Effect of Saccharomyces cerevisiae and Candida zemplinina on quercetin, vitisin A and hydroxytyrosol contents in Sangiovese wines.

Quercetins, vitisin A and hydroxytyrosol are phenolic compounds possessing several positive properties to human health. This paper refers on the possible effects of two wine yeast species, Saccharomyces cerevisiae and Starmerella bacillaris (synonym Candida zemplinina) on the accumulation of these compounds in experimental Sangiovese wines. A single lot of Sangiovese grapes was fermented by S. cerevisiae alone or by sequential inoculum of C. zemplinina and S. cerevisiae under two aeration conditions. The accumulation of quercetin and its glycosides resulted only influenced by must aeration. However, yeast species occurring in the fermentative process affected the relative abundances among the different forms of quercetin. Vitisin A contents were higher in wines produced in the presence of C. zemplinina. Finally, higher concentrations of hydroxytyrosol and tyrosol were found in wines produced by S. cerevisiae alone under non-aerated condition. The fermentation of different Sangiovese grape musts carried out by the assayed S. cerevisiae strain pointed out that slow fermentation kinetics lead to higher levels of hydroxytyrosol and tyrosol. The study underlines the role of yeast species in determining the accumulation of bioactive compounds in Sangiovese wine.

A new proposal for urease mechanism based on the crystal structures of the native and inhibited enzyme from Bacillus pasteurii: why urea hydrolysis costs two nickels.

BACKGROUND: Urease catalyzes the hydrolysis of urea, the final step of organic nitrogen mineralization, using a bimetallic nickel centre. The role of the active site metal ions and amino acid residues has not been elucidated to date. Many pathologies are associated with the activity of ureolytic bacteria, and the efficiency of soil nitrogen fertilization with urea is severely decreased by urease activity. Therefore, the development of urease inhibitors would lead to a reduction of environmental pollution, to enhanced efficiency of nitrogen uptake by plants, and to improved therapeutic strategies for treatment of infections due to ureolytic bacteria. Structure-based design of urease inhibitors would require knowledge of the enzyme mechanism at the molecular level. RESULTS: The structures of native and inhibited urease from Bacillus pasteurii have been determined at a resolution of 2.0 A by synchrotron X-ray cryogenic crystallography. In the native enzyme, the coordination sphere of each of the two nickel ions is completed by a water molecule and a bridging hydroxide. A fourth water molecule completes a tetrahedral cluster of solvent molecules. The enzyme crystallized in the presence of phenylphosphorodiamidate contains the tetrahedral transition-state analogue diamidophosphoric acid, bound to the two nickel ions in an unprecedented mode. Comparison of the native and inhibited structures reveals two distinct conformations of the flap lining the active-site cavity. CONCLUSIONS: The mode of binding of the inhibitor, and a comparison between the native and inhibited urease structures, indicate a novel mechanism for enzymatic urea hydrolysis which reconciles the available structural and biochemical data.

Crystal structure of the complex between human carbonic anhydrase II and the aromatic inhibitor 1,2,4-triazole.

The X-ray crystal structure of the complex between human carbonic anhydrase II and the inhibitor 1,2,4-triazole has been refined at 1.9 A resolution to a final R-factor of 0.153. Triazole is an analogue of the competitive inhibitor imidazole, but the crystal structure shows a different type of binding to the enzyme. 1,2,4-Triazole is directly bound to the zinc(II) ion through the nitrogen in position 4, replacing the native water/hydroxyl (Wat263) in a distorted four-co-ordinated complex. The interaction of the inhibitor with the active site is completed by two hydrogen bonds to O gamma of Thr200 and to the amide nitrogen atom of Thr199 through the two adjacent N-1 and N-2 atoms. The binding site of triazole overlaps the proposed binding sites for the substrates, explaining the observed competitive behaviour of the inhibitor towards CO2/HCO3- under equilibrium conditions.

Crystal structure of the complex between carboxypeptidase A and the biproduct analog inhibitor L-benzylsuccinate at 2.0 A resolution.

The X-ray crystal structure of the carboxypeptidase A-L-benzylsuccinate complex has been refined at 2.0 A resolution to a final R-factor of 0.166. One molecule of the inhibitor binds to the enzyme active site. The terminal carboxylate forms a salt link with the guanidinium group of Arg145 and hydrogen bonds with Tyr248 and Asn144. The second carboxylate group binds to the zinc ion in an asymmetric bidentate fashion replacing the water molecule of the native structure. The zinc ion moves 0.5 A from its position in the native structure to accommodate the inhibitor binding. The overall stereochemistry around the zinc can be considered a distorted tetrahedron, although six atoms of the co-ordinated groups lie within 3.0 A from the zinc ion. The key for the strong inhibitory properties of L-benzylsuccinate can be found in its ability both to co-ordinate the zinc and to form a short carboxyl-carboxylate-type hydrogen bond (2.5 A) with Glu270.

Crystal structure of reduced bovine erythrocyte superoxide dismutase at 1.9 A resolution.

Cu,Zn superoxide dismutase was investigated crystallographically in the reduced form. Co-ordinate errors were estimated by comparing two independently refined models, based on two different data sets. This gave a detailed error estimation as opposed to the standard sigma A and Luzzati plots, which estimate only the overall error. The high quality of the final model, obtained after scaling together the two data sets, combined with the error estimates allowed a detailed analysis of the protein and solvent structures. An automatic procedure for building and refining solvent structure was tested and found to give reproducible results. Contrary to results obtained from spectroscopic studies, the co-ordination of the metal ions in the catalytic site is preserved in the crystal structure of the reduced enzyme, as compared with the crystal structure of the oxidised form. Analysis of the solvent reveals a well-defined chain of closely packed, hydrogen-bonded water molecules filling the active site groove. This structural feature could serve as a hydrogen bond relay for efficient delivery of protons to the active centre. Analysis of electron density suggests that Glu119 is covalently modified. The modification, if originated in vivo, could have a role in the catalytic mechanism and could affect the overall electrostatic field in the active site. There are significant differences between the active sites of the two crystallographically independent monomers. They are explained in terms of local differences in the crystal environment.

Crystal structure of the catalytic domain of human matrix metalloproteinase 10.

The catalytic domain of matrix metalloproteinase-10 (MMP-10) has been expressed in Escherichia coli and its crystal structure solved at 2.1 A resolution. The availability of this structure allowed us to critically examine the small differences existing between the catalytic domains of MMP-3 and MMP-10, which show the highest sequence identity among all MMPs. Furthermore, the binding mode of N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid (NNGH), which is one of the most known commercial inhibitors of MMPs, is described for the first time.

The crystal structure of the monomeric human SOD mutant F50E/G51E/E133Q at atomic resolution. The enzyme mechanism revisited.

The crystal structure of the engineered monomeric human Cu,ZnSOD triple mutant F50E/G51E/E133Q (Q133M2SOD) is reported at atomic resolution (1.02 A). This derivative has about 20 % of the wild-type activity. Crystals of Q133M2SOD have been obtained in the presence of CdCl2. The metal binding site is disordered, with both cadmium and copper ions simultaneously binding to the copper site. The cadmium (II) ions occupy about 45 % of the copper sites by binding the four histidine residues which ligate copper in the native enzyme, and two further water molecules to complete octahedral coordination. The copper ion is tri-coordinate, and the fourth histidine (His63) is detached from copper and bridges cadmium and zinc. X-ray absorption spectroscopy performed on the crystals suggests that the copper ion has undergone partial photoreduction upon exposure to the synchrotron light. The structure is also disordered in the disulfide bridge region of loop IV that is located at the subunit/subunit interface in the native SOD dimer. As a consequence, the catalytically relevant Arg143 residue is disordered. The present structure has been compared to other X-ray structures on various isoenzymes and to the solution structure of the same monomeric form. The structural results suggest that the low activity of monomeric SOD is due to the disorder in the conformation of the side-chain of Arg143 as well as of loop IV. It is proposed that the subunit-subunit interactions in the multimeric forms of the enzyme are needed to stabilize the correct geometry of the cavity and the optimal orientation of the charged residues in the active channel. Furthermore, the different coordination of cadmium and copper ions, contemporaneously present in the same site, are taken as models for the oxidized and reduced copper species, respectively. These properties of the structure have allowed us to revisit the enzymatic mechanism.

Substrate, substrate analogue, and inhibitor interactions with the ferrous active site of catechol 2,3-dioxygenase monitored through XAS studies.

The interactions of catechol (substrate), 2-hydroxy-pyridine-N-oxide (substrate analogue), and 2-bromophenol (inhibitor) with the extradiol cleaving catechol-2,3-dioxygenase from Pseudomonas putida mt-2 have been monitored through X-ray absorption spectroscopy (XAS). The analysis of the data provides details about the mode of coordination of the substrate and of the inhibitors to the active site of the enzyme.

XAS characterization of the active sites of novel intradiol ring-cleaving dioxygenases: hydroxyquinol and chlorocatechol dioxygenases.

The intradiol cleaving dioxygenases hydroxyquinol 1,2-dioxygenase (HQI,20) from Nocardiodes simplex 3E, chlorocatechol 1,2-dioxygenase (CIC1,20) from Rhodococcus erythropolis ICP, and their anaerobic substrate adducts (hydroxyquinol-HQ1,20 and 4-chlorocatechol-CIC1,20) have been characterized through X-ray absorption spectroscopy. In both enzymes the iron(III) is pentacoordinated and the distance distribution inside the Fe(III) first coordination shell is close to that already found in the extensively characterized protocatechuate 3,4-dioxygenase. The coordination number and the bond lengths are not significantly affected by the substrate binding. Therefore it is confirmed that the displacement of a protein donor upon substrate binding has to be considered a general step valid for all intradiol dioxygenases.

Theoretical and experimental study of DNA helix-coil transition in acidic and alkaline medium.

The theoretical approach to the calculation of the influence of selective binding of small ligands on DNA helix-coil transition has been described in the previous paper (Lando D. Yu., J. Biomol. Struct. Dyn., (1994)). In the present paper that method is used for the study of DNA protonation and deprotonation in acidic and alkaline medium by theoretical analysis of pH effect on DNA heat denaturation. The mechanism of DNA protonation in acidic medium and pK values of nucleotides are well known. It gave us an opportunity to check the theory without any fitting of pK values. A good agreement between experimental and calculated functions Tm(pH) and delta T(pH) (melting temperature and melting range width) obtained for acidic medium proved the validity of the theory. However, for alkaline medium there was not even qualitative agreement when the agreed-upon mechanism of deprotonation was considered. Looking into the cause of the discrepancy, we have studied the DNA melting for different mechanisms of deprotonation by calculation of Tm(pH) and delta T(pH). As a result, it has been established that the discrepancy is due to deprotonation of bonded GC base pairs of helical DNA regions (pK = 11). It was shown that the early known protonation and newly found deprotonation of helical DNA essentially stabilised double helix in alkaline and acidic medium.

Short duration of breastfeeding and early introduction of cow's milk as a result of mothers' low level of education.

Inappropriate infant feeding including a lack of breastfeeding and the early introduction of cow's milk are the most common forms of infant feeding malpractice. To evaluate the hypothesis that infant feeding malpractices are associated with mothers' low level of education, questionnaires were administered to 400 mothers of infants below 12 mo of age divided into 3 groups according to their various educational levels. Items included the type of milk given at birth and at 1, 3 and 6 mo of age. To investigate the efficacy of paediatricians in orienting infant feeding, the same questionnaire was given to 30 paediatricians in primary paediatric healthcare, in hospitals or in private practices. Initiation of breastfeeding was similar in the three groups. An analysis of the data showed that an increasing number of infants born to mothers of low and intermediate educational level did not receive exclusive breastfeeding compared with those with a higher level of education, a difference that was significant as early as 1 mo of age. In infants aged 3 mo, the prevalence of exclusive breastfeeding was 37%, 40% and 65% in the three groups, respectively, in relation to progressively increasing levels of education. In infants of 6 mo, the respective prevalence rates were 13%, 15% and 48%. Early introduction of cow's milk showed a similar correlation with educational level. A greater number of infants born to mothers with a low level of education received cow's milk at 3 mo of age compared with those born to mothers with an intermediate education (12% vs 5%). A similar difference was observed between the latter group and infants born to mothers with a high educational level (0%). This pattern was supported by data for infants at 6 mo of age with prevalence rates for cow's milk feeding of 39%, 20% and 0% in the three groups in association with progressively increasing level of educational (p < 0.05). The analysis of the paediatricians' response to the questionnaire showed that while physicians know and correctly prescribe age-related infant nutrition regimens, they are unaware that a substantial number of mothers do not comply with what they prescribe. Overall, these data support the relationship between a low educational level and infant feeding malpractice and suggest that a more effective role should be played by paediatricians in supporting an adequate duration of breastfeeding and the use of formula rather than cow's milk protein.

Homodimeric enzymes as drug targets.

Many enzymes and proteins are regulated by their quaternary structure and/or by their association in homo- and/or hetero-oligomer complexes. Thus, these protein-protein interactions can be good targets for blocking or modulating protein function therapeutically. The large number of oligomeric structures in the Protein Data Bank (http://www.rcsb.org/) reflects growing interest in proteins that function as multimeric complexes. In this review, we consider the particular case of homodimeric enzymes as drug targets. There is intense interest in drugs that inhibit dimerization of a functionally obligate homodimeric enzyme. Because amino acid conservation within enzyme interfaces is often low compared to conservation in active sites, it may be easier to achieve drugs that target protein interfaces selectively and specifically. Two main types of dimerization inhibitors have been developed: peptides or peptidomimetics based on sequences involved in protein-protein interactions, and small molecules that act at hot spots in protein-protein interfaces. Examples include inhibitors of HIV protease and HIV integrase. Studying the mechanisms of action and locating the binding sites of such inhibitors requires different techniques for different proteins. For some enzymes, ligand binding is only detectable in vivo or after unfolding of the complexes. Here, we review the structural features of dimeric enzymes and give examples of inhibition through interference in dimer stability. Several techniques for studying these complex phenomena will be presented.

Crystallographic studies of the binding of protonated and unprotonated inhibitors to carbonic anhydrase using hydrogen sulphide and nitrate anions.

The structures of human carbonic-anhydrase-II complexes with the anionic inhibitors hydrogen sulphide (HS-) and nitrate (NO3-) have been determined by X-ray diffraction at 0.19-nm resolution from crystals soaked at pH 7.8 and 6.0, respectively. The modes of binding of these two anions differ markedly from each other. The strong inhibitor HS- replaces the native zinc-bound water/hydroxide (Wat263) leaving the tetrahedral metal geometry unaltered and acts as a hydrogen-bonding donor towards Thr199 gamma. The weak NO3- inhibitor does not displace Wat263 from the metal coordination but occupies a fifth binding site changing the zinc coordination polyhedron into a slightly distorted trigonal bipyramid. The interaction of NO3- with the metal is weak; the nearest of its oxygen atoms being at a distance of 0.28 nm from the zinc ion. The binding of nitrate to the enzyme is completed by a hydrogen bond to the metal coordinated Wat263 and a second one to a water molecule of the active-site cavity. The structures of the two complexes help to rationalize the binding of anionic inhibitors to carbonic anhydrase and the binding mode displayed by NO39 may be relevant to the catalytic mechanism.

X-ray diffraction study of the interaction between carboxypeptidase A and (S)-(+)-1-amino-2-phenylethyl phosphonic acid.

The structure of the carboxypeptidase A complex with the inhibitor (S)-(+)-1-amino-2-phenylethylphosphonic acid has been determined at 0.23 nm resolution. The delta F map shows electron-density peaks both in the S1 and S'1 sites, where the inhibitor molecule can be modeled in two different orientations with approximate 50% occupancy. In the proposed model, the phosphonate group binds to the zinc ion in a monodentate fashion. Other anchoring groups for the inhibitor molecule are Arg127 (hydrogen bonds with the phosphonate oxygen atoms) and Glu270 (hydrogen bond with the amino group in one of the two orientations). A recent spectroscopic investigation of the complex between cobalt(II) carboxypeptidase A and (S)-(+)-1-amino-2-phenylethylphosphonic acid is essentially in agreement with our results.

Changes in the perception of biological X-ray absorption spectroscopy between ICBIC 1 and ICBIC 10/BIOXAS 2001: how far have we travelled?

The proceedings of BIOXAS 2001 (Siena, Italy, 2001) and both current and planned activities in the field of biological X-ray absorption spectroscopy are discussed against the perspective of changes in the perception of the technique since ICBIC 1 (Florence, Italy, 1983).

High-resolution structure of the complex between carboxypeptidase A and L-phenyl lactate.

The X-ray structures of native carboxypeptidase A and of the enzyme-inhibitor complex with L-phenyl lactate have been refined at 1.54 and 1.45 A resolution to R factors of 0.151 and 0.161, respectively. Crystals of the complex were isomorphous with the native crystals (space group P2(1), a = 51.60, b = 60.27, c = 47.25 A, beta = 97.27 degrees ). The high-resolution electron density allowed correction of many side-chain positions in the classical carboxypeptidase A model. This reflects the advantages of the high-quality complete synchrotron data collected with an imaging plate detector. The conformational changes in the active centre of the enzyme upon binding of the inhibitor are restricted to only two residues, Tyr248 and Arg145. L-Phenyl lactate is bound in the S1' pocket and forms hydrogen bonds to Arg145, Glu270 and to the zinc-bound water molecule. The present structure provides an explanation for the higher stability of the complexes with the products of esterolysis in comparison with those of amidolysis. This is consistent with the finding that product release is rate limiting for esters but not for peptides.

Reduction of vanadate to vanadyl by a strain of Saccharomyces cerevisiae.

Three strains of Saccharomyces cerevisiae, SC-1, DBVPG 6173 and DBVPG 6037, were studied for vanadate resistance in complex Sabouraud medium since they did not thrive in different minimal media (yeast nitrogen base with and without amino acids). The strain SC-1 was resistant up to 16 mM of vanadate, whereas the strains DBVPG 6173 and DBVPG 6037 were inhibited by 8 mM and 4 mM vanadate, respectively. The vanadate resistance in strain SC-1 was constitutive and due to the reduction of this oxyanion to vanadyl, which was detected by EPR spectroscopy and visible spectroscopy. The transformation of vanadate to vanadyl took place during the exponential growth phase; 10 mM of vanadate was reduced to vanadyl outside the cells since the oxyanion was not detected in the cell biomass and only a negligible concentration of vanadyl (25 nmoles mg-1 cells dry weight) was found in the biomass. The other two vanadate-sensitive yeast strains only accumulated vanadate and did not reduce the oxyanion to vanadyl.

X-ray structure of the ternary complex Zn(II)-ATP-2,2'-bipyridyl and possible model for ATP transport.

Ternary complexes between metal ions, nucleotides and aromatic heterocyclic amines are being investigated by X-ray diffraction as possible models for enzyme-metal-substrate interactions. Kennard and coworkers have reported the crystal structure of the ATP disodium salt. Although more recently several crystal structures of binary and ternary complexes between metal ions and nucleoside monophosphates have been reported, no structure combining metal complexes with nucleoside di- or triphosphates has been reported. Most divalent metal ions catalyse the nonenzymatic transfer of phosphate from nucleoside polyphosphates to various acceptors. Therefore, these compounds in water solution undergo extensive hydrolysis in the presence of metal ions, leading to the formation of the monophosphates; for instance, at pH 6.5, Cu2+ accelerates the hydrolysis of ATP by a factor of about 300 (ref. 5). We report here the crystal and molecular structure of the complex [Zn(II)-H2ATP-2,2'-bipyridyl]2.4H2O, determined by single crystal X-ray analysis. The complex molecule provides a possible model for ATP transport and phosphate group transfer mechanism.

X-ray absorption spectroscopy study of native and phenylphosphorodiamidate-inhibited Bacillus pasteurii urease.

X-ray absorption spectroscopy (XAS) has been applied to urease from Bacillus pasteurii, a highly ureolytic soil bacterium, with the aim of elucidating the structural details of the nickel-containing active site. The results indicate the presence of octahedrally coordinated Ni2+, in a sphere of six N/O donors at an average distance of 0.203 nm. An average of two histidine residues are bound to nickel. The experimental evidence suggests direct binding of the urease inhibitor phenylphosphorodiamidate to Ni2+. These spectroscopic results are in agreement with previous findings on both plant and microbial ureases, but differ in some respect from the results obtained by X-ray crystallography analysis of Klebsiella aerogenes urease.

X-ray absorption studies on catechol 2,3-dioxygenase from Pseudomonas putida mt2.

X-ray absorption spectroscopy has been utilized to investigate the structure of the active site of iron(II) catechol 2,3-dioxygenase from Pseudomonas putida mt2 both in the native and the 2-chlorophenol inhibited forms. XANES (X-ray absorption near edge structure) and EXAFS (extended X-ray absorption fine structure) results allow us to discuss the coordination number and geometry of the ferrous ion in the native enzyme. The metal geometry is not significantly affected by the binding of the inhibitor. The EXAFS spectrum is consistent with an iron(II) bound to six N/O atoms at an average distance of 2.05 A or to five N/O at an average distance of 2.04 A. The stimulation of the experimental data is greatly enhanced by considering the iron ligands divided in two different shells. Analysis of the outer shells performed using multiple scattering theory shows that there are histidines in the coordination sphere. The best fitting is obtained assuming the presence of two of them. Similar results are obtained for the inhibited enzyme, which, however, are indicative of a slight shortening of the average metal-donor bond distances. The direct binding of inhibitors to the metal center is confirmed by 1H NMR data.