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Daniel J Rigden

Publications and source records attributed to Daniel J Rigden.

At least 37 records · Page 2Linked to original sources
PubMed2004

Characterization of the ligandin site of maize glutathione S-transferase I.

Cytosolic GSTs (glutathione S-transferases) are a major reserve of high-capacity binding proteins and exhibit ligand-binding properties for a large variety of compounds. In the present study, the binding of two non-substrate anthraquinone dyes VBAR (Vilmafix Blue A-R) and CB3GA (Cibacron Blue 3GA) to maize (Zea mays) GST I was investigated. The results showed that the enzyme was specifically and irreversible inactivated by VBAR with a K(d) of 35.5+/-2.2 microM and a k(3) of 0.47 min(-1). Proteolytic cleavage of the VBAR-modified enzyme and subsequent separation of peptides gave only one modified peptide. Sequencing of the modified peptide revealed the target site of VBAR reaction to be Lys(41). CB3GA binds reversibly to GST I and behaves as a competitive inhibitor towards CDNB (1-chloro-2,4-dinitrobenzene) and glutathione. CB3GA binding to GST I is accompanied by a characteristic spectral change in the absorption at positive maximum (670 nm) which exhibited a hyperbolic dependence on dye concentration with a K(d) of 12.1+/-0.5 microM. Site-directed mutagenesis of selected residues (Trp(12), Phe(35), Lys(41), Asn(49), Gln(53), Ser(67) and Ile(118)) was employed, and the mutated enzymes were assessed for CB3GA binding. These results, together with molecular-modelling studies, established that the ligandin-binding site of GST I is located mainly in the hydrophobic binding site. The ability of VBAR to specifically inactivate GST I was exploited further to demonstrate the specific binding of several plant hormones and flavonoids to GST I. The inactivation of other GST isoenzymes by VBAR was also investigated, and it was concluded that VBAR may have wide applicability as an affinity label for probing structure-function relationships of GST isoenzymes.

Amino Acid Sequence↗
PubMed2004

L-Ascorbic acid 6-hexadecanoate, a potent hyaluronidase inhibitor. X-ray structure and molecular modeling of enzyme-inhibitor complexes.

Hyaluronidases are enzymes that degrade hyaluronan, an important component of the extracellular matrix. The mammalian hyaluronidases are considered to be involved in many (patho)physiological processes like fertilization, tumor growth, and metastasis. Bacterial hyaluronidases, also termed hyaluronate lyases, contribute to the spreading of microorganisms in tissues. Such roles for hyaluronidases suggest that inhibitors could be useful pharmacological tools. Potent and selective inhibitors are not known to date, although L-ascorbic acid has been reported to be a weak inhibitor of Streptococcus pneumoniae hyaluronate lyase (SpnHL). The x-ray structure of SpnHL complexed with L-ascorbic acid has been elucidated suggesting that additional hydrophobic interactions might increase inhibitory activity. Here we show that L-ascorbic acid 6-hexadecanoate (Vcpal) is a potent inhibitor of both streptococcal and bovine testicular hyaluronidase (BTH). Vcpal showed strong inhibition of Streptococcus agalactiae hyaluronate lyase with an IC(50) of 4 microM and weaker inhibition of SpnHL and BTH with IC(50) values of 100 and 56 microM, respectively. To date, Vcpal has proved to be one of the most potent inhibitors of hyaluronidase. We also determined the x-ray structure of the SpnHL-Vcpal complex and confirmed the hypothesis that additional hydrophobic interactions with Phe-343, His-399, and Thr-400 in the active site led to increased inhibition. A homology structural model of BTH was also generated to suggest binding modes of Vcpal to this hyaluronidase. The long alkyl chain seemed to interact with an extended, hydrophobic channel formed by mostly conserved amino acids Ala-84, Leu-91, Tyr-93, Tyr-220, and Leu-344 in BTH.

Amino Acid Sequence↗
PubMed2004

A new type of antimicrobial protein with multiple histidines from the hard tick, Amblyomma hebraeum.

A novel 11 kDa antimicrobial protein, named as hebraein, and having a unique amino acid sequence, was purified from the hemolymph of fed female Amblyomma hebraeum ticks. A full-length cDNA clone encoding hebraein was isolated from a cDNA library made from tick synganglia. Hebraein consists of 102 amino acids, including 6 cysteine residues; has 9 histidines in its C-terminal domain that are mainly present as HX repeats; and has no significant similarity to any known protein. The secondary structure prediction is very clearly all alpha-helical (4-6 helices) except for a very short extension at the C terminus. Such high alpha-helical content is quite different from known antimicrobial proteins. Recombinant hebraein and a mutant lacking the histidine residues in the C-terminal domain were constructed and expressed. Assayed at the slightly acidic pH equivalent of fed female tick hemolymph, the wild-type and the histidine-rich recombinant hebraein had stronger antimicrobial activities than the histidine-deficient mutant. The pH-dependent properties of histidine-rich antimicrobial proteins may allow the design of agents that would function selectively in specific pH environments. The results from protein profiling of hemolymph, analyzed by surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry combined with ProteinChip technology and RT-PCR analysis suggested that this antimicrobial protein was up-regulated by blood feeding. Our findings describe a new type of antimicrobial protein with multiple cysteine and histidine residues, and with unique secondary structure.

Amino Acid Sequence↗
PubMed2004

A distant evolutionary relationship between GPI-specific phospholipase D and bacterial phosphatidylcholine-preferring phospholipase C.

In eukaryotes some surface proteins are attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. A GPI-specific phospholipase D (GPI-PLD) activity has been characterized and implicated in the regulation of anchoring, thereby influencing the dispersal of anchored proteins or their maintenance on the cell surface, and possibly in cell signalling. Despite its biological and medical importance, little is known of the structure of GPI-PLD. Here, a distant relationship between the catalytic domains of GPI-PLD and some bacterial phospholipases C is demonstrated. A model of the GPI-PLD catalytic site sheds light on catalysis and highlights possibilities for design of improved and more specific GPI-PLD inhibitors. The databases contain hitherto unnoticed close homologues of GPI-PLD from yeast and Dictyostelium discoideum.

Amino Acid Sequence↗
PubMed2004

Engineering the pH-dependence of kinetic parameters of maize glutathione S-transferase I by site-directed mutagenesis.

The optimisation of enzymes for particular application or conditions remains an important target in all protein engineering endeavours. Here, we report a successful strategy for altering the pH-profile of kinetic parameters and to define in detail the molecular mechanism of maize glutathione S-transferase I (GST I). To accomplish this, selected residues from the glutathione binding site (His40, Ser11, Lys41, Asn49, Gln53 and Ser67) were mutated to Ala, and the pH-dependence of the catalytic parameters V(max), and V(max)/K(GSH)(m) of the mutated forms were analysed. The pH-dependence of V(max) for the wild-type enzyme exhibits two transitions in the acidic pH range with pK(a1) of 5.7 and pK(a2) of 6.6. Based on thermodynamic data, site-directed mutagenesis and UV deference spectroscopy, it was concluded that pK(a1) corresponds to GSH carboxylates, whereas the pK(a2) has a conformational origin of the protein. The pH-dependence of V(max)/K(GSH)(m) for the wild-type enzyme exhibits a single transition with pK(a) of 6.28 which was attributed to the thiol ionisation of bound GSH. These findings complement the conclusions about the catalytic mechanism deduced from the crystal structure of the enzyme and provide the basis for rationally designing engineered forms of GST I with valuable properties.

Amino Acid Substitution↗
PubMed2004

A diverse family of serine proteinase genes expressed in cotton boll weevil (Anthonomus grandis): implications for the design of pest-resistant transgenic cotton plants.

Fourteen different cDNA fragments encoding serine proteinases were isolated by reverse transcription-PCR from cotton boll weevil (Anthonomus grandis) larvae. A large diversity between the sequences was observed, with a mean pairwise identity of 22% in the amino acid sequence. The cDNAs encompassed 11 trypsin-like sequences classifiable into three families and three chymotrypsin-like sequences belonging to a single family. Using a combination of 5' and 3' RACE, the full-length sequence was obtained for five of the cDNAs, named Agser2, Agser5, Agser6, Agser10 and Agser21. The encoded proteins included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Southern blotting analysis suggested that one or two copies of these serine proteinase genes exist in the A. grandis genome. Northern blotting analysis of Agser2 and Agser5 showed that for both genes, expression is induced upon feeding and is concentrated in the gut of larvae and adult insects. Reverse northern analysis of the 14 cDNA fragments showed that only two trypsin-like and two chymotrypsin-like were expressed at detectable levels. Under the effect of the serine proteinase inhibitors soybean Kunitz trypsin inhibitor and black-eyed pea trypsin/chymotrypsin inhibitor, expression of one of the trypsin-like sequences was upregulated while expression of the two chymotrypsin-like sequences was downregulated.

Amino Acid Sequence↗
PubMed2004

Sequence analysis and characterization of a novel fibronectin-binding repeat domain from the surface of Streptococcus pneumoniae.

Streptococcus pneumoniae open reading frame SP0082 encodes a surface protein that contains four copies of a novel conserved repeat domain that bears no significant sequence similarity to proteins of known function. Homologous sequences from other streptococci contain two to six of these repeats, designated the SSURE (streptococcal surface repeat) domain. To investigate the functional role(s) of this domain, the third SSURE repeat of SP0082 sequence has been expressed in Escherichia coli, purified to homogeneity and characterized by biochemical and immunological methods. The expressed protein fragment was found to bind to fibronectin, but not to collagen or submaxillary mucin. Anti-SSURE antibodies recognized the corresponding protein on the surface of pneumococcal cells. These data identify S. pneumoniae SP0082 protein and its homologs in other streptococci as fibronectin-binding surface adhesins. The SSURE domain is likely to contain a novel protein fold, which was tentatively modeled using ab initio modeling methods.

Adhesins, Bacterial↗
PubMed2004

The structure-function relationship in the clostripain family of peptidases.

In this study we investigate the active-site structure and the catalytic mechanism of clostripain by using a combination of three separate techniques: affinity labelling, site-directed mutagenesis and molecular modelling. A benzamidinyl-diazo dichlorotriazine dye (BDD) was shown to act as an efficient active site-directed affinity label for Clostridium histolyticum clostripain. The enzyme, upon incubation with BDD in 0.1 m Hepes/NaOH buffer pH 7.6, exhibits a time-dependent loss of activity. The rate of inactivation exhibits a nonlinear dependence on the BDD concentration, which can be described by reversible binding of dye to the enzyme prior to the irreversible reaction. The dissociation constant of the reversible formation of an enzyme-BDD complex is KD = 74.6 +/- 2.1 micro m and the maximal rate constant of inactivation is k3 = 0.21 x min(-1). Effective protection against inactivation by BDD is provided by the substrate N-benzoyl-L-arginine ethyl ester (BAEE). Cleavage of BDD-modified enzyme with trypsin and subsequent separation of peptides by reverse-phase HPLC gave only one modified peptide. Amino acid sequencing of the modified tryptic peptide revealed the target site of BDD reaction to be His176. Site-directed mutagenesis was used to study further the functional role of His176. The mutant His176Ala enzyme exhibited zero activity against BAEE. Together with previous data, these results confirm that a catalytic dyad of His176 and Cys231 is responsible for cysteine peptidase activity in the C11 peptidase family. A molecular model of the catalytic domain of clostripain was constructed using a manually extended fold recognition-derived alignment with caspases. A rigorous iterative modelling scheme resulted in an objectively sound model which points to Asp229 as responsible for defining the strong substrate specificity for Arg at the P1 position. Two possible binding sites for the calcium required for auto-activation could be located. Database searches show that clostripain homologues are not confined to bacterial lineages and reveal an intriguing variety of domain architectures.

Affinity Labels↗
PubMed2003

Structures of Streptococcus pneumoniae hyaluronate lyase in complex with chondroitin and chondroitin sulfate disaccharides. Insights into specificity and mechanism of action.

Streptococcus pneumoniae hyaluronate lyase is a surface enzyme of this Gram-positive bacterium. The enzyme degrades hyaluronan and chondroitin/chondroitin sulfates by cleaving the beta1,4-glycosidic linkage between the glycan units of these polymeric substrates. This degradation helps spreading of this bacterial organism throughout the host tissues and facilitates the disease process caused by pneumococci. The mechanism of this degradative process is based on beta-elimination, is termed proton acceptance and donation, and involves selected residues of a well defined catalytic site of the enzyme. The degradation of hyaluronan alone is thought to proceed through a processive mode of action. The structures of complexes between the enzyme and chondroitin as well as chondroitin sulfate disaccharides allowed for the first detailed insights into these interactions and the mechanism of action on chondroitins. This degradation of chondroitin/chondroitin sulfates is nonprocessive and is selective for the chondroitin sulfates only with certain sulfation patterns. Chondroitin sulfation at the 4-position on the nonreducing site of the linkage to be cleaved or 2-sulfation prevent degradation due to steric clashes with the enzyme. Evolutionary studies suggest that hyaluronate lyases evolved from chondroitin lyases and still retained chondroitin/chondroitin sulfate degradation abilities while being specialized in the degradation of hyaluronan. The more efficient processive degradation mechanism has come to be preferred for the unsulfated substrate hyaluronan.

Binding Sites↗
PubMed2003

Use of phage display to select novel cystatins specific for Acanthoscelides obtectus cysteine proteinases.

Cysteine proteinases from larvae of the common bean weevil, Acanthoscelides obtectus (Coleoptera: Bruchidae), were isolated by ion exchange affinity chromatography on a CM-Cellulose column and used to select mutant cystatins from a library made with the filamentous M13 phage display system. The library contained variant cystatins derived from the nematode Onchocerca volvulus cystatin through mutagenesis of loop 1, which contains the QVVAG motif that is involved in binding to proteinases. After three rounds of selection, the activity of variant cystatins against papain and cysteine proteinases from A. obtectus was assayed by ELISA. Two different variant cystatins (presenting amino acids DVVSA and NTSSA at positions 65-69) bound to A. obtectus cysteine proteinases more tightly than to papain. In contrast, the wild type had similar affinity for A. obtectus proteinases and for papain. These two selected variants cystatins have greater specificity towards A. obtectus cysteine proteinases than the original sequence and could represent good candidate genes for the production of transgenic plants resistant to this insect pest.

Animals↗
PubMed2003

The crystal structure of Trypanosoma brucei enolase: visualisation of the inhibitory metal binding site III and potential as target for selective, irreversible inhibition.

The glycolytic enzymes of the trypanosomatids, that cause a variety of medically and agriculturally important diseases, are validated targets for drug design. Design of species-specific inhibitors is facilitated by the availability of structural data. Irreversible inhibitors, that bound covalently to the parasite enzyme alone, would be potentially particularly effective. Here we determine the crystal structure of enolase from Trypanosoma brucei and show that two cysteine residues, located in a water-filled cavity near the active-site, are modified by iodoacetamide leading to loss of catalytic activity. Since these residues are specific to the Trypanosomatidae lineage, this finding opens the way for the development of parasite-specific, irreversibly binding enolase inhibitors. In the present structure, the catalytic site is partially occupied by sulphate and two zinc ions. Surprisingly, one of these zinc ions illustrates the existence of a novel enolase-binding site for divalent metals. Evidence suggests that this is the first direct visualization of the elusive inhibitory metal site, whose existence has hitherto only been inferred from kinetic data.

Amino Acid Sequence↗
PubMed2003

Genome-based identification of a carbohydrate binding module in Streptococcus pneumoniae hyaluronate lyase.

Hyaluronate lyase enzymes degrade hyaluronan, the main polysaccharide component of the connective tissues of higher animals, thereby destroying the normal connective tissue structure and exposing the host tissue cells to various endo- and exogenous factors, including bacterial toxins. The 3D crystal structures of functionally active but truncated Streptococcus pneumoniae and S. agalactiae hyaluronate lyases, along with their substrate and product complexes, have been determined. The enzymes are multidomain proteins with helical barrel-like catalytic domains and two types of beta-sheet domains. Here, through genome-based bioinformatics studies we identify an additional beta-sheet domain present in the most N-terminal part of streptococcal hyaluronate lyases. Fold recognition and modeling studies show that the domain is structurally similar to carbohydrate binding modules and is therefore likely to be directly involved in hyaluronan binding. Likely carbohydrate binding residues were identified and electrostatic complementarity of the hyaluronate lyase domain with hyaluronan demonstrated. The newly identified presumed hyaluronan binding domain likely improves catalytic efficiency by colocalizing the enzyme and its substrate. Other possible functions are discussed. Two contacting aromatic residues are conserved in the hydrophobic core of the hyaluronate lyase domain and in many, perhaps all, families in the superfamily in which they may be placed. This observation may help the identification and classification of other carbohydrate binding modules.

Amino Acid Sequence↗
PubMed2003

Fold recognition analysis of glycosyltransferase families: further members of structural superfamilies.

Glycosyltransferases (GTs) are diverse enzymes organized into 65 families. X-ray crystallography and in silico studies have shown many of these to belong to two structural superfamilies: GT-A and GT-B. Through application of fold recognition and iterated sequence searches, we demonstrate that families 60, 62, and 64 may also be grouped into the GT-A fold superfamily. Analysis of conserved acidic residues suggests that catalytic sites are better conserved in superfamily GT-B than in GT-A. Although 26% and 29% of GT families may now be confidently placed in superfamilies GT-A and GT-B, respectively, the remaining 45% of families bear no discernible resemblance to either superfamily, which, given the sensitivity of modern fold recognition methods, suggests the existence of novel structural scaffolds associated with GT activity. Furthermore, bioinformatics studies indicate the apparent ease with which mechanism-inverting or retaining-may change during evolution.

Amino Acids↗
PubMed2003

Identification and analysis of catalytic TIM barrel domains in seven further glycoside hydrolase families.

Fold recognition results allocate catalytic triose phosphate isomerase (TIM) barrels to seven previously unassigned glycoside hydrolase (GH) families, numbers 29, 44, 50, 71, 84, 85 and 89, enabling prediction of catalytic residues. Modelling of GH family 50 suggests that it may be the common evolutionary ancestor of families 42 and 14. TIM barrels now comprise the catalytic domains of more than half of the assigned GH families, and catalyse a much larger variety of GH reactions than any other catalytic domain architecture. Only 327 GH sequences still have no structurally identified catalytic domain.

Amino Acid Sequence↗
PubMed2003

Insights into the catalytic mechanism of cofactor-independent phosphoglycerate mutase from X-ray crystallography, simulated dynamics and molecular modeling.

Phosphoglycerate mutases catalyze the isomerization of 2 and 3-phosphoglycerates, and are essential for glucose metabolism in most organisms. Here, we further characterize the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGM) from Bacillus stearothermophilus by determination of a high-resolution (1.4A) crystal structure of the wild-type enzyme and the crystal structure of its S62A mutant. The mutant structure surprisingly showed the replacement of one of the two catalytically essential manganese ions with a water molecule, offering an additional possible explanation for its lack of catalytic activity. Crystal structures invariably show substrate phosphoglycerate to be entirely buried in a deep cleft between the two iPGM domains. Flexibility analyses were therefore employed to reveal the likely route of substrate access to the catalytic site through an aperture created in the enzyme's surface during certain stages of the catalytic process. Several conserved residues lining this aperture may contribute to orientation of the substrate as it enters. Factors responsible for the retention of glycerate within the phosphoenzyme structure in the proposed mechanism are identified by molecular modeling of the glycerate complex of the phosphoenzyme. Taken together, these results allow for a better understanding of the mechanism of action of iPGMs. Many of the results are relevant to a series of evolutionarily related enzymes. These studies will facilitate the development of iPGM inhibitors which, due to the demonstrated importance of this enzyme in many bacteria, would be of great potential clinical significance.

Alanine↗
PubMed2003

An extracellular calcium-binding domain in bacteria with a distant relationship to EF-hands.

Extracellular Ca(2+)-dependent nuclease YokF from Bacillus subtilis and several other surface-exposed proteins from diverse bacteria are encoded in the genomes in two paralogous forms that differ by a approximately 45 amino acid fragment, which comprises a novel conserved domain. Sequence analysis of this domain revealed a conserved DxDxDGxxCE motif, which is strikingly similar to the Ca(2+)-binding loop of the calmodulin-like EF-hand domains, suggesting an evolutionary relationship between them. Functions of many of the other proteins in which the novel domain, named Excalibur (extracellular calcium-binding region), is found, as well as a structural model of its conserved motif are consistent with the notion that the Excalibur domain binds calcium. This domain is but one more example of the diversity of structural contexts surrounding the EF-hand-like calcium-binding loop in bacteria. This loop is thus more widespread than hitherto recognized and the evolution of EF-hand-like domains is probably more complex than previously appreciated.

Amino Acid Sequence↗