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C J Schofield

Publications and source records attributed to C J Schofield.

At least 37 records · Page 2Linked to original sources
PubMed2001

Alteration of the co-substrate selectivity of deacetoxycephalosporin C synthase. The role of arginine 258.

Deacetoxycephalosporin C synthase is an iron(II) 2-oxoglutaratedependent oxygenase that catalyzes the oxidative ring-expansion of penicillin N to deacetoxycephalosporin C. The wild-type enzyme is only able to efficiently utilize 2-oxoglutarate and 2-oxoadipate as a 2-oxoacid co-substrate. Mutation of arginine 258, the side chain of which forms an electrostatic interaction with the 5-carboxylate of the 2-oxoglutarate co-substrate, to a glutamine residue reduced activity to about 5% of the wild-type enzyme with 2-oxoglutarate. However, other aliphatic 2-oxoacids, which were not co-substrates for the wild-type enzyme, were utilized by the R258Q mutant. These 2-oxoacids "rescued" catalytic activity to the level observed for the wild-type enzyme as judged by penicillin N and G conversion. These co-substrates underwent oxidative decarboxylation as observed for 2-oxoglutarate in the normal reaction with the wild-type enzyme. Crystal structures of the iron(II)- 2-oxo-3-methylbutanoate (1.5 A), and iron(II)-2-oxo-4-methylpentanoate (1.6 A) enzyme complexes were obtained, which reveal the molecular basis for this "chemical co-substrate rescue" and help to rationalize the co-substrate selectivity of 2-oxoglutaratedependent oxygenases.

Intramolecular Transferases↗
PubMed2001

The ITS-2 of the nuclear rDNA as a molecular marker for populations, species, and phylogenetic relationships in Triatominae (Hemiptera: Reduviidae), vectors of Chagas disease.

The nucleotide sequences of the rDNA second internal transcribed spacer (ITS-2) of 31 populations of 12 and 3 species of the two main Triatominae tribes Triatomini and Rhodniini, including the most important Chagas disease vectors, were obtained. Sequence comparisons and parsimony, distance, and maximum-likelihood analyses indicate that ITS-2 is a useful marker for resolving supraspecific, specific, subspecific, and even sometimes population-level relationships in Triatominae. Results were markedly different between species of Triatomini and Rhodniini, suggesting polyphyly. Phylogenetic trees support an old divergence between South American and North-Central American Triatomini and query the validity of some genera (Dipetalogaster, Psammolestes). The very low sequence variation between species of the phyllosoma complex suggests that subspecific ranking would be more appropriate. Triatoma dimidiata proves to be a clearly differentiated species, with several populations evidencing a clinal variation along a north-south axis and a population from Yucatan showing differences consistent with specific status.

Animals↗
PubMed2001

Probing the penicillin sidechain selectivity of recombinant deacetoxycephalosporin C synthase.

Deacetoxycephalosporin C synthase from Streptomyces clavuligerus catalyses the conversion of the five-membered penicillin ring to the unsaturated six-membered cephem ring of deacetoxycephalosporin C. The effects on enzyme activity of the penicillin substrate sidechain and various cofactors were investigated using a continuous spectrophotometric assay. The conversion of penicillin G to phenylacetyl-7-aminodeacetoxycephalo sporanic acid (G-7-ADCA) was confirmed, and further details of the reaction were elucidated. The conversion of ampicillin to cephalexin was faster than that of acetyl-6-APA to acetyl-7-ADCA kcat = 0.120 +/- 0.001 s(-1) versus 0.035 +/- 0.001 s(-1), but they had similar Km values: 4.86 +/- 0.12 and 3.28 +/- 0.26 mM, respectively. Amoxycillin and penicillin V were also converted at low levels. Conversion was not detected for penicillanate, 6-aminopenicillanate, carbenicillin, temocillin, ticarcillin or benzylpenicilloic acid, suggesting that the enzyme has a relatively strict selectivity for the sidechain of the penicillin substrate.

Amoxicillin↗
PubMed2001

The molecular evolution of Trypanosomatidae.

In the absence of a fossil record, theories relating to the evolution of protozoa have, for most of the twentieth century, been based on morphological and life cycle data despite their known limitations. However, recent advances in molecular methodology, notably the wide availability of accurate, automated DNA sequencing, have made it possible to deduce the evolutionary relationships of extant species from their genes. This paper focuses on new findings concerning the evolution of the Trypanosomatidae, based on the ever-expanding body of molecular data now available. Classically, the evolution of digenetic parasitism in kinetoplastids has centred around two opposing theories--invertebrate first or vertebrate first--depending on which was the original host of the monogenetic parasite. However, data supporting a close phylogenetic relationship between genera of monogenetic insect parasites and digenetic vertebrate parasites challenge the simplicity of these hypotheses and suggest that the transition may not have been a major evolutionary barrier. The implications of these observations for the evolution of parasitism within the group are discussed. Phylogenetic analysis of a diverse selection of trypanosomatid species suggests that the genus Trypanosoma is monophyletic and that the human parasites, T. brucei, T. cruzi and Leishmania spp., have fundamentally different patterns of evolution. T. brucei clusters with mammalian trypanosomes of African origin, suggesting an evolutionary history confined to Africa. T. cruzi shows association with trypanosomes from bats, T. rangeli, and trypanosomes from a range of South American mammals and an Australian kangaroo. The origins of most parasites within this clade lie in South America and Australia, suggesting an ancient southern super-continent origin for T. cruzi, possibly in marsupials. The divergence between the Leishmania and Trypanosoma lineages is also ancient. The topology of Leishmania phylogenies suggests an independent transition to digenetic parasitism, a neotropical origin and an early tertiary radiation of the parasite.

Animals↗
PubMed2001

Hydrocarbons of Rhodnius prolixus, a Chagas disease vector.

The surface hydrocarbons of the blood-sucking insect, Rhodnius prolixus, a major Chagas disease vector in Venezuela, Colombia and Central America, were characterized by capillary gas chromatography coupled to mass spectrometry (CGC-MS). A total of 54 single or multicomponent peaks of saturated, straight-chain and methyl-branched hydrocarbons were identified. Major n-alkanes were n-C27, n-C29, n-C31 and n-C33 hydrocarbons. In the branched fraction, methyl groups were at positions 3, 5, 7, 11, 13, 15 and 17- for monomethyl isomers, and separated by three or five methylene groups for the trimethyl or tetramethyl derivatives. For the higher molecular weight components of 37, 39 and 41 atoms in the carbon skeleton, the di-, tri- and tetramethyl branches were usually separated by three or five, and sometimes 7, 11 or 13, methylene groups. The internal hydrocarbon pool contained larger amounts of the higher molecular weight methyl-branched components. Qualitative differences among epicuticular and internal hydrocarbon compositions were detected, both in adult and nymphal stages. No significant sexual dimorphism was detected, but a significant shift in the major n-alkane components was evident from the nymphal to the adult stage, differing also in the relative amounts of the higher molecular weight methyl-branched chains. Comparison of the hydrocarbon components to that of other Chagas disease vectors is discussed.

Alkanes↗
PubMed2001

Genetic studies of Psammolestes tertius (hemiptera: reduviidae: triatominae) using male genital morphology, morphometry, isoenzymes, and random amplified polymorphic DNA.

Two Brazilian populations of Psammolestes tertius (Ceará and Minas Gerais) collected from thornbird nests (Furnariidae) were compared by male genital morphology, morphometry, isoenzymes, and random amplified polymorphic DNA (RAPD). Male genitalia showed no difference between the populations. In contrast, morphometry, isoenzyme, and RAPD clearly distinguished the two populations. Possible mechanisms of dispersal and the origin of Psammolestes are discussed.

Animals↗
PubMed2001

X-ray snapshots of serine protease catalysis reveal a tetrahedral intermediate.

Studies on the catalytic mechanism and inhibition of serine proteases are widely used as paradigms for teaching enzyme catalysis. Ground-breaking work on the structures of chymotrypsin and subtilisin led to the idea of a conserved catalytic triad formed by the active site Ser, His and Asp residues. An oxyanion hole, consisting of the peptide amide of the active site serine and a neighbouring glycine, was identified, and hydrogen bonding in the oxyanion hole was suggested to stabilize the two proposed tetrahedral intermediates on the catalytic pathway. Here we show electron density changes consistent with the formation of a tetrahedral intermediate during the hydrolysis of an acyl-enzyme complex formed between a natural heptapeptide and elastase. No electron density for an enzyme-product complex was observed. The structures also suggest a mechanism for the synchronization of hydrolysis and peptide release triggered by the conversion of the sp2 hybridized carbonyl carbon to an sp3 carbon in the tetrahedral intermediate. This affects the location of the peptide in the active site cleft, triggering the collapse of a hydrogen bonding network between the peptide and the beta-sheet of the active site.

Acylation↗
PubMed2001

Structure of proline 3-hydroxylase. Evolution of the family of 2-oxoglutarate dependent oxygenases.

Iron (II)/2-oxoglutarate (2-OG)-dependent oxygenases catalyse oxidative reactions in a range of metabolic processes including the hydroxylation of proline and lysine residues during the post-translational modification of collagen. 2-OG oxygenases commonly require ascorbate for full activity. In the vitamin C deficient disease, scurvy, reduced activity of 2-OG oxygenases results in impaired formation of collagen. Here we report the crystal structure of bacterial proline 3-hydroxylase from Streptomyces sp., an enzyme which hydroxylates proline at position 3, the first of a 2-OG oxygenase catalysing oxidation of a free alpha-amino acid. Structures were obtained for the enzyme in the absence of iron (to 2.3A resolution, R=20.2%, Rfree=25.3%) and that complexed to iron (II) (to 2.4A resolution, R=19.8%, Rfree=22.6%). The structure contains conserved motifs present in other 2-OG oxygenases including a 'jelly roll' beta strand core and residues binding iron and 2-oxoglutarate, consistent with divergent evolution within the extended family. The structure differs significantly from many other 2-OG oxygenases in possessing a discrete C-terminal helical domain. Analysis of the structure suggests a model for proline binding and a mechanism for uncoupling of proline and 2-OG turnover.

Amino Acid Sequence↗
PubMed2001

Population morphometric analysis of the tropicopolitan bug Triatoma rubrofasciata and relationships with old world species of Triatoma: evidence of New World ancestry.

Quantitative analysis of morphological characters of the head was used to reconstruct the evolutionary history of the tropicopolitan bug Triatoma rubrofasciata (De Geer) (Hemiptera: Reduviidae) and seven species of Old World Triatoma. Multivariate analysis demonstrates that T. rubrofasciata and the Old World species have a high degree of similarity with Nearctic Triatoma species, particularly T. rubida (Uhler). We interpret this to imply a common ancestry for these groups. Dissemination of T. rubrofasciata and subsequent derivation of the Old World species of Triatoma is deduced to have occurred over a period of not more than 350 years.

Animals↗
PubMed2001

Kinetic and crystallographic analysis of complexes formed between elastase and peptides from beta-casein.

Human beta-casomorphin-7 (NH2-Tyr-Pro-Phe-Val-Glu-Pro-Ile-CO2H) is a naturally occurring peptide inhibitor of elastase that has been shown to form an acyl-enzyme complex stable enough for X-ray crystallographic analysis at pH 5. To investigate the importance of the N-terminal residues of the beta-casomorphin-7 peptide for the inhibition of elastase, kinetic and crystallographic analyses were undertaken to identify the minimum number of residues required for effective formation of a stable complex between truncated beta-casomorphin-7 peptides and porcine pancreatic elastase (PPE). The results clearly demonstrate that significant inhibition of PPE can be effected by simple tri-, tetra-and pentapeptides terminating in a carboxylic acid. These results also suggest that in vivo regulation of protease activity could be mediated via short peptides as well as by proteins. Crystallographic analysis of the complex formed between N-acetyl-Val-Glu-Pro-Ile-CO2H and PPE at pH 5 (to 1.67 A resolution) revealed an active site water molecule in an analogous position to that observed in the PPE/beta-casomorphin-7 structure supportive of its assignment as the 'hydrolytic water' in the deacylation step of serine protease catalysis.

Animals↗
PubMed2001

Purification, crystallization and preliminary X-ray diffraction of anthocyanidin synthase from Arabidopsis thaliana.

Anthocyanidin synthase (ANS) from Arabidopsis thaliana is a non-haem iron(II)-dependent dioxygenase reported to catalyse the conversion of leucoanthocyanidins to anthocyanidins. Anthocyanidins are precursors of anthocyanins, which are a major family of pigments in higher plants. ANS was crystallized by the vapour-diffusion method using polyethylene glycol as a precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 61.0, b = 73.2, c = 87.0 A, and diffract to 2.4 A using Cu Kalpha radiation.

Arabidopsis↗
PubMed2000

Mutagenesis studies on the iron binding ligands of clavaminic acid synthase.

Mutagenesis studies on conserved histidine residues identified as possible metal binding ligands in clavaminic acid synthase isozyme 2 were consistent with His-145 and His-280 acting as iron ligands, in support of crystallographic and previous mutagenesis studies. Mutagenesis of the four cysteines and a glutamine residue, conserved in both clavaminic acid synthase isozymes 1 and 2, demonstrated that none of these residues is essential for activity.

Amino Acid Substitution↗
PubMed2000

Studies of isopenicillin N synthase enzymatic properties using a continuous spectrophotometric assay.

Isopenicillin N synthase (IPNS) from Aspergillus nidulans is a no-heme iron(II)-dependent oxygenase which catalyses, in a single reaction, the bicyclisation of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine into isopenicillin N, the precursor of all other penicillins, cephalosporins and cephamycins. The IPNS reaction can be followed directly and continuously by a new assay which monitors the absorbance increase at 235 nm characteristic of penicillin nucleus formation. Using this assay, the effects of influential factors affecting the in vitro IPNS enzymatic reaction were investigated. Even under optimal conditions, enzyme inactivation occurred during catalysis. Iron(II) depletion and product inhibition were not the cause of this phenomenon, the addition of antioxidants or reducing agents failed to slow down inactivation or reactivate the enzyme. Therefore, this phenomenon appears to be irreversible and is attributed to oxidative damage caused to the enzyme by reactive oxygen species generated in solution during catalysis. Nevertheless, the steady-state kinetic parameters for the IPNS reaction were determined.

Antioxidants↗
PubMed2000

'pH-jump' crystallographic analyses of gamma-lactam-porcine pancreatic elastase complexes.

beta-Lactams inhibit a range of enzymes via acylation of nucleophilic serine residues. Certain gamma-lactam analogues of monocyclic beta-lactams have also been shown to be reversible inhibitors of porcine pancreatic elastase (PPE), forming acyl-enzyme complexes that are stable with respect to hydrolysis. Crystallographic analysis at pH 5 of an acyl-enzyme complex formed with PPE and one of these inhibitors revealed the ester carbonyl located in the oxyanion hole in a similar conformation to that observed in the structure of a complex formed between a heptapeptide (beta-casomorphin-7) and PPE. Only weak electron density was observed for the His-57 side chain in its 'native' conformation. Instead, the His-57 side chain predominantly adopted a conformation rotated approx. 90 degrees from its normal position. PPE-gamma-lactam crystals were subjected to 'pH-jumps' by placing the crystals in a buffer of increased pH prior to freezing for data collection. The results indicate that the conformation of the gamma-lactam-derived acyl-enzyme species in the PPE active site is dependent on pH, a result having implications for the analysis of other serine protease-inhibitor structures at non-catalytic pH values. The results help to define the stereoelectronic relationship between the ester of the acyl-enzyme complex, the side chain of His-57 and the incoming nucleophile during the reversible (de)acylation steps, implying it is closely analogous to the hydrolytic deacylation step during catalytic peptide hydrolysis.

Animals↗
PubMed2000

Mass spectrometry reveals elastase inhibitors from the reactive centre loop of alpha1-antitrypsin.

Peptides derived from the reactive centre loop of alpha1-antitrypsin, a serpin, were screened as potential elastase inhibitors by mass spectrometry. An octapeptide, MFLEAIPM, formed a 'stable' ternary complex with porcine elastase: one MFLEAIPM molecule reacted covalently with loss of water, whilst an additional peptide was bound non-covalently. Kinetic analyses suggested that MFLEAIPM may act as an uncompetitive inhibitor and that the activity was associated with the four N-terminal residues.

Amino Acid Sequence↗
PubMed2000

Structural origins of the selectivity of the trifunctional oxygenase clavaminic acid synthase.

Clavaminate synthase (CAS), a remarkable Fe(II)/2-oxoglutarate oxygenase, catalyzes three separate oxidative reactions in the biosynthesis of clavulanic acid, a clinically used inhibitor of serine beta-lactamases. The first CAS-catalyzed step (hydroxylation) is separated from the latter two (oxidative cyclization/desaturation) by the action of an amidinohydrolase. Here, we describe crystal structures of CAS in complex with Fe(II), 2-oxoglutarate (2OG) and substrates (N-alpha-acetyl-L-arginine and proclavaminic acid). They reveal how CAS catalyzes formation of the clavam nucleus, via a process unprecedented in synthetic organic chemistry, and suggest how it discriminates between substrates and controls reaction of its highly reactive ferryl intermediate. The presence of an unpredicted jelly roll beta-barrel core in CAS implies divergent evolution within the family of 2OG and related oxygenases. Comparison with other non-heme oxidases/oxygenases reveals flexibility in the position which dioxygen ligates to the iron, in contrast to the analogous heme-using enzymes.

Arginine↗