The oral motor capacity and feeding performance of preterm newborns at the time of transition to oral feeding.

The objective of the present study was to determine the oral motor capacity and the feeding performance of preterm newborn infants when they were permitted to start oral feeding. This was an observational and prospective study conducted on 43 preterm newborns admitted to the Neonatal Intensive Care Unit of UFSM, RS, Brazil. Exclusion criteria were the presence of head and neck malformations, genetic disease, neonatal asphyxia, intracranial hemorrhage, and kernicterus. When the infants were permitted to start oral feeding, non-nutritive sucking was evaluated by a speech therapist regarding force (strong vs weak), rhythm (rapid vs slow), presence of adaptive oral reflexes (searching, sucking and swallowing) and coordination between sucking, swallowing and respiration. Feeding performance was evaluated on the basis of competence (defined by rate of milk intake, mL/min) and overall transfer (percent ingested volume/total volume ordered). The speech therapist's evaluation showed that 33% of the newborns presented weak sucking, 23% slow rhythm, 30% absence of at least one adaptive oral reflex, and 14% with no coordination between sucking, swallowing and respiration. Mean feeding competence was greater in infants with strong sucking fast rhythm. The presence of sucking-swallowing-respiration coordination decreased the days for an overall transfer of 100%. Evaluation by a speech therapist proved to be a useful tool for the safe indication of the beginning of oral feeding for premature infants.

The oral motor capacity and feeding performance of preterm newborns at the time of transition to oral feeding

The objective of the present study was to determine the oral motor capacity and the feeding performance of preterm newborn infants when they were permitted to start oral feeding. This was an observational and prospective study conducted on 43 preterm newborns admitted to the Neonatal Intensive Care Unit of UFSM, RS, Brazil. Exclusion criteria were the presence of head and neck malformations, genetic disease, neonatal asphyxia, intracranial hemorrhage, and kernicterus. When the infants were permitted to start oral feeding, non-nutritive sucking was evaluated by a speech therapist regarding force (strong vs weak), rhythm (rapid vs slow), presence of adaptive oral reflexes (searching, sucking and swallowing) and coordination between sucking, swallowing and respiration. Feeding performance was evaluated on the basis of competence (defined by rate of milk intake, mL/min) and overall transfer (percent ingested volume/total volume ordered). The speech therapist's evaluation showed that 33 percent of the newborns presented weak sucking, 23 percent slow rhythm, 30 percent absence of at least one adaptive oral reflex, and 14 percent with no coordination between sucking, swallowing and respiration. Mean feeding competence was greater in infants with strong sucking fast rhythm. The presence of sucking-swallowing-respiration coordination decreased the days for an overall transfer of 100 percent. Evaluation by a speech therapist proved to be a useful tool for the safe indication of the beginning of oral feeding for premature infants.

Structures of adenylosuccinate synthetase from Triticum aestivum and Arabidopsis thaliana.

Catalyzing the first step in the de novo synthesis of adenylmonophosphate, adenylosuccinate synthetase (AdSS) is a known target for herbicides and antibiotics. We have purified and crystallized recombinant AdSS from Arabidopsis thaliana and Tritium aestivum, expressed in Escherichia coli. The structures of A. thaliana and T. aestivum AdSS in complex with GDP were solved at 2.9 A and 3.0 A resolution, respectively. Comparison with the known structures from E. coli reveals that the overall fold is very similar to that of the E. coli protein. The longer N terminus in the plant sequences is at the same place as the longer C terminus of the E. coli sequence in the 3D structure. The GDP-binding sites have one additional hydrogen-bonding partner, which is a plausible explanation for the lower K(m) value. Due to its special position, this partner may also enable GTP to initiate a conformational change, which was, in E. coli AdSS, exclusively activated by ligands at the IMP-binding site. The dimer interfaces show up to six hydrogen bonds and six salt-bridges more than in the E. coli structure, although the contact areas have approximately the same size.

An Enzyme-Bound Bisubstrate Hybrid Inhibitor of Adenylosuccinate Synthetase.

Two relatively weak herbicides, hydantocidin phosphate and hadacidin were linked by a C(3) chain to afford a potent inhibitor (the 2S hybrid is shown) of the enzyme adenylosuccinate synthetase. The crystal structures of the bisubstrate-enzyme complexes were determined.

Crystal structure of Escherichia coli cystathionine gamma-synthase at 1.5 A resolution.

The transsulfuration enzyme cystathionine gamma-synthase (CGS) catalyses the pyridoxal 5'-phosphate (PLP)-dependent gamma-replacement of O-succinyl-L-homoserine and L-cysteine, yielding L-cystathionine. The crystal structure of the Escherichia coli enzyme has been solved by molecular replacement with the known structure of cystathionine beta-lyase (CBL), and refined at 1.5 A resolution to a crystallographic R-factor of 20.0%. The enzyme crystallizes as an alpha4 tetramer with the subunits related by non-crystallographic 222 symmetry. The spatial fold of the subunits, with three functionally distinct domains and their quaternary arrangement, is similar to that of CBL. Previously proposed reaction mechanisms for CGS can be checked against the structural model, allowing interpretation of the catalytic and substrate-binding functions of individual active site residues. Enzyme-substrate models pinpoint specific residues responsible for the substrate specificity, in agreement with structural comparisons with CBL. Both steric and electrostatic designs of the active site seem to achieve proper substrate selection and productive orientation. Amino acid sequence and structural alignments of CGS and CBL suggest that differences in the substrate-binding characteristics are responsible for the different reaction chemistries. Because CGS catalyses the only known PLP-dependent replacement reaction at Cgamma of certain amino acids, the results will help in our understanding of the chemical versatility of PLP.

Structures of herbicides in complex with their detoxifying enzyme glutathione S-transferase - explanations for the selectivity of the enzyme in plants.

BACKGROUND: Glutathione S-transferases (GSTs) are detoxifying enzymes present in all aerobic organisms. These enzymes catalyse the conjugation of glutathione with a variety of electrophilic compounds. In plants, GSTs catalyse the first step in the degradation of several herbicides, such as triazines and acetamides, thus playing an important role in herbicide tolerance. RESULTS: We have solved the structures of GST-I from maize in complex with an atrazine-glutathione conjugate (at 2.8 A resolution) and GST from Arabidopsis thaliana (araGST) in complex with an FOE-4053-glutathione conjugate (at 2.6 A resolution). These ligands are products of the detoxifying reaction and are well defined in the electron density. The herbicide-binding site (H site) is different in the two structures. The architecture of the glutathione-binding site (G site) of araGST is different to that of the previously described structure of GST in complex with two S-hexylglutathione molecules, but is homologous to that of GST-I. CONCLUSIONS: Three features are responsible for the differences in the H site of the two GSTs described here: the exchange of hydrophobic residues of different degrees of bulkiness; a slight difference in the location of the H site; and a difference in the degree of flexibility of the upper side of the H site, which is built up by the loop between helices alpha4 and alpha5. Taking these two structures as a model, the different substrate specificities of other plant GSTs may be explained. The structures reported here provide a basis for the design of new, more selective herbicides.

Lactate dehydrogenase from the hyperthermophilic bacterium thermotoga maritima: the crystal structure at 2.1 A resolution reveals strategies for intrinsic protein stabilization.

BACKGROUND: L(+)-Lactate dehydrogenase (LDH) catalyzes the last step in anaerobic glycolysis, the conversion of pyruvate to lactate, with the concomitant oxidation of NADH. Extensive physicochemical and structural investigations of LDHs from both mesophilic and thermophilic organisms have been undertaken in order to study the temperature adaptation of proteins. In this study we aimed to determine the high-resolution structure of LDH from the hyperthermophilic bacterium Thermotoga maritima (TmLDH), the most thermostable LDH to be isolated so far. It was hoped that the structure of TmLDH would serve as a model system to reveal strategies of protein stabilization at temperatures near the boiling point of water. RESULTS: The crystal structure of the extremely thermostable TmLDH has been determined at 2.1 A resolution as a quaternary complex with the cofactor NADH, the allosteric activator fructose-1,6-bisphosphate, and the substrate analog oxamate. The structure of TmLDH was solved by Patterson search methods using a homology-based model as a search probe. The native tetramer shows perfect 222 symmetry. Structural comparisons with five LDHs from mesophilic and moderately thermophilic organisms and with other ultrastable enzymes from T. maritima reveal possible strategies of protein thermostabilization. CONCLUSIONS: Structural analysis of TmLDH and comparison of the enzyme to moderately thermophilic and mesophilic homologs reveals a strong conservation of both the three-dimensional fold and the catalytic mechanism. Going from lower to higher physiological temperatures a variety of structural differences can be observed: an increased number of intrasubunit ion pairs; a decrease of the ratio of hydrophobic to charged surface area, mainly caused by an increased number of arginine and glutamate sidechains on the protein surface; an increased secondary structure content including an additional unique 'thermohelix' (alphaT) in TmLDH; more tightly bound intersubunit contacts mainly based on hydrophobic interactions; and a decrease in both the number and the total volume of internal cavities. Similar strategies for thermal adaptation can be observed in other enzymes from T. maritima.

Rack-induced metal binding vs. flexibility: Met121His azurin crystal structures at different pH.

The rack-induced bonding mechanism of metals to proteins is a useful concept for explaining the generation of metal sites in electron transfer proteins, such as the blue copper proteins, that are designed for rapid electron transfer. The trigonal pyramidal structure imposed by the protein with three strong equatorial ligands (one Cys and two His) provides a favorable geometry for both cuprous and cupric oxidation states. However, the crystal structures of the Met121His mutant of azurin from Alcaligenes denitrificans at pH 6.5 (1.89- and 1.91-A resolutions) and pH 3.5 (2.45-A resolution) show that the preformed metal binding cavity in the protein is more flexible than expected. At high pH (6.5), the Cu site retains the same three equatorial ligands as in the wild-type azurin and adds His121 as a fourth strong ligand, creating a tetrahedral copper site geometry with a green color referred to as 1.5 type. In the low pH (3.5) structure, the protonation of His121 causes a conformational change in residues 117-123, moving His121 away from the copper. The empty coordination site is occupied by an oxygen atom of a nitrate molecule of the buffer solution. This axial ligand is coordinated less strongly, generating a distorted tetrahedral copper geometry with a blue color and spectroscopic properties of a type-1 site. These crystal structures demonstrate that blue copper proteins are flexible enough to permit a range of movement of the Cu atom along the axial direction of the trigonal pyramid.

Structures of class pi glutathione S-transferase from human placenta in complex with substrate, transition-state analogue and inhibitor.

BACKGROUND: Glutathione S-transferases (GSTs) are detoxification enzymes, found in all aerobic organisms, which catalyse the conjugation of glutathione with a wide range of hydrophobic electrophilic substrates, thereby protecting the cell from serious damage caused by electrophilic compounds. GSTs are classified into five distinct classes (alpha, mu, pi, sigma and theta) by their substrate specificity and primary structure. Human GSTs are of interest because tumour cells show increased levels of expression of single classes of GSTs, which leads to drug resistance. Structural differences between classes of GST can therefore be utilised to develop new anti-cancer drugs. Many mutational and structural studies have been carried out on the mu and alpha classes of GST to elucidate the reaction mechanism, whereas knowledge about the pi class is still limited. RESULTS: We have solved the structures of the pi class GST hP1-1 in complex with its substrate, glutathione, a transition-state complex, the Meisenheimer complex, and an inhibitor, S-(rho-bromobenzyl)-glutathione, and refined them to resolutions of 1.8 A, 2.0 A and 1.9 A, respectively. All ligand molecules are well-defined in the electron density. In all three structures, an additionally bound N-morpholino-ethansulfonic acid molecule from the buffer solution was found. CONCLUSIONS: In the structure of the GST-glutathione complex, two conserved water molecules are observed, one of which hydrogen bonds directly to the sulphur atom of glutathione and the other forms hydrogen bonds with residues around the glutathione-binding site. These water molecules are absent from the structure of the Meisenheimer complex bound to GST, implicating that deprotonation of the cysteine occurs during formation of the ternary complex which involves expulsion of the inner bound water molecule. The comparison of our structures with known mu class GST structures show differences in the location of the electrophile-binding site (H-site), explaining the different substrate specificities of the two classes. Fluorescence measurements are in agreement with the position of the N-morpholino-ethansulfonic acid, close to Trp28, identifying a possible ligandin-substrate binding site.

Cloning, purification, crystallization, and preliminary X-ray diffraction analysis of cystathionine gamma-synthase from E. coli.

The Escherichia coli metB gene has been PCR-extracted from genomic DNA and placed under the control of a tac and a T7 promoter in plasmids pCYB1 and pET22b(+), respectively, to produce overexpressing bacterial strains for the gene product, cystathionine gamma-synthase. Efficient purification procedures have been developed for a C-terminally intein-tagged version and the wild-type target protein, yielding the product in a quantity and homogeneity amenable to high-resolution single-crystal X-ray analysis. Crystals have been obtained in space group P1 with unit cell constants a=82.2 A, b=84.2 A, c=116.2 A, alpha=107.0 degrees, beta=96.3 degrees, gamma=108.0 degrees, suggesting eight monomers per asymmetric unit (V[M]=2.23 A3/Da). Crystals diffract to beyond 2.6 A resolution and a data set complete to 2.8 A resolution has been collected using a rotating anode X-ray source. A cryogenic buffer system has been developed to allow synchrotron data collection. Patterson self rotation searches reveal the presence of two independent tetramers with local 222 symmetry in an asymmetric unit. The crystallographic results corroborate and extend previous solution studies regarding the quaternary organization of the enzyme.

Bioincorporation of telluromethionine into proteins: a promising new approach for X-ray structure analysis of proteins.

A simple and efficient method for the specific and quantitative replacement of the naturally occurring amino acid methionine by its isosteric analogue telluromethionine in the expression of recombinant proteins has been developed. The method requires a controlable and competitive expression system like the bacteriophage T7 polymerase/promoter in a methionine-auxotrophic host. Using methionine-auxotrophic Escherichia coli strains, incorporation of telluromethionine at high yields has been achieved for human recombinant annexin V, human mitochondrial transamidase, Arabidopsis glutathione-S-transferase and the N-terminal domain of Salmonella tailspike adhesion protein as confirmed by amino acid, mass-spectrometric and X-ray analyses. Expressed and purified telluromethionine-proteins and native proteins were found to crystallise isomorphously. In terms of efficient bio-expression, isomorphism of crystals and relative abundance of methionine residues, the production of telluromethionine-proteins as heavy-atom derivatives offers a valid and general approach in X-ray analysis by the method of multiple isomorphous replacement.

Implications for the catalytic mechanism of the vanadium-containing enzyme chloroperoxidase from the fungus Curvularia inaequalis by X-ray structures of the native and peroxide form.

Implications for the catalytic mechanism of the vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis have been obtained from the crystal structures of the native and peroxide forms of the enzyme. The X-ray structures have been solved by difference Fourier techniques using the atomic model of the azide chloroperoxidase complex. The 2.03 A crystal structure (R = 19.7%) of the native enzyme reveals the geometry of the intact catalytic vanadium center. The vanadium is coordinated by four non-protein oxygen atoms and one nitrogen (NE2) atom from histidine 496 in a trigonal bipyramidal fashion. Three oxygens are in the equatorial plane and the fourth oxygen and the nitrogen are at the apexes of the bipyramid. In the 2.24 A crystal structure (R = 17.7%) of the peroxide derivate the peroxide is bound to the vanadium in an eta2-fashion after the release of the apical oxygen ligand. The vanadium is coordinated also by 4 non-protein oxygen atoms and one nitrogen (NE2) from histidine 496. The coordination geometry around the vanadium is that of a distorted tetragonal pyramid with the two peroxide oxygens, one oxygen and the nitrogen in the basal plane and one oxygen in the apical position. A mechanism for the catalytic cycle has been proposed based on these X-ray structures and kinetic data.

Dimer interface of glutathione S-transferase from Arabidopsis thaliana: influence of the G-site architecture on the dimer interface and implications for classification.

The three-dimensional structure of glutathione S-transferase from Arabidopsis thaliana has been solved at 2.2 A resolution (Reinemer et al., 1996). The enzyme forms a dimer of two identical subunits. The structure shows a new G-site architecture and a novel and unique dimer interface. Each monomer of the protein forms a separate G-site. Therefore, the requirements on the dimer interface are reduced. As a consequence, the interactions between the monomers are weaker and residues at the dimer interface are more variable. Thus, the dimer interface looses its relevance for a classification of plant glutathione S-transferases and the formation of heterodimers becomes even more difficult to predict.

Crystal structure of herbicide-detoxifying maize glutathione S-transferase-I in complex with lactoylglutathione: evidence for an induced-fit mechanism.

Glutathione S-transferases (GSTs) -I and -III are involved in herbicide metabolism in maize and have been intensively studied. Starting with plant tissue from Zea mays var. mutin recombinant GST-I was prepared by heterologous expression in Escherichia coli. The enzyme was crystallized in the presence of lactoylglutathione, a ligand formerly never observed in a GST structure and known as an intermediate of the pharmacologically relevant glyoxalase system. The crystal structure of GST-I has been determined at 2.5 A resolution and exhibits the GST-typical dimer of two identical subunits, each consisting of 214 residues. Compared with other plant GSTs the three-dimensional structure of GST-I primarily shows structural differences in the hydrophobic substrate binding site, the linker segment and the C-terminal region. Furthermore, a comparison of the ligand-bound GST-I structure with the apo structure of GST-III indicates the movement of a ten-residue loop upon binding of the ligand to the active site. This is the first structure-based evidence for an induced fit mechanism of glutathione S-transferases, which has previously been postulated for class pi enzymes. Together with GST-III, GST-I may explain herbicide resistance and selectivity in maize as well as in other agronomic relevant crops.

Cloning, sequencing, crystallization and X-ray structure of glutathione S-transferase-III from Zea mays var. mutin: a leading enzyme in detoxification of maize herbicides.

Glutathione S-transferases (GSTs) are enzymes that inactivate toxic compounds by conjugation with glutathione and are involved in resistance towards drugs, antibiotics, insecticides and herbicides. Their ability to confer herbicide tolerance in plants provides a tool to control weeds in a wide variety of agronomic crops. GST-III was prepared from Zea mays var. mutin and its amino acid sequence was determined from two sets of peptides obtained by cleavage with endoprotease Asp-N and with trypsin, respectively. Recombinant GST-III was prepared by extraction of mRNA from plant tissue, transcription into cDNA, amplification by PCR and expression. It was crystallized and the crystal structure of the unligated form was determined at 2.2 A resolution. The enzyme forms a GST-typical dimer with one subunit consisting of 220 residues. Each subunit is formed of two distinct domains, an N-terminal domain consisting of a beta-sheet flanked by two helices, and a C-terminal domain, entirely helical. The dimeric molecule is globular with a large cleft between the two subunits. The amino acid sequence of GST-III and its cDNA sequence determined here show differences from sequences published earlier.

Staurosporine-induced conformational changes of cAMP-dependent protein kinase catalytic subunit explain inhibitory potential.

BACKGROUND: Staurosporine inhibits most protein kinases at low nanomolar concentrations. As most tyrosine kinases, along with many serine/threonine kinases, are either proto oncoproteins or are involved in oncogenic signaling, the development of protein kinase inhibitors is a primary goal of cancer research. Staurosporine and many of its derivatives have significant biological effects, and are being tested as anticancer drugs. To understand in atomic detail the mode of inhibition and the parameters of high-affinity binding of staurosporine to protein kinases, the molecule was cocrystallized with the catalytic subunit of cAMP-dependent protein kinase. RESULTS: The crystal structure of the protein kinase catalytic subunit with staurosporine bound to the adenosine pocket shows considerable induced-fit rearrangement of the enzyme and a unique open conformation. The inhibitor mimics several aspects of adenosine binding, including both polar and nonpolar interactions with enzyme residues, and induces conformational changes of neighboring enzyme residues. CONCLUSIONS: The results explain the high inhibitory potency of staurosporine, and also illustrate the flexibility of the protein kinase active site. The structure, therefore, is not only useful for the design of improved anticancer therapeutics and signaling drugs, but also provides a deeper understanding of the conformational flexibility of the protein kinase.

Three-dimensional structure of glutathione S-transferase from Arabidopsis thaliana at 2.2 A resolution: structural characterization of herbicide-conjugating plant glutathione S-transferases and a novel active site architecture.

Glutathione S-transferases (GST) are a family of multifunctional enzymes involved in the metabolization of a broad variety of xenobiotics and reactive endogenous compounds. The interest in plant glutathione S-transferases may be attributed to their agronomic value, since it has been demonstrated that glutathione conjugation for a variety of herbicides is the major resistance and selectivity factor in plants. The three-dimensional structure of glutathione S-transferase from the plant Arabidopsis thaliana has been solved by multiple isomorphous replacement and multiwavelength anomalous dispersion techniques at 3 A resolution and refined to a final crystallographic R-factor of 17.5% using data from 8 to 2.2 A resolution. The enzyme forms a dimer of two identical subunits each consisting of 211 residues. Each subunit is characterized by the GST-typical modular structure with two spatially distinct domains. Domain I consists of a central four-stranded beta-sheet flanked on one side by two alpha-helices and on the other side by an irregular segment containing three short 3(10)-helices, while domain II is entirely helical. The dimeric molecule is globular with a prominent large cavity formed between the two subunits. The active site is located in a cleft situated between domains I and II and each subunit binds two molecules of a competitive inhibitor S-hexylglutathione. Both hexyl moieties are oriented parallel and fill the H-subsite of the enzyme's active site. The glutathione peptide of one inhibitor, termed productive binding, occupies the G-subsite with multiple interactions similar to those observed for other glutathione S-transferases, while the glutathione backbone of the second inhibitor, termed unproductive binding, exhibits only weak interactions mediated by two polar contacts. A most striking difference from the mammalian glutathione S-transferases, which share a conserved catalytic tyrosine residue, is the lack of this tyrosine in the active site of the plant glutathione S-transferase.