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COVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight the current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful "tool" to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterization of the native RBD produced in three major model systems: Escherichia coli , insect and HEK-293 cells. Circular dichroism, gel filtration chromatography and thermal denaturation experiments indicated that recombinant SARS-CoV-2 RBD proteins are stable and correctly folded. In addition, their functionality and receptor-binding ability were further evaluated through ELISA, flow cytometry assays and bio-layer interferometry.

Background: Fungal glucose dehydrogenases (GDHs) are FAD-dependent enzymes belonging to the glucose-methanol-choline oxidoreductase superfamily. These enzymes are classified in the "Auxiliary Activity" family 3 (AA3) of the Carbohydrate-Active enZymes database, and more specifically in subfamily AA3_2, that also includes the closely related flavoenzymes aryl-alcohol oxidase and glucose 1-oxidase. Based on sequence similarity to known fungal GDHs, an AA3_2 enzyme active on glucose was identified in the genome of Pycnoporus cinnabarinus, a model Basidiomycete able to completely degrade lignin., Results: In our work, substrate screening and functional characterization showed an unexpected preferential activity of this enzyme toward oligosaccharides containing a β(1→3) glycosidic bond, with the highest efficiency observed for the disaccharide laminaribiose. Despite its sequence similarity to GDHs, we defined a novel enzymatic activity, namely oligosaccharide dehydrogenase (ODH), for this enzyme. The crystallographic structures of ODH in the sugar-free form and in complex with glucose and laminaribiose unveiled a peculiar saccharide recognition mechanism which is not shared with previously characterized AA3 oxidoreductases and accounts for ODH preferential activity toward oligosaccharides. The sugar molecules in the active site of ODH are mainly stabilized through CH-π interactions with aromatic residues rather than through hydrogen bonds with highly conserved residues, as observed instead for the fungal glucose dehydrogenases and oxidases characterized to date. Finally, three sugar-binding sites were identified on ODH external surface, which were not previously observed and might be of importance in the physiological scenario., Conclusions: Structure-function analysis of ODH is consistent with its role as an auxiliary enzyme in lignocellulose degradation and unveils yet another enzymatic function within the AA3 family of the Carbohydrate-Active enZymes database. Our findings allow deciphering the molecular determinants of substrate binding and provide insight into the physiological role of ODH, opening new perspectives to exploit biodiversity for lignocellulose transformation into fuels and chemicals., (© 2021. The Author(s).)

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. In silico 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.

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 40-48 and Gly-loop 44-47 ), the F106A mutant, and the double Gly-loop 44-47 /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 44-47 /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., (© 2020 Federation of European Biochemical Societies.)

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 in crystallo , 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 2 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., (© Chiara Ardiccioni et al. 2019.)

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.

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.

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.

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., (© 2017 The Protein Society.)

Internal cavities are crucial for conformational flexibility of proteins and can be mapped through noble gas diffusion and docking. Here we investigate the hydrophobic cavities and tunnel network in neuroglobin (Ngb), a hexacoordinated heme protein likely to be involved in neuroprotection, using crystallography under noble gas pressure, mostly at room temperature. In murine Ngb, a large internal cavity is involved in the heme sliding mechanism to achieve binding of gaseous ligands through coordination to the heme iron. In this study, we report that noble gases are hosted by two major sites within the internal cavity. We propose that these cavities could store oxygen and allow its relay in the heme proximity, which could correspond to NO location in the nitrite-reductase function of Ngb. Thanks to a recently designed pressurization cell using krypton at high pressure, a new gas binding site has been characterized that reveals an alternate pathway for gaseous ligands. A new gas binding site on the proximal side of the heme has also been characterized, using xenon pressure on a Ngb mutant (V140W) that binds CO with a similar rate and affinity to the wild-type, despite a reshaping of the internal cavity. Moreover, this study, to our knowledge, provides new insights into the determinants of the heme sliding mechanism, suggesting that the shift at the beginning of helix G precedes and drives this process., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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., (© 2017 Federation of European Biochemical Societies.)

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 4 -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.

Investigating the effect of pressure sheds light on the dynamics and plasticity of proteins, intrinsically correlated to functional efficiency. Here we detail the structural response to pressure of neuroglobin (Ngb), a hexacoordinate globin likely to be involved in neuroprotection. In murine Ngb, reversible coordination is achieved by repositioning the heme more deeply into a large internal cavity, the "heme sliding mechanism". Combining high pressure crystallography and coarse-grain simulations on wild type Ngb as well as two mutants, one (V101F) with unaffected and another (F106W) with decreased affinity for CO, we show that Ngb hinges around a rigid mechanical nucleus of five hydrophobic residues (V68, I72, V109, L113, Y137) during its conformational transition induced by gaseous ligand, that the intrinsic flexibility of the F-G loop appears essential to drive the heme sliding mechanism, and that residue Val 101 may act as a sensor of the interaction disruption between the heme and the distal histidine.

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.

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.

Neuroglobin is a globin present in the brain and retina of mammals. This hexacoordinated hemoprotein binds small diatomic molecules, albeit with lower affinity compared with other globins. We report here the resonance assignment of murine met-Neuroglobine, free and in complex with cyanide.

Background: The mechanisms by which general anesthetics, including xenon and nitrous oxide, act are only beginning to be discovered. However, structural approaches revealed weak but specific protein-gas interactions., Methods: To improve knowledge, we performed x-ray crystallography studies under xenon and nitrous oxide pressure in a series of 10 binding sites within four proteins., Results: Whatever the pressure, we show (1) hydrophobicity of the gas binding sites has a screening effect on xenon and nitrous oxide binding, with a threshold value of 83% beyond which and below which xenon and nitrous oxide, respectively, binds to their sites preferentially compared to each other; (2) xenon and nitrous oxide occupancies are significantly correlated respectively to the product and the ratio of hydrophobicity by volume, indicating that hydrophobicity and volume are binding parameters that complement and oppose each other's effects; and (3) the ratio of occupancy of xenon to nitrous oxide is significantly correlated to hydrophobicity of their binding sites., Conclusions: These data demonstrate that xenon and nitrous oxide obey different binding mechanisms, a finding that argues against all unitary hypotheses of narcosis and anesthesia, and indicate that the Meyer-Overton rule of a high correlation between anesthetic potency and solubility in lipids of general anesthetics is often overinterpreted. This study provides evidence that the mechanisms of gas binding to proteins and therefore of general anesthesia should be considered as the result of a fully reversible interaction between a drug ligand and a receptor as this occurs in classical pharmacology.

Equilibrium unfolding experiments provide access to protein thermodynamic stability revealing basic aspects of protein structure-function relationships. A limitation of these experiments stands on the availability of large amounts of protein samples. Here we present the use of the NanoDrop for monitoring guanidinium chloride-induced unfolding by Soret absorbance of monomeric heme proteins. Unfolding experiments using 2 μl of reactant are validated by fluorescence and circular dichroism spectroscopy and supported with five heme proteins including neuroglobin, cytochrome b5, and cyanoglobin. This work guarantees 2 orders of magnitude reduction in protein expense. Promising low-cost protein unfolding experiments following other chromophores and high-throughput screenings are discussed., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

The acknowledged success of the Monod-Wyman-Changeux (MWC) allosteric model stems from its efficacy in accounting for the functional behavior of many complex proteins starting with hemoglobin (the paradigmatic case) and extending to channels and receptors. The kinetic aspects of the allosteric model, however, have been often neglected, with the exception of hemoglobin and a few other proteins where conformational relaxations can be triggered by a short and intense laser pulse, and monitored by time-resolved optical spectroscopy. Only recently the application of time-resolved wide-angle X-ray scattering (TR-WAXS), a direct structurally sensitive technique, unveiled the time scale of hemoglobin quaternary structural transition. In order to test the generality of the MWC kinetic model, we carried out a TR-WAXS investigation in parallel on adult human hemoglobin and on a recombinant protein (HbYQ) carrying two mutations at the active site [Leu(B10)Tyr and His(E7)Gln]. HbYQ seemed an ideal test because, although exhibiting allosteric properties, its kinetic and structural properties are different from adult human hemoglobin. The structural dynamics of HbYQ unveiled by TR-WAXS can be quantitatively accounted for by the MWC kinetic model. Interestingly, the main structural change associated with the R-T allosteric transition (i.e., the relative rotation and translation of the dimers) is approximately 10-fold slower in HbYQ, and the drop in the allosteric transition rate with ligand saturation is steeper. Our results extend the general validity of the MWC kinetic model and reveal peculiar thermodynamic properties of HbYQ. A possible structural interpretation of the characteristic kinetic behavior of HbYQ is also discussed.

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.

Polarized Fe K-edge X-ray absorption near-edge structure (XANES) spectra of murine carbonmonoxy-neuroglobin (NgbCO) single crystals have been collected and compared with a number of derivatives of sperm whale myoglobin (Mb), that is, the nitrosyl (MbNO) and deoxy (Mb) derivatives, the previously reported cyanomet (MbCN) and carbonmonoxy (MbCO) derivatives, and the cryogenic photoproduct of MbCO (Mb·CO). The single crystals under study exhibit a strong XANES angular dichroism which allows the heme geometry of each sample to be analyzed with extremely high accuracy via the full multiple scattering (MS) approach. The results of two alternative methods to undergo the MS analysis have been compared with high resolution X-ray diffraction (XRD) data and with X-ray absorption spectroscopy (XAS) data in solution. As a result of the present analysis, the Fe-heme structure in solution and in the cryo-trapped NgbCO single crystal (which cracks at room temperature) are the same. Accordingly, the residual energy involved in the protein relaxation responsible of crystal cracking at room temperature after CO binding does not reside in the heme pocket. A combined approach (polarized XANES and XRD) is suggested to be applied on the same single crystals of metalloproteins at opportunely equipped synchrotron beamlines.

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.

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.

Neuroglobin (Ngb) is a hexacoordinate globin expressed in the brain of vertebrates. Ferrous Ngb binds dioxygen with high affinity and the O(2) adduct is able to scavenge NO. Convincing in vitro and in vivo data indicate that Ngb is involved in neuroprotection during hypoxia and ischemia. The 3D structure of Ngb reveals the presence of a wide internal cavity connecting its heme active site with the bulk. To explore the role of this "tunnel" in the control of ligand binding, we determined the structure of metNgb and NgbCO equilibrated with Xe or Kr. We show four docking sites for Xe (only two for Kr); two of the four Xe sites are within the large cavity. They are only partially conserved in globins, since the two proximal Xe sites identified in myoglobin (Xe1 and Xe2) are absent in Ngb, as well as in cytoglobin. The Xe docking sites in Ngb map a pathway within the protein matrix, leading to the heme, which becomes more accessible in the ligand-bound species. This may be of significance in connection with the redox chemistry that may be the primary function of this hexacoordinate globin.

Neuroglobin (Ngb) is a monomeric protein that, despite the small sequence similarity with other globins, displays the typical globin fold. In the absence of exogenous ligands, the ferric and the ferrous forms of Ngb are both hexacoordinated to the distal and proximal histidines. In the ferrous form, oxygen, nitric oxide or carbon monoxide can displace the distal histidine, yielding a reversible adduct. Crystallographic data show that the binding of an exogenous ligand is associated to structural changes involving heme sliding and a topological reorganization of the internal cavities. Molecular dynamics (MD) simulations in solution show that the heme oscillates between two positions, much as the ones observed in the crystal structure, although the occupancy is different. The simulations also suggest that ligand binding in solution can affect the flexibility and conformation of residues connecting the C and D helices, referred to as the CD corner, which is coupled to the configuration adopted by the distal histidine. In this study, we report the results of 30 ns MD simulations of CO-bound Ngb in the crystal. Our goal was to compare the protein dynamical behavior in the crystal with the results supplied by the previous MD simulation of CO-bound Ngb in solution and the x-ray experimental data. The results show that the different environments (crystal or solution) affect the dynamics of the heme group and of the CD corner.

Neuroglobin (Ngb) is a member of the globin family expressed in the vertebrate brain, involved in neuroprotection. A combined approach of X-ray diffraction (XRD) on single crystal and X-ray absorption spectroscopy (XAS) in solution, allows to determine the oxidation state and the structure of the Fe-heme both in the bis-histidine and the CO-bound (NgbCO) states. The overall data demonstrate that under X-ray the iron is photoreduced fairly rapidly, and that the previously reported X-ray structure of ferric Ngb [B. Vallone, K. Nienhaus, M. Brunori, G.U. Nienhaus, Proteins 56 (2004) 85-92] very likely refers to a photoreduced species indistinguishable from the dithionite reduced protein. Results from the XAS analysis of NgbCO in solution are in good agreement with XRD data on the crystal. However prolonged X-ray exposure at 15K determines CO release. This preliminary result paves the way to experiments aimed at the characterization of pentacoordinate ferrous Ngb, the only species competent in binding external ligands such as O2, CO or NO.

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.

When polychromatic X-rays are shined onto crystalline material, they generate a Laue diffraction pattern. At third generation synchrotron radiation sources, a single X-ray pulse of approximately 100 ps duration is enough to produce interpretable Laue data from biomolecular crystals. Thus, by initiating biological turnover in a crystalline protein, structural changes along the reaction pathway may be filmed by ultra-fast Laue diffraction. Using laser-light as a trigger, transient species in photosensitive macromolecules can be captured at near atomic resolution with sub-nanosecond time-resolution. Such pump-probe Laue experiments have now reached an outstanding level of sophistication and have found a domain of excellence in the investigation of light-sensitive proteins undergoing cyclic photo-reactions and producing stiff crystals. The main theoretical concepts of Laue diffraction and the challenges associated with time-resolved experiments on biological crystals are recalled. The recent advances in the design of experiments are presented in terms of instrumental choices, data collection strategy and data processing, and some of the inherent difficulties of the method are highlighted. The discussion is based on the example of myoglobin, a protein that has traversed the whole history of pump-probe Laue diffraction, and for which a massive amount of data have provided considerable insight into the understanding of protein dynamics.

Globins are respiratory proteins that reversibly bind dioxygen and other small ligands at the iron of a heme prosthetic group. Hemoglobin and myoglobin are the most prominent members of this protein family. Unexpectedly a few years ago a new member was discovered and called neuroglobin (Ngb), being predominantly expressed in the brain. Ngb is a single polypeptide of 151 amino acids and despite the small sequence similarity with other globins, it displays the typical globin fold. Oxygen, nitric oxide, or carbon monoxide can displace the distal histidine which, in ferrous Ngb as well as in ferric Ngb, is bound to the iron, yielding a reversible adduct. Recent crystallographic data on carboxy Ngb show that binding of an exogenous ligand is associated to structural changes involving heme sliding and a topological reorganization of the internal cavities; in particular, the huge internal tunnel that connects the bulk with the active site, peculiar to Ngb, is heavily reorganized. We report the results of extended (90 ns) molecular dynamics simulations in water of ferrous deoxy and carboxy murine neuroglobin, which are both coordinated on the distal site, in the latter case by CO and in the former one by the distal His(64)(E7). The long timescale of the simulations allowed us to characterize the equilibrated protein dynamics and to compare protein structure and dynamical behavior coupled to the binding of an exogenous ligand. We have characterized the heme sliding motion, the topological reorganization of the internal cavities, the dynamics of the distal histidine, and particularly the conformational change of the CD loop, whose flexibility depends ligand binding.

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.

Despite their similar fold topologies, anthozoan fluorescent proteins (FPs) can exhibit widely different optical properties, arising either from chemical modification of the chromophore itself or from specific interactions of the chromophore with the surrounding protein moiety. Here we present a structural and spectroscopic investigation of the green FP asFP499 from the sea anemone Anemonia sulcata var. rufescens to explore the effects of the protein environment on the chromophore. The optical absorption and fluorescence spectra reveal two discrete species populated in significant proportions over a wide pH range. Moreover, multiple protonation reactions are evident from the observed pH-dependent spectral changes. The x-ray structure of asFP499, determined by molecular replacement at a resolution of 1.85 A, shows the typical beta-barrel fold of the green FP from Aequorea victoria (avGFP). In its center, the chromophore, formed from the tripeptide Gln(63)-Tyr(64)-Gly(65), is tightly held by multiple hydrogen bonds in a polar cage that is structurally quite dissimilar to that of avGFP. The x-ray structure provides interesting clues as to how the spectroscopic properties are fine tuned by the chromophore environment.

Autofluorescent proteins of the GFP family all share the same three-dimensional beta-can fold; yet they exhibit widely different optical properties, arising either from chemical modification of the chromophore itself or from specific interactions of the chromophore with the surrounding protein moiety. Here we present a structural and spectroscopic characterization of the green fluorescent protein cmFP512 from Cerianthus membranaceus, a nonbioluminescent, azooxanthellate cnidarian, which has only approximately 22% sequence identity with Aequorea victoria GFP. The X-ray structure, obtained by molecular replacement at a resolution of 1. 35 A, shows the chromophore, formed from the tripeptide Gln-Tyr-Gly, in a hydrogen-bonded cage in the center of an 11-stranded beta-barrel, tightly restrained by adjacent residues and structural water molecules. It exists in a neutral (A) and an anionic (B) species, with absorption/emission maxima at 392/460 (pH 5) and 503/512 nm (pH 7). Their fractional populations and peak positions depend sensitively on pH, reflecting protonation of groups adjacent to the chromophore. The pH dependence of the spectra is explained by a protonation mechanism involving a hydrogen-bonded cluster of charged/polar groups. Cryospectroscopy at 12 K was also performed to analyze the vibronic coupling of the electronic transitions.

Small nucleolar RNAs (snoRNAs) play a key role in eukaryotic ribosome biogenesis. In most cases, snoRNAs are encoded in introns and are released through the splicing reaction. Some snoRNAs are, instead, produced by an alternative pathway consisting of endonucleolytic processing of pre-mRNA. XendoU, the endoribonuclease responsible for this activity, is a U-specific, metal-dependent enzyme that releases products with 2'-3' cyclic phosphate termini. XendoU is broadly conserved among eukaryotes, and it is a genetic marker of nidoviruses, including the severe acute respiratory syndrome coronavirus, where it is essential for replication and transcription. We have determined by crystallography the structure of XendoU that, by refined search methodologies, appears to display a unique fold. Based on sequence conservation, mutagenesis, and docking simulations, we have identified the active site. The conserved structural determinants of this site may provide a framework for attempting to design antiviral drugs to interfere with the infectious nidovirus life cycle.

Work carried out over the last 30 years unveiled the role of structural dynamics in controlling protein function. Cavity networks modulate structural dynamics trajectories and are functionally relevant; in globins they have been assigned a role in ligand migration and docking. These findings raised renewed interest for time-resolved structural investigations of myoglobin (Mb), a simple heme protein displaying a photosensitive iron-ligand bond. Photodissociation of MbCO generates a nonequilibrium population of protein structures relaxing over a time range extending from picoseconds to milliseconds. This process triggers ligand migration to matrix cavities with clear-cut effects on the rate and yield of geminate rebinding. Here, we report subnanosecond time-resolved Laue diffraction data on the triple mutant YQR-Mb [Leu-29(B10)Tyr, His-64(E7)Gln, Thr-67(E10)Arg] that depict the sequence of structural events associated with heme and protein relaxation from 100 ps to 316 ns and above. The photodissociated ligand rapidly (<0.1 ns) populates the Xe-binding cavity distal to the heme. Moreover, the heme relaxation toward the deoxy configuration is heterogeneous, with a slower phase ( approximately ns) evident in these experiments. Damping of the heme response appears to result from a strain exerted by the E-helix via the CD-turn; Phe-43(CD1), in close contact with heme, opposes tilt until the strain is relieved. A comparison with crystallographic data on wild-type Mb and mutants Leu(29)Phe or Leu(29)Trp suggests that the internal structure controls the rate and amplitude of the relaxation events. A correlation between structural dynamics as unveiled by Laue crystallography and functional properties of Mb is presented.

EosFP is a fluorescent protein from the coral Lobophyllia hemprichii that changes its fluorescence emission from green to red upon irradiation with near-UV light. Here we present the spectroscopic properties of wild-type EosFP and a variety of monomeric and dimeric mutants and provide a structural interpretation of its oligomerization and photoconversion, which is based on X-ray structure analysis of the green and red species that we reported recently. Because functional expression of the monomeric EosFP variant is limited to temperatures of 30 degrees C, we have developed a tandem dimer. This construct, in which two EosFP subunits are connected by a flexible 12 amino acid linker, expresses well after fusion with the androgen and endothelin A receptors at 37 degrees C. A variety of applications in cellular imaging, developmental biology and automated high-content screening applications are presented, which demonstrate that EosFP is a powerful tool for in vivo monitoring of cellular processes.

After over a century of extensive research, hemoglobin has become the prototype of allosteric and cooperative proteins. Its molecular structure, known in great detail, has allowed the design of hundreds of site directed mutations, aimed at interfering with its function, and thus at testing our hypotheses on the molecular mechanisms of allostery. The wealth of information thus obtained is difficult to read except for specialists, not only because it makes use of many different technical approaches, but also because of its intrinsically patchy nature. Moreover, several researchers have tried to assign specific roles to segments of the polypeptide chains, rather than to single residues, and have tested their hypotheses by multiple point mutations or by complete replacement with the homologous segment from a different hemoglobin to produce chimeric macromolecules. This approach is in great need of a revision since putative functionally relevant segments partially overlap. This review briefly describes the structure and function of hemoglobin, and analyzes the effect of point mutations, multiple mutations and segment replacement, with special attention to possible biotechnological applications, ranging from pharmacology (Hb solutions as resuscitating fluids and sources of the protein found in hemoglobinopathies for biochemical studies) to bioreactors. Occasional reference is made to site directed mutants of myoglobin, whenever this helps clarifying perplexing results obtained on hemoglobin.

The results of extended (80-ns) molecular dynamics simulations of wild-type and YQR triple mutant of sperm whale deoxy myoglobin in water are reported and compared with the results of the simulation of the intermediate(s) obtained by photodissociation of CO in the wild-type protein. The opening/closure of pathways between preexistent cavities is different in the three systems. For the photodissociated state, we previously reported a clear-cut correlation between the opening probability and the presence of the photolyzed CO in the proximity of the passage; here we show that in wild-type deoxy myoglobin, opening is almost random. In wild-type deoxy myoglobin, the passage between the distal pocket and the solvent is strictly correlated to the presence/absence of a water molecule that simultaneously interacts with the distal histidine side chain and the heme iron; conversely, in the photodissociated myoglobin, the connection with the bulk solvent is always open when CO is in the vicinity of the A pyrrole ring. In YQR deoxy myoglobin, the mutated Gln(E7)64 is stably H-bonded with the mutated Tyr(B10)29. The essential dynamics analysis unveils a different behavior for the three systems. The motion amplitude is progressively restricted in going from wild-type to YQR deoxy myoglobin and to wild-type myoglobin photoproduct. In all cases, the principal motions involve mainly the same regions, but their directions are different. Analysis of the dynamics of the preexisting cavities indicates large fluctuations and frequent connections with the solvent, in agreement with the earlier hypothesis that some of the ligand may escape from the protein through these pathways.

The red fluorescent protein (FP) eqFP611 from the sea anemone Entacmaea quadricolor shows favorable properties for applications as a molecular marker. Like other anthozoan FPs, it forms tetramers at physiological concentrations. The interactions among the monomers, however, are comparatively weak, as inferred from the dissociation into monomers in the presence of sodium dodecyl sulfate (SDS) or at high dilution. Analysis at the single-molecule level revealed that the monomers are highly fluorescent. For application as fusion markers, monomeric FPs are highly desirable. Therefore, we examine the monomer interfaces in the x-ray structure of eqFP611 to provide a basis for the rational design of monomeric variants. The arrangement of the four beta cans is very similar to that of other green fluorescent protein (GFP-like) proteins such as DsRed and RTMS5. A variety of structural features of the tetrameric interfaces explain the weak subunit interactions in eqFP611. We produce functional dimeric variants by introducing single point mutations in the A/B interface (Thr122Arg, Val124Thr). By contrast, structural manipulations in the A/C interface result in essentially complete loss of fluorescence, suggesting that A/C interfacial interactions play a crucial role in the folding of eqFP611 into its functional form., (Copyright 2005 Society of Photo-Optical Instrumentation Engineers)

Neuroglobin (Ngb), a globular heme protein expressed in the brain of vertebrates, binds oxygen reversibly, with an affinity comparable to myoglobin (Mb). Despite low sequence identity, the overall 3D fold of Ngb and Mb is very similar. Unlike in Mb, in Ngb the sixth coordination position of the heme iron is occupied by the distal histidine, in the absence of an exogenous ligand. Endogenous ligation has been proposed as a unique mechanism for affinity regulation and ligand discrimination in heme proteins. This peculiarity might be related to the still-unknown physiological function of Ngb. Here, we present the x-ray structure of CO-bound ferrous murine Ngb at 1.7 A and a comparison with the 1.5-A structure of ferric bis-histidine Ngb. We have also used Fourier transform IR spectroscopy of WT and mutant CO-ligated Ngb to examine structural heterogeneity in the active site. Upon CO binding, the distal histidine retains (by and large) its position, whereas the heme group slides deeper into a preformed crevice, thereby reshaping the large cavity ( approximately 290 A(3)) connecting the distal and proximal heme sides with the bulk. The heme relocation is accompanied by a significant decrease of structural disorder, especially of the EF loop, which may be the signal whereby Ngb communicates hypoxic conditions. This unexpected structural change unveils a heme-sliding mechanism of affinity control that may be of significance to understanding Ngb's role in the pathophysiology of the brain.

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.

Neuroglobin, a recently discovered globin predominantly expressed in neuronal tissue of vertebrates, binds small, gaseous ligands at the sixth coordination position of the heme iron. In the absence of an exogenous ligand, the distal histidine (His64) binds to the heme iron in the ferrous and ferric states. The crystal structure of murine ferric (met) neuroglobin at 1.5 A reveals interesting features relevant to the ligand binding mechanism. Only weak selectivity is observed for the two possible heme orientations, the occupancy ratio being 70:30. Two small internal cavities are present on the heme distal side, which enable the His64(E7) side chain to move out of the way upon exogenous ligand binding. Moreover, a third, huge cavity (volume approximately 290 A3) connecting both sides of the heme, is open towards the exterior and provides a potential passageway for ligands. The CD and EF corners exhibit substantial flexibility, which may assist ligands in entering the protein and accessing the active site. Based on this high-resolution structure, further structure-function studies can be planned to elucidate the role of neuroglobin in physiological responses to hypoxia., (Copyright 2004 Wiley-Liss, Inc.)

We report the results of an extended molecular dynamics simulation on the migration of photodissociated carbon monoxide in wild-type sperm whale myoglobin. Our results allow following one possible ligand migration dynamics from the distal pocket to the Xe1 cavity via a path involving the other xenon binding cavities and momentarily two additional packing defects along the pathway. Comparison with recent time resolved structural data obtained by Laue crystallography with subnanosecond to millisecond resolution shows a more than satisfactory agreement. In fact, according to time resolved crystallography, CO, after photolysis, can occupy the Xe1 and Xe4 cavities. However, no information on the trajectory of the ligand from the distal pocket to the Xe1 is available. Our results clearly show one possible path within the protein. In addition, although our data refer to a single trajectory, the local dynamics of the ligand in each cavity is sufficiently equilibrated to obtain local structural and thermodynamic information not accessible to crystallography. In particular, we show that the CO motion and the protein fluctuations are strictly correlated: free energy calculations of the migration between adjacent cavities show that the migration is not a simple diffusion but is kinetically or thermodynamically driven by the collective motions of the protein; conversely, the protein fluctuations are influenced by the ligand in such a way that the opening/closure of the passage between adjacent cavities is strictly correlated to the presence of CO in its proximity. The compatibility between time resolved crystallographic experiments and molecular dynamics simulations paves the way to a deeper understanding of the role of internal dynamics and packing defects in the control of ligand binding in heme proteins.

Although conformational changes are essential for the function of proteins, little is known about their structural dynamics at atomic level resolution. Myoglobin (Mb) is the paradigm to investigate conformational dynamics because it is a simple globular heme protein displaying a photosensitivity of the iron-ligand bond. Upon laser photodissociation of carboxymyoglobin Mb a nonequilibrium population of protein structures is generated that relaxes over a broad time range extending from picoseconds to milliseconds. This process is associated with migration of the ligand to cavities in the matrix and with a reduction in the geminate rebinding rate by several orders of magnitude. Here we report nanosecond time-resolved Laue diffraction data to 1.55-A resolution on a Mb mutant, which depicts the sequence of structural events associated with this extended relaxation. Motions of the distal E-helix, including the mutated residue Gln-64(E7), and of the CD-turn are found to lag significantly (100-300 ns) behind local rearrangements around the heme such as heme tilting, iron motion out of the heme plane, and swinging of the mutated residue Tyr-29(B10), all of which occur promptly (< or =3 ns). Over the same delayed time range, CO is observed to migrate from a cavity distal to the heme known to bind xenon (called Xe4) to another such cavity proximal to the heme (Xe1). We propose that the extended relaxation of the globin moiety reflects reequilibration among conformational substates known to play an essential role in controlling protein function.

A novel red fluorescent protein, eqFP611, from the sea anemone Entacmaea quadricolor has been cloned in Escherichia coli. With excitation and emission maxima at 559 and 611 nm, this protein shows the most red-shifted emission and the largest Stokes shift of all non-modified proteins in the green fluorescent protein (GFP) family. The protein fluoresces over a wide pH range (4-10) with high quantum yield (0.45). Its photophysical properties make eqFP611 an excellent marker protein for in vivo labeling in eukaryotic systems as was shown by expression in a mammalian cell culture. eqFP611 has been crystallized in space group P6(5)22, with unit-cell parameters a = b = 77.26, c = 329.49 A. The unit cell contains 12 asymmetric units, with two monomers in each. A molecular-replacement solution has been obtained using the 48.4% homologous red fluorescent protein from Discosoma coral (DsRed).

Crystals of the Met derivative of the sperm whale myoglobin triple mutant Mb-YQR [L(B10)Y, H(E7)Q and T(E10)R] were grown under microgravity conditions and on earth by vapour diffusion. A comparison of crystal quality after complete data collection and processing shows how microgravity-grown crystals diffract to better resolution and lead to considerably improved statistics for X-ray diffraction data compared with crystals grown on earth under the same conditions. The same set of experiments was reproduced on two different Spacelab missions (ISS 6A and ISS 8A) in 2001 and 2002. The structure of this mutant myoglobin, refined using data collected at ELETTRA (Trieste, Italy) from both kinds of crystals, shows that X-ray diffraction from microgravity-grown crystals leads to better defined electron-density maps as well as improved geometrical quality of the refined model. Improvement of the stereochemical parameters of a protein structure is fundamental to quantitative analysis of its function and dynamics and hence to thorough understanding of the molecular mechanisms of action.

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.

The fine structural properties of the distal heme pocket have been probed by infrared spectroscopy of ferrous carbon monoxy human hemoglobin mutants carrying the mutations LeuB10-->Tyr and HisE7-->Gln on the alpha, beta, and both chains, respectively. The stretching frequency of iron-bound carbon monoxide occurs as a single broad band around 1943 cm(-1) in both the alpha and the beta mutated chains. Such a frequency value indicates that no direct hydrogen bonding exists between the bound CO molecule and the TyrB10 phenolic oxygen, at variance with other naturally occurring TyrB10, GlnE7 nonvertebrate hemoglobins. The rates of carbon monoxide release have been determined for the first time by a Fourier transform infrared spectroscopy stopped-flow technique that allowed us to single out the heterogeneity in the kinetics of CO release in the alpha and beta chains for the mutated proteins and for native HbA. The rates of CO release are 15- to 20-fold faster for the mutated alpha or beta chains with respect to the native ones consistent with the lack of distal stabilization on the iron-bound CO molecule. The present results demonstrate that residues in key topological positions (namely E7 and B10) for the distal steric control of the iron-bound ligand are not interchangeable among hemoglobins from different species.

A quadruple mutant of sperm whale myoglobin was constructed to mimic the structure found in Ascaris suum hemoglobin. The replacements include His(E7)-->Gln, Leu(B10)-->Tyr, Thr(E10)--> Arg, and Ile(G8)-->Phe. Single, double, and triple mutants were characterized to dissect out the effects of the individual substitutions. The crystal structures of the deoxy and oxy forms of the quadruple mutant were determined and compared with that of native Ascaris hemoglobin. Tyr(B10) myoglobin displays low O(2) affinity, high dissociation rate constants, and heterogeneous kinetic behavior, suggesting unfavorable steric interactions between the B10 phenol side chain and His(E7). In contrast, all mutants containing the Tyr(B10)/Gln(E7) pair show high O(2) affinity, low dissociation rate constants, and simple, monophasic kinetic behavior. Replacement of Ile(107) with Phe enhances nanosecond geminate recombination singly and in combination with the Tyr(B10)/Gln(E7)/Arg(E10) mutation by limiting access to the Xe4 site. These kinetic results and comparisons with native Ascaris hemoglobin demonstrate the importance of distal pocket cavities in governing the kinetics of ligand binding. The approximately 150-fold higher O(2) affinity of Ascaris hemoglobin compared with that for Tyr(B10)/Gln(E7)-containing myoglobin mutants appears to be the result of favorable proximal effects in the Ascaris protein, due to a staggered orientation of His(F8), the lack of a hydrogen bonding lattice between the F4, F7, and F8 residues, and the presence of a large polar Trp(G5) residue in the interior portion of the proximal heme pocket.

The effect of mutagenesis on O(2), CO, and NO binding to mutants of human hemoglobin, designed to modify some features of the reactivity that hinder use of hemoglobin solutions as blood substitute, has been extensively investigated. The kinetics may be interpreted in the framework of the Monod-Wyman-Changeux two-state allosteric model, based on the high-resolution crystallographic structures of the mutants and taking into account the control of heme reactivity by the distal side mutations. The mutations involve residues at topological position B10 and E7, i.e., Leu (B10) to Tyr and His (E7) to Gln, on either the alpha chains alone (yielding the hybrid tetramer Hbalpha(YQ)), the beta chains alone (hybrid tetramer Hbbeta(YQ)), or both types of chains (Hb(YQ)). Our data indicate that the two mutations affect ligand diffusion into the pocket, leading to proteins with low affinity for O(2) and CO, and especially with reduced reactivity toward NO, a difficult goal to achieve. The observed kinetic heterogeneity between the alpha(YQ) and beta(YQ) chains in Hb(YQ) has been rationalized on the basis of the three-dimensional structure of the active site. Furthermore, we report for the first time an experiment of partial CO binding, selective for the beta chains, to high salt crystals of the mutant Hb(YQ) in the T-state; these crystallographic data may be interpreted as "snapshots" of the initial events possibly occurring on ligand binding to the T-allosteric state of this peculiar mutant Hb.