provide the molecular basis of the CD71 ectodomain-human ferritin interaction by determining the 3.9 Å resolution single-particle cryo-electron microscopy structure of their complex and by validating our structural findings in a cellular context. The contact surfaces between the heavy-chain ferritin and CD71 largely overlap with arenaviruses and Plasmodium vivax binding regions in the apical part of the receptor ectodomain. Our data account for transferrin-independent binding of ferritin to CD71 and suggest that select pathogens may have adapted to enter cells by mimicking the ferritin access gate.
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http://dx.doi.org/10.1038/s41467-019-09098-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6408514PMC
March 2019

Substrate-induced conformational change in cytochrome P450 OleP.

FASEB J 2019 02 12;33(2):1787-1800. Epub 2018 Sep 12.

Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Sapienza Università di Roma, Rome, Italy.

The regulation of cytochrome P450 activity is often achieved by structural transitions induced by substrate binding. We describe the conformational transition experienced upon binding by the P450 OleP, an epoxygenase involved in oleandomycin biosynthesis. OleP bound to the substrate analog 6DEB crystallized in 2 forms: one with an ensemble of open and closed conformations in the asymmetric unit and another with only the closed conformation. Characterization of OleP-6DEB binding kinetics, also using the P450 inhibitor clotrimazole, unveiled a complex binding mechanism that involves slow conformational rearrangement with the accumulation of a spectroscopically detectable intermediate where 6DEB is bound to open OleP. Data reported herein provide structural snapshots of key precatalytic steps in the OleP reaction and explain how structural rearrangements induced by substrate binding regulate activity.-Parisi, G., Montemiglio, L. C., Giuffrè, A., Macone, A., Scaglione, A., Cerutti, G., Exertier, C., Savino, C., Vallone, B. Substrate-induced conformational change in cytochrome P450 OleP.
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http://dx.doi.org/10.1096/fj.201800450RRDOI Listing
February 2019

Engineered ferritin for lanthanide binding.

PLoS One 2018 13;13(8):e0201859. Epub 2018 Aug 13.

Department of Biochemical Sciences "Alessandro Rossi Fanelli", Sapienza University of Rome, Rome Italy.

Ferritin H-homopolymers have been extensively used as nanocarriers for diverse applications in the targeted delivery of drugs and imaging agents, due to their unique ability to bind the transferrin receptor (CD71), highly overexpressed in most tumor cells. In order to incorporate novel fluorescence imaging properties, we have fused a lanthanide binding tag (LBT) to the C-terminal end of mouse H-chain ferritin, HFt. The HFt-LBT possesses one high affinity Terbium binding site per each of the 24 subunits provided by six coordinating aminoacid side chains and a tryptophan residue in its close proximity and is thus endowed with strong FRET sensitization properties. Accordingly, the characteristic Terbium emission band at 544 nm for the HFt-LBT Tb(III) complex was detectable upon excitation of the tag enclosed at two order of magnitude higher intensity with respect to the wtHFt protein. X-ray data at 2.9 Å and cryo-EM at 7 Å resolution demonstrated that HFt-LBT is correctly assembled as a 24-mer both in crystal and in solution. On the basis of the intrinsic Tb(III) binding properties of the wt protein, 32 additional Tb(III) binding sites, located within the natural iron binding sites of the protein, were identified besides the 24 Tb(III) ions coordinated to the LBTs. HFt-LBT Tb(III) was demonstrated to be actively uptaken by selected tumor cell lines by confocal microscopy and FACS analysis of their FITC derivatives, although direct fluorescence from Terbium emission could not be singled out with conventional, 295-375 nm, fluorescence excitation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0201859PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6089422PMC
February 2019

Effects of Y361-auto-phosphorylation on structural plasticity of the HIPK2 kinase domain.

Protein Sci 2018 03 28;27(3):725-737. Epub 2017 Dec 28.

Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, P.le A. Moro 5, 00185, Italy, Rome.

The dual-specificity activity of the homeodomain interacting protein kinase 2 (HIPK2) is regulated by cis-auto-phosphorylation of tyrosine 361 (Y361) on the activation loop. Inhibition of this process or substitution of Y361 with nonphosphorylatable amino acid residues result in aberrant HIPK2 forms that show altered functionalities, pathological-like cellular relocalization, and accumulation into cytoplasmic aggresomes. Here, we report an in vitro characterization of wild type HIPK2 kinase domain and of two mutants, one at the regulating Y361 (Y361F, mimicking a form of HIPK2 lacking Y361 phosphorylation) and another at the catalytic lysine 228 (K228A, inactivating the enzyme). Gel filtration and thermal denaturation analyzes along with equilibrium binding experiments and kinase assays performed in the presence or absence of ATP-competitors were performed. The effects induced by mutations on overall stability, oligomerization and activity support the existence of different conformations of the kinase domain linked to Y361 phosphorylation. In addition, our in vitro data are consistent with both the cross-talk between the catalytic site and the activation loop of HIPK2 and the aberrant activities and accumulation previously reported for the Y361 nonphosphorylated HIPK2 in mammalian cells.
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http://dx.doi.org/10.1002/pro.3367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818748PMC
March 2018

Subcellular localization of the five members of the human steroid 5α-reductase family.

Biochim Open 2017 Jun 21;4:99-106. Epub 2017 Mar 21.

Dept. of Biochemical Sciences, Sapienza University of Rome, P.le A.Moro 5, 00185, Rome, Italy.

In humans the steroid 5alpha-reductase (SRD5A) family comprises five integral membrane enzymes that carry out reduction of a double bond in lipidic substrates: Δ-3-keto steroids, polyprenol and trans-enoyl CoA. The best-characterized reaction is the conversion of testosterone into the more potent dihydrotestosterone carried out by SRD5A1-2. Some controversy exists on their possible nuclear or endoplasmic reticulum localization. We report the cloning and transient expression in HeLa cells of the five members of the human steroid 5α-reductase family as both N- and C-terminus green fluorescent protein tagged protein constructs. Following the intrinsic fluorescence of the tag, we have determined that the subcellular localization of these enzymes is in the endoplasmic reticulum, upon expression in HeLa cells. The presence of the tag at either end of the polypeptide chain can affect protein expression and, in the case of trans enoyl-CoA reductase, it induces the formation of protein aggregates.
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http://dx.doi.org/10.1016/j.biopen.2017.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5656259PMC
June 2017

Structure of the adenylation domain Thr1 involved in the biosynthesis of 4-chlorothreonine in Streptomyces sp. OH-5093-protein flexibility and molecular bases of substrate specificity.

FEBS J 2017 09 7;284(18):2981-2999. Epub 2017 Aug 7.

Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, Italy.

We determined the crystal structure of Thr1, the self-standing adenylation domain involved in the nonribosomal-like biosynthesis of free 4-chlorothreonine in Streptomyces sp. OH-5093. Thr1 shows two monomers in the crystallographic asymmetric unit with different relative orientations of the C- and N-terminal subdomains both in the presence of substrates and in the unliganded form. Cocrystallization with substrates, adenosine 5'-triphosphate and l-threonine, yielded one monomer containing the two substrates and the other in complex with l-threonine adenylate, locked in a postadenylation state. Steady-state kinetics showed that Thr1 activates l-Thr and its stereoisomers, as well as d-Ala, l- and d-Ser, albeit with lower efficiency. Modeling of these substrates in the active site highlighted the molecular bases of substrate discrimination. This work provides the first crystal structure of a threonine-activating adenylation enzyme, a contribution to the studies on conformational rearrangement in adenylation domains and on substrate recognition in nonribosomal biosynthesis.

Database: Structural data are available in the Protein Data Bank under the accession number 5N9W and 5N9X.
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http://dx.doi.org/10.1111/febs.14163DOI Listing
September 2017

Humanized archaeal ferritin as a tool for cell targeted delivery.

Nanoscale 2017 Jan;9(2):647-655

Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, V.le Regina Elena 291, Rome 00161, Italy.

Human ferritins have been extensively studied to be used as nanocarriers for diverse applications and could represent a convenient alternative for targeted delivery of anticancer drugs and imaging agents. However, the most relevant limitation to their applications is the need for highly acidic experimental conditions during the initial steps of particle/cargo assembly, a process that could affect both drug stability and the complete reassembly of the ferritin cage. To overcome this issue the unique assembly of Archaeoglobus fulgidus ferritin was genetically engineered by changing a surface exposed loop of 12 amino acids connecting B and C helices to mimic the sequence of the analogous human H-chain ferritin loop. This new chimeric protein was shown to maintain the unique, cation linked, association-dissociation properties of Archaeoglobus fulgidus ferritin occurring at neutral pH values, while exhibiting the typical human H-homopolymer recognition by the transferrin receptor TfR1. The chimeric protein was confirmed to be actively and specifically internalized by HeLa cells, thus representing a unique nanotechnological tool for cell-targeted delivery of possible payloads for diagnostic or therapeutic purposes. Moreover, it was demonstrated that the 12 amino acids' loop is necessary and sufficient for binding to the transferrin receptor. The three-dimensional structure of the humanized Archaeoglobus ferritin has been obtained both as crystals by X-ray diffraction and in solution by cryo-EM.
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http://dx.doi.org/10.1039/c6nr07129eDOI Listing
January 2017

Functional analysis and crystallographic structure of clotrimazole bound OleP, a cytochrome P450 epoxidase from Streptomyces antibioticus involved in oleandomycin biosynthesis.

Biochim Biophys Acta 2016 Mar 22;1860(3):465-75. Epub 2015 Oct 22.

Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, P.le A. Moro 5, 00185 Rome, Italy; Department of Biochemical Sciences, "Sapienza" University of Rome, P.le A. Moro 5, 00185 Rome, Italy; CNR Institute of Molecular Biology and Pathology, P.le A. Moro 5, 00185 Rome, Italy. Electronic address:

Background: OleP is a cyt P450 from Streptomyces antibioticus carrying out epoxigenation of the antibiotic oleandomycin during its biosynthesis. The timing of its reaction has not been fully clarified, doubts remain regarding its substrate and catalytic mechanism.

Methods: The crystal structure of OleP in complex with clotrimazole, an inhibitor of P450s used in therapy, was solved and the complex formation dynamics was characterized by equilibrium and kinetic binding studies and compared to ketoconazole, another azole differing for the N1-substituent.

Results: Clotrimazole coordinates the heme and occupies the active site. Most of the residues interacting with clotrimazole are conserved and involved in substrate binding in MycG, the P450 epoxigenase with the highest homology with OleP. Kinetic characterization of inhibitor binding revealed OleP to follow a simple bimolecular reaction, without detectable intermediates.

Conclusions: Clotrimazole-bound OleP adopts an open form, held by a π-π stacking chain that fastens helices F and G and the FG loop. Affinity is affected by the interactions of the N1 substituent within the active site, given the one order of magnitude difference of the off-rate constants between clotrimazole and ketoconazole. Based on structural similarities with MycG, we propose a binding mode for both oleandomycin intermediates, that are the candidate substrates of OleP.

General Significance: Among P450 epoxigenases OleP is the only one that introduces an epoxide on a non-activated C–C bond. The data here presented are necessary to understand the rare chemistry carried out by OleP, to engineer it and to design more selective and potent P450-targeted drugs.
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http://dx.doi.org/10.1016/j.bbagen.2015.10.009DOI Listing
March 2016

Effects of microwaves (900 MHz) on peroxidase systems: A comparison between lactoperoxidase and horseradish peroxidase.

Electromagn Biol Med 2016 12;35(2):126-33. Epub 2015 Jan 12.

f Department of Biochemistry , "Sapienza" University of Rome , Rome , Italy.

This work shows the effects of exposure to an electromagnetic field at 900 MHz on the catalytic activity of the enzymes lactoperoxidase (LPO) and horseradish peroxidase (HRP). Experimental evidence that irradiation causes conformational changes of the active sites and influences the formation and stability of the intermediate free radicals is documented by measurements of enzyme kinetics, circular dichroism spectroscopy (CD) and cyclic voltammetry.
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http://dx.doi.org/10.3109/15368378.2014.1002135DOI Listing
February 2017

Redirecting P450 EryK specificity by rational site-directed mutagenesis.

Biochemistry 2013 May 16;52(21):3678-87. Epub 2013 May 16.

Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma , Piazzale A. Moro 5, 00185 Rome, Italy.

The C-12 hydroxylase EryK is a bacterial cytochrome P450, active during one of the final tailoring steps of erythromycin A (ErA) biosynthesis. Its tight substrate specificity, restricted to the metabolic intermediate ErD, leads to the accumulation in the culture broth of a shunt metabolite, ErB, that originates from the competitive action of a methyltranferase on the substrate of EryK. Although the methylation of the mycarosyl moiety represents the only difference between the two metabolites, EryK exhibits very low conversion of ErB in ErA via a parallel pathway. Given its limited antimicrobial activity and its moderate toxicity, contamination by such a byproduct decreases the yield and purity of the antibiotic. In this study, EryK has been redesigned to make it suitable for industrial application. Taking advantage of the three-dimensional structure of the enzyme in complex with ErD, three single active-site mutants of EryK (M86A, H88E, and E89L) have been designed to allow hydroxylation of the nonphysiological substrate ErB. The binding and catalytic properties of these three variants on both ErD and ErB have been analyzed. Interestingly, we found the mutation of Met 86 to Ala to yield enzymatic activity on both ErB and ErD. The three-dimensional structure of the complex of mutated EryK with ErB revealed that the mutation allows ErB to accommodate in the active site of the enzyme and to induce its closure, thus assuring the progress of the catalytic reaction. Therefore, by single mutation the fine substrate recognition, active site closure, and locking were recovered.
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http://dx.doi.org/10.1021/bi400223jDOI Listing
May 2013

Azole drugs trap cytochrome P450 EryK in alternative conformational states.

Biochemistry 2010 Nov;49(43):9199-206

Department of Biochemical Sciences, Sapienza University of Rome and CNR Institute of Molecular Biology and Pathology, Piazzale A. Moro 5, Rome, Italy.

EryK is a bacterial cytochrome P450 that catalyzes the last hydroxylation occurring during the biosynthetic pathway of erythromycin A in Streptomyces erythraeus. We report the crystal structures of EryK in complex with two widely used azole inhibitors: ketoconazole and clotrimazole. Both of these ligands use their imidazole moiety to coordinate the heme iron of P450s. Nevertheless, because of the different chemical and structural properties of their N1-substituent group, ketoconazole and clotrimazole trap EryK, respectively, in a closed and in an open conformation that resemble the two structures previously described for the ligand-free EryK. Indeed, ligands induce a distortion of the internal helix I that affects the accessibility of the binding pocket by regulating the kink of the external helix G via a network of interactions that involves helix F. The data presented thus constitute an example of how a cytochrome P450 may be selectively trapped in different conformational states by inhibitors.
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http://dx.doi.org/10.1021/bi101062vDOI Listing
November 2010