Publications by authors named "Carmelinda Savino"

23 Publications

  • Page 1 of 1

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

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

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

A Practical Approach to Protein Crystallography.

Methods Mol Biol 2017 ;1525:47-78

C.N.R.-Istituto di Biologia e Patologia Molecolari c/o Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Università "Sapienza", P.le Aldo Moro, 5, 00185, Rome, Italy.

Macromolecular crystallography is a powerful tool for structural biology. The resolution of a protein crystal structure is becoming much easier than in the past, thanks to developments in computing, automation of crystallization techniques and high-flux synchrotron sources to collect diffraction datasets. The aim of this chapter is to provide practical procedures to determine a protein crystal structure, illustrating the new techniques, experimental methods, and software that have made protein crystallography a tool accessible to a larger scientific community.It is impossible to give more than a taste of what the X-ray crystallographic technique entails in one brief chapter and there are different ways to solve a protein structure. Since the number of structures available in the Protein Data Bank (PDB) is becoming ever larger (the protein data bank now contains more than 100,000 entries) and therefore the probability to find a good model to solve the structure is ever increasing, we focus our attention on the Molecular Replacement method. Indeed, whenever applicable, this method allows the resolution of macromolecular structures starting from a single data set and a search model downloaded from the PDB, with the aid only of computer work.
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http://dx.doi.org/10.1007/978-1-4939-6622-6_3DOI Listing
January 2018

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

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

Flexibility of the Cu,Zn superoxide dismutase structure investigated at 0.57 GPa.

Acta Crystallogr D Biol Crystallogr 2010 Jun 15;66(Pt 6):654-63. Epub 2010 May 15.

ENSCP, UMR CNRS 7223, 11 Rue Pierre et Marie Curie, 75231 Paris CEDEX 05, France.

The 2 A resolution crystal structure of bovine erythrocyte Cu,Zn superoxide dismutase (CuZnSOD) has been determined by X-ray diffraction at high pressure (0.57 GPa) and room temperature. At 0.57 GPa the secondary, tertiary and quaternary structures are similar to other previously determined bovine erythrocyte CuZnSOD structures. Nevertheless, pressure has a localized impact on the atomic coordinates of C(alpha) atoms and on side chains. The compression of the crystal and of the protein backbone is anisotropic. This anisotropy is discussed, taking into account intermolecular contacts and protein conformation. Pressure perturbation highlights the more flexible zones in the protein such as the electrostatic loop. At 0.57 GPa, a global shift of the dimetallic sites in both subunits and changes in the oxidation state of Cu were observed. The flexibility of the electrostatic loop may be useful for the interaction of different metal carriers in the copper-uptake process, whereas the flexibility of the metal sites involved in the activity of the protein could contribute to explaining the ubiquitous character of CuZnSODs, which are found in organisms living in very different conditions, including the deep-sea environment. This work illustrates the potential of combining X-ray crystallography with high pressure to promote and stabilize higher energy conformational substates.
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http://dx.doi.org/10.1107/S0907444910012321DOI Listing
June 2010

Glyphosate resistance by engineering the flavoenzyme glycine oxidase.

J Biol Chem 2009 Dec 28;284(52):36415-36423. Epub 2009 Oct 28.

Dipartimento di Biotecnologie e Scienze Molecolari and the Centro Interuniversitario di Ricerca in Biotecnologie Proteiche "The Protein Factory," Politecnico di Milano, Università degli Studi dell'Insubria, 21100 Varese, Italy. Electronic address:

Glycine oxidase from Bacillus subtilis is a homotetrameric flavoprotein of great potential biotechnological use because it catalyzes the oxidative deamination of various amines and d-isomer of amino acids to yield the corresponding alpha-keto acids, ammonia/amine, and hydrogen peroxide. Glyphosate (N-phosphonomethylglycine), a broad spectrum herbicide, is an interesting synthetic amino acid: this compound inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids in plants and certain bacteria. In recent years, transgenic crops resistant to glyphosate were mainly generated by overproducing the plant enzyme or by introducing a 5-enolpyruvylshikimate-3-phosphate synthase insensitive to this herbicide. In this work, we propose that the enzymatic oxidation of glyphosate could be an effective alternative to this important biotechnological process. To reach this goal, we used a rational design approach (together with site saturation mutagenesis) to generate a glycine oxidase variant more active on glyphosate than on the physiological substrate glycine. The glycine oxidase containing three point mutations (G51S/A54R/H244A) reaches an up to a 210-fold increase in catalytic efficiency and a 15,000-fold increase in the specificity constant (the k(cat)/K(m) ratio between glyphosate and glycine) as compared with wild-type glycine oxidase. The inspection of its three-dimensional structure shows that the alpha2-alpha3 loop (comprising residues 50-60 and containing two of the mutated residues) assumes a novel conformation and that the newly introduced residue Arg(54) could be the key residue in stabilizing glyphosate binding and destabilizing glycine positioning in the binding site, thus increasing efficiency on the herbicide.
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http://dx.doi.org/10.1074/jbc.M109.051631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2794757PMC
December 2009

Investigating the structural plasticity of a cytochrome P450: three-dimensional structures of P450 EryK and binding to its physiological substrate.

J Biol Chem 2009 Oct 22;284(42):29170-9. Epub 2009 Jul 22.

CNR Institute of Molecular Biology and Pathology, P.le A. Moro 5, 00185 Rome, Italy.

Cytochrome P450s are heme-containing proteins that catalyze the oxidative metabolism of many physiological endogenous compounds. Because of their unique oxygen chemistry and their key role in drug and xenobiotic metabolism, particular attention has been devoted in elucidating their mechanism of substrate recognition. In this work, we analyzed the three-dimensional structures of a monomeric cytochrome P450 from Saccharopolyspora erythraea, commonly called EryK, and the binding kinetics to its physiological ligand, erythromycin D. Three different structures of EryK were obtained: two ligand-free forms and one in complex with its substrate. Analysis of the substrate-bound structure revealed the key structural determinants involved in substrate recognition and selectivity. Interestingly, the ligand-free structures of EryK suggested that the protein may explore an open and a closed conformation in the absence of substrate. In an effort to validate this hypothesis and to investigate the energetics between such alternative conformations, we performed stopped-flow absorbance experiments. Data demonstrated that EryK binds erythromycin D via a mechanism involving at least two steps. Contrary to previously characterized cytochrome P450s, analysis of double jump mixing experiments confirmed that this complex scenario arises from a pre-existing equilibrium between the open and closed subpopulations of EryK, rather than from an induced-fit type mechanism.
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http://dx.doi.org/10.1074/jbc.M109.003590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781461PMC
October 2009

Pattern of cavities in globins: the case of human hemoglobin.

Biopolymers 2009 Dec;91(12):1097-107

University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy.

Our aim is to shed light on the conservation of potential ligand docking sites that play an important role in ligand dynamics of globins by using the technique of filling internal cavities naturally present in hemoglobin and myoglobin with xenon atoms. In particular, we present the high resolution structures of the Xe-adduct of deoxygenated wild type human hemoglobin and a quadruple mutant (L(B10)Y and H(E7)Q in alpha and beta chains). For the sake of comparison we also determined under the same experimental conditions the xenon complex of wild type sperm whale myoglobin. The analysis revealed that the number and position of Xe binding cavities are different in the alpha and beta subunits, the latter being more similar to myoglobin. Notably, no proximal Xe docking site was detected in hemoglobin, at variance with myoglobin. The pattern of internal cavities accessibility and affinity for xenon suggests a different role for the dynamics of ligand migration in the two types of hemoglobin chains as compared to myoglobin. The number and position of hydrophobic cavities in hemoglobin are briefly discussed also in comparison with the data available for other members of the globin superfamily.
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http://dx.doi.org/10.1002/bip.21201DOI Listing
December 2009

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of C-12 hydroxylase EryK from Saccharopolyspora erythraea.

Protein Pept Lett 2008 ;15(10):1138-41

Istituto di Biologia e Patologia Molecolari, CNR, and Dipartimento di Scienze Biochimiche, "Sapienza" Università di Roma, P.le A. Moro 5, I-00185 Rome, Italy.

Erythromycin A is produced by Saccharopolyspora erythraea via a secondary metabolic pathway using several steps including glycosylations and hydroxylations of the first macrolide intermediate 6-deoxyerythronolide B. Erythromycin C-12 hydroxylase (CYP113A1), the P450 cytochrome active in the final stages of erythromycin biosynthesis, was cloned and expressed in E. coli. Different crystal forms were harvested from distinct crystallization conditions: two ligand-free forms, one substrate bound and two inhibitors-bound. All crystals belong either to the monoclinc P2(1)or to the orthorhombic P2(1)2(1)2(1) space groups, and exhibit diffraction limits ranging from 2.3 to 1.6 A. The structures will be determined by molecular replacement.
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http://dx.doi.org/10.2174/092986608786071201DOI Listing
January 2009

Protein structure determination by x-ray crystallography.

Methods Mol Biol 2008 ;452:63-87

CNR Institute of Molecular Biology and Pathology (IBPM), Department of Biochemical Sciences, University of Rome, Sapienza,, Roma, Italy.

X-ray biocrystallography is the most powerful method to obtain a macromolecular structure. The improvement of computational technologies in recent years and the development of new and powerful computer programs together with the enormous increment in the number of protein structures deposited in the Protein Data Bank, render the resolution of new structures easier than in the past. The aim of this chapter is to provide practical procedures useful for solving a new structure. It is impossible to give more than a flavor of what the x-ray crystallographic technique entails in one brief chapter; therefore, this chapter focuses its attention on the Molecular Replacement method. Whenever applicable, this method allows the resolution of macromolecular structures starting from a single data set and a search model downloaded from the PDB, with the aid only of computer work.
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http://dx.doi.org/10.1007/978-1-60327-159-2_3DOI Listing
July 2008

X-ray structure analysis of a metalloprotein with enhanced active-site resolution using in situ x-ray absorption near edge structure spectroscopy.

Proc Natl Acad Sci U S A 2007 Apr 2;104(15):6211-6. Epub 2007 Apr 2.

Department of Biochemical Sciences and Consiglio Nazionale delle Ricerche, Institute of Molecular Biology and Pathology, University of Rome La Sapienza, P.le A. Moro 5, 00185 Rome, Italy.

X-ray absorption spectroscopy is exquisitely sensitive to the coordination geometry of an absorbing atom and therefore allows bond distances and angles of the surrounding atomic cluster to be measured with atomic resolution. By contrast, the accuracy and resolution of metalloprotein active sites obtainable from x-ray crystallography are often insufficient to analyze the electronic properties of the metals that are essential for their biological functions. Here, we demonstrate that the combination of both methods on the same metalloprotein single crystal yields a structural model of the protein with exceptional active-site resolution. To this end, we have collected an x-ray diffraction data set to 1.4-A resolution and Fe K-edge polarized x-ray absorption near edge structure (XANES) spectra on the same cyanomet sperm whale myoglobin crystal. The XANES spectra were quantitatively analyzed by using a method based on the multiple scattering approach, which yielded Fe-heme structural parameters with +/-(0.02-0.07)-A accuracy on the atomic distances and +/-7 degrees on the Fe-CN angle. These XANES-derived parameters were subsequently used as restraints in the crystal structure refinement. By combining XANES and x-ray diffraction, we have obtained an cyanomet sperm whale myoglobin structural model with a higher precision of the bond lengths and angles at the active site than would have been possible with crystallographic analysis alone.
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http://dx.doi.org/10.1073/pnas.0608411104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1851025PMC
April 2007

Insights into DNA replication: the crystal structure of DNA polymerase B1 from the archaeon Sulfolobus solfataricus.

Structure 2004 Nov;12(11):2001-8

Department of Biochemical Sciences and Consiglio Nazionale delle Ricerche, Institute of Molecular Biology and Pathology, University of Rome La Sapienza, P.le A. Moro 5, 00185 Roma, Italy.

To minimize the large number of mispairs during genome duplication owing to the large amount of DNA to be synthesized, many replicative polymerases have accessory domains with complementary functions. We describe the crystal structure of replicative DNA polymerase B1 from the archaeon Sulfolobus solfataricus. Comparison between other known structures indicates that although the protein is folded into the typical N-terminal, editing 3'-5'exonuclease, and C-terminal right-handed polymerase domains, it is characterized by the unusual presence of two extra alpha helices in the N-terminal domain interacting with the fingers helices to form an extended fingers subdomain, a structural feature that can account for some functional features of the protein. We explore the structural basis of specific lesion recognition, the initial step in DNA repair, describing how the N-terminal subdomain pocket of archaeal DNA polymerases could allow specific recognition of deaminated bases such as uracil and hypoxanthine in addition to the typical DNA bases.
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http://dx.doi.org/10.1016/j.str.2004.09.007DOI Listing
November 2004

The structure of ActVA-Orf6, a novel type of monooxygenase involved in actinorhodin biosynthesis.

EMBO J 2003 Jan;22(2):205-15

Dipartimento di Scienze Biochimiche and CNR, Centro di Studi sulla Biologia Molecolare, Università di Roma 'La Sapienza', Piazzale A.Moro, 5, 00185 Roma, Italy.

ActVA-Orf6 monooxygenase from Streptomyces coelicolor that catalyses the oxidation of an aromatic intermediate of the actinorhodin biosynthetic pathway is a member of a class of small monooxygenases that carry out oxygenation without the assistance of any of the prosthetic groups, metal ions or cofactors normally associated with activation of molecular oxygen. The overall structure is a ferredoxin-like fold with a novel dimeric assembly, indicating that the widely represented ferredoxin fold may sustain yet another functionality. The resolution (1.3 A) of the enzyme structure and its complex with substrate and product analogues allows us to visualize the mechanism of binding and activation of the substrate for attack by molecular oxygen, and utilization of two gates for the reaction components including a proton gate and an O(2)/H(2)O gate with a putative protein channel. This is the first crystal structure of an enzyme involved in the tailoring of a type II aromatic polyketide and illustrates some of the enzyme-substrate recognition features that may apply to a range of other enzymes involved in modifying a polyketide core structure.
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http://dx.doi.org/10.1093/emboj/cdg031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC140106PMC
January 2003