Isabel Bento

Crystal Structure of Cardosin A, a Glycosylated and Arg-Gly-Asp-containing Aspartic Proteinase from the Flowers of Cynara cardunculus L.*

Aspartic proteinases (AP) have been widely studied within the living world, but so far no plant AP have been structurally characterized. The refined cardosin A crystallographic structure includes two molecules, built up by two glycosylated peptide chains (31 and 15 kDa each). The fold of cardosin A is typical within the AP family. The glycosyl content is described by 19 sugar rings attached to Asn-67 and Asn-257. They are localized on the molecular surface away from the conserved active site and show a new glycan of the plant complex type. A hydrogen bond between Gln-126 and Manβ4 renders the monosaccharide oxygen O-2 sterically inaccessible to accept a xylosyl residue, therefore explaining the new type of the identified plant glycan. The Arg-Gly-Asp sequence, which has been shown to be involved in recognition of a putative cardosin A receptor, was found in a loop between two β-strands on the molecular surface opposite the active site cleft. Based on the crystal structure, a possible mechanism whereby cardosin A might be orientated at the cell surface of the style to interact with its putative receptor from pollen is proposed. The biological implications of these findings are also discussed.

Reduction of dioxygen by enzymes containing copper

The reduction of dioxygen is a key step in many important biological processes including respiration and ligand oxidation. Enzymes containing either iron or copper or, indeed, both elements are often involved in this process, yet the catalytic mechanisms employed are not fully understood at the current time despite intensive biochemical, spectroscopic and structural studies. The aim of this article is to highlight the current structural knowledge regarding the process of dioxygen reduction using examples of copper-containing enzymes.

Ceruloplasmin revisited: structural and functional roles of various metal cation-binding sites

The three-dimensional molecular structure of human serum ceruloplasmin has been reinvestigated using X-ray synchrotron data collected at 100 K from a crystal frozen to liquid-nitrogen temperature.

Copper-Catalyzed Regioselective Intramolecular Oxidative α-Functionalization of Tertiary Amines: An Efficient Synthesis of Dihydro-1,3-Oxazines†

Oxidative coupling reactions, especially through cross-dehydrogenative coupling (CDC) of C H bonds, which avoids the prefunctionalization of substrates, have recently been in focus for being more atom economical, directive, and environmentally benign than other cross-coupling reactions. Among the CDC reactions, the functionalization of sp C H bonds adjacent to a nitrogen atom has generally been achieved utilizing transition metal catalysts with co-oxidants, such as tert-butylhydroperoxide (TBHP), H2O2, molecular oxygen, and others, to generate iminium ion species, which in turn react with various nucleophiles. For this purpose, metals such as Ru, Fe, Rh, V, and others are often used. However, copper salts retain many advantages, such as ready availability, low cost, high efficiency and low toxicity, and have turned out to be the most efficient catalysts for the a-functionalization of tertiary amines. More recently, homogeneous copper catalysis have also been used to achieve the selective aerobic oxidative functionalization of C H bonds. However, such copper-catalyzed functionalization, whether under aerobic or anaerobic conditions, is mainly confined to the benzylic position of N-phenyltetrahydroisoquinoline. Examples relating to the activation of non-benzylic methyl/methylene C H, are scarce. Hence, despite significant development in recent years, still more research in this area is required to enhance the selectivity and substrate scope. Looking for synthetic advances in regiocontrolled C H functionalization, we became interested in the design of a synthetic strategy for a copper-catalyzed aerobic oxidative/ dehydrogenative a-functionalization of tertiary amines with subsequent intramolecular sp C O bond formation. Dihydro-1,3-oxazine derivatives are important heterocyclic molecules, which exhibit a wide range of pharmacological activity, including antibacterial, fungicidal, antitumor, antituberculosis, and anti-HIV. Naphthoxazine derivatives have therapeutic potential for the treatment of Parkinson s disease, and are also used as potent nonsteroidal progesterone receptor agonists. They can be used as intermediates in the synthesis of N-substituted aminoalcohols, bioactive natural products, or in asymmetric catalysis. Justifiably, the intrinsic versatility and synthetic utility of these heterocyclic systems has attracted great interest from chemists, who have developed several exquisite methods for their synthesis. However, except for a few examples, most of the reported strategies are primarily based on Mannich type condensations, which are restricted to the use of aliphatic alicyclic primary amines. Hence, regardless of these advances, investigations in search of more efficient routes for the synthesis of these compounds are still highly desirable for drug discovery and medicinal chemistry. Herein, we disclose an efficient, environmentally friendly, diastereoselective copper-catalyzed synthesis of naphtho and benzo2,3-dihydro-1,3-oxazines through regioselective C H bond activation and cyclization (Scheme 1).

Dioxygen reduction by multi-copper oxidases; a structural perspective.

The multi-copper oxidases oxidise substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and, following the transfer of four electrons, is reduced to two molecules of water. The precise mechanism of this reduction has been unclear, but recent X-ray structural studies using the CotA endospore coat protein from Bacillus subtilis have given further insights into the principal stages. It is proposed that the mechanism involves binding of the dioxygen into the trinuclear centre so that it is sited approximately symmetrically between the two type 3 copper ions with one oxygen atom close to the type 2 copper ion. Further stages involve the formation of a peroxide intermediate and following the splitting of this intermediate, the migration of the hydroxide moieties towards the solvent exit channel. The migration steps are likely to involve a movement of the type 2 copper ion and its environment. Details of a putative mechanism are described herein based both on structures already reported in the literature and on structures of the CotA protein in the oxidised and reduced states and with the addition of peroxide and the inhibitor, azide.

Condensed phase behaviour of ionic liquid–benzene mixtures: congruent melting of a [emim][NTf2]·C6H6 inclusion crystal

The solid-liquid phase diagram of the (1-ethyl-3-methylimidazolium bis{(trifluoromethyl) sulfonyl} amide + benzene) system was determined and allowed us to identify and characterize an equimolar inclusion compound, [emim][NTf2].C6H6, with a congruent melting temperature: this type of behaviour, reported here for the first time, together with the X-ray structure of the inclusion compound lays emphasis upon the interactions that are responsible for the existence of liquid clathrates at higher benzene concentrations.

Proximal mutations at the type 1 copper site of CotA laccase: spectroscopic, redox, kinetic and structural characterization of I494A and L386A mutants.

In the present study the CotA laccase from Bacillus subtilis has been mutated at two hydrophobic residues in the vicinity of the type 1 copper site. The mutation of Leu(386) to an alanine residue appears to cause only very subtle alterations in the properties of the enzyme indicating minimal changes in the structure of the copper centres. However, the replacement of Ile(494) by an alanine residue leads to significant changes in the enzyme. Thus the major visible absorption band is upshifted by 16 nm to 625 nm and exhibits an increased intensity, whereas the intensity of the shoulder at approx. 330 nm is decreased by a factor of two. Simulation of the EPR spectrum of the I494A mutant reveals differences in the type 1 as well as in the type 2 copper centre reflecting modifications of the geometry of these centres. The intensity weighted frequencies , calculated from resonance Raman spectra are 410 cm(-1) for the wild-type enzyme and 396 cm(-1) for the I494A mutant, indicating an increase of the Cu-S bond length in the type 1 copper site of the mutant. Overall the data clearly indicate that the Ile(494) mutation causes a major alteration of the structure near the type 1 copper site and this has been confirmed by X-ray crystallography. The crystal structure shows the presence of a fifth ligand, a solvent molecule, at the type 1 copper site leading to an approximate trigonal bipyramidal geometry. The redox potentials of the L386A and I494A mutants are shifted downwards by approx. 60 and 100 mV respectively. These changes correlate well with decreased catalytic efficiency of both mutants compared with the wild-type.

Mechanisms underlying dioxygen reduction in laccases. Structural and modelling studies focusing on proton transfer

BackgroundLaccases are enzymes that couple the oxidation of substrates with the reduction of dioxygen to water. They are the simplest members of the multi-copper oxidases and contain at least two types of copper centres; a mononuclear T1 and a trinuclear that includes two T3 and one T2 copper ions. Substrate oxidation takes place at the mononuclear centre whereas reduction of oxygen to water occurs at the trinuclear centre.ResultsIn this study, the CotA laccase from Bacillus subtilis was used as a model to understand the mechanisms taking place at the molecular level, with a focus in the trinuclear centre. The structures of the holo-protein and of the oxidised form of the apo-protein, which has previously been reconstituted in vitro with Cu(I), have been determined. The former has a dioxygen moiety between the T3 coppers, while the latter has a monoatomic oxygen, here interpreted as a hydroxyl ion. The UV/visible spectra of these two forms have been analysed in the crystals and compared with the data obtained in solution. Theoretical calculations on these and other structures of CotA were used to identify groups that may be responsible for channelling the protons that are needed for reduction of dioxygen to water.ConclusionsThese results present evidence that Glu 498 is the only proton-active group in the vicinity of the trinuclear centre. This strongly suggests that this residue may be responsible for channelling the protons needed for the reduction. These results are compared with other data available for these enzymes, highlighting similarities and differences within laccases and multicopper oxidases.

New Evidence for the Role of Calcium in the Glycosidase Reaction of Gh43 Arabinanases.

Endo‐1,5‐α‐l‐arabinanases are glycosyl hydrolases that are able to cleave the glycosidic bonds of α‐1,5‐l‐arabinan, releasing arabino‐oligosaccharides and l‐arabinose. Two extracellular endo‐1,5‐α‐l‐arabinanases have been isolated from Bacillus subtilis, BsArb43A and BsArb43B (formally named AbnA and Abn2, respectively). BsArb43B shows low sequence identity with previously characterized 1,5‐α‐l‐arabinanases and is a much larger enzyme. Here we describe the 3D structure of native BsArb43B, biochemical and structure characterization of two BsArb43B mutant proteins (H318A and D171A), and the 3D structure of the BsArb43B D171A mutant enzyme in complex with arabinohexose. The 3D structure of BsArb43B is different from that of other structurally characterized endo‐1,5‐α‐l‐arabinanases, as it comprises two domains, an N‐terminal catalytic domain, with a 3D fold similar to that observed for other endo‐1,5‐α‐l‐arabinanases, and an additional C‐terminal domain. Moreover, this work also provides experimental evidence for the presence of a cluster containing a calcium ion in the catalytic domain, and the importance of this calcium ion in the enzymatic mechanism of BsArb43B.

Purification, crystallization and preliminary X-ray study of the fungal laccase from Cerrena maxima

The crystallization and preliminary X-ray structure at 1.9 Å resolution of the fungal laccase from C. maxima are presented.