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C J Schofield

Publications and source records attributed to C J Schofield.

At least 19 recordsLinked to original sources
PubMed1995

Delta-L-(alpha-aminoadipoyl)-L-cysteinyl-D-valine synthetase: isolation of L-cysteinyl-D-valine, a 'shunt' product, and implications for the order of peptide bond formation.

L-Cysteinyl-D-valine was isolated from incubations of L-glutamate, L-cysteine and L-valine with delta-L-(alpha-aminoadipoyl)-L-cysteinyl-D-valine synthetase and identified by 1H NMR and electrospray ionization MS. This is entirely consistent with our prior proposal (Shiau, C.-Y., Baldwin, J.E., Byford, M.F., Sobey, W.J. and Schofield, C.J. (1995) FEBS Lett. 358, 97-100) that the alpha-peptide bond between cysteine and valine is formed before the delta-peptide bond between alpha-aminoadipate and cysteine. The inclusion of L-glutamate, an analogue of L-alpha-aminoadipate, did not result in a detectable amount of tripeptide product, but did increase apparent yields of L-cysteinyl-D-valine. Conceivably, formation of the L-glutamyladenylate stimulates synthesis of the cysteinyl-valine dipeptide indirectly via a conformational change in the enzyme.

Acremonium↗
PubMed1995

Crystal structure of isopenicillin N synthase is the first from a new structural family of enzymes.

Penicillin antibiotics are all produced from fermentation-derived penicillins because their chemical synthesis is not commercially viable. The key step in penicillin biosynthesis, in which both the beta-lactam and thiazolidine rings of the nucleus are created, is mediated by isopenicillin N synthase (IPNS), which binds ferrous iron and uses dioxygen as a cosubstrate. In a unique enzymatic step, with no chemical precedent, IPNS catalyses the transfer of four hydrogen atoms from its tripeptide substrate to dioxygen forming, in a single reaction, the complete bicyclic nucleus of the penicillins. We now report the structure of IPNS complexed with manganese, which reveals the active site is unusually buried within a 'jelly-roll' motif and lined by hydrophobic residues, and suggest how this structure permits the process of penicillin formation. Sequence analyses indicate IPNS, 1-aminocyclopropane-1-carboxylic acid oxidase and many of the 2-oxo-acid-dependent oxygenases contain a conserved jelly-roll motif, forming a new structural family of enzymes.

Acremonium↗
PubMed1995

Expression, purification and characterization of 1-aminocyclopropane-1-carboxylate oxidase from tomato in Escherichia coli.

1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyses the final step in the biosynthesis of the plant hormone ethylene. The successful overexpression and characterization of active ACC oxidase from tomato has been achieved. PCR was used to insert the corrected cDNA coding for the tomato ACC oxidase into the pET-11a expression vector. Cloning of the resultant construct in Escherichia coli BL21(DE3)pLysE gave transformants which expressed ACC oxidase at levels greater than 30% of soluble protein under optimized conditions. When induced by addition of isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37 degrees C the ACC oxidase expressed was less soluble and less active than when induced at 27 degrees C. The enzyme was purified to near homogeneity by a three-step chromatographic procedure. The specific activity of the purified recombinant ACC oxidase was typically 1.3-1.9 mol of ethylene/mol of enzyme per min, higher than values reported for native enzyme. Like the native enzyme it displayed a requirement for ferrous iron and ascorbate, and CO2 was an activator. The ability to discriminate between racemic diastereomers of 1-amino-2-ethyl cyclopropane-1-carboxylic acid was demonstrated. The enzyme was found to have a loose specificity for ascorbate, showing apparent preference for D-ascorbate and 5,6-O-isopropylidene L-ascorbate rather than L-ascorbate. The addition of catalase, dithiothreitol and BSA to incubation mixtures all resulted in significant increases in activity. When treated with diethylpyrocarbonate (DEPC) under mildly acidic conditions, the enzyme rapidly lost activity. Comparison of the rate of inactivation with the increase in absorbance at 240 nm gave results consistent with the modification of two to three histidine residues at the active site, although the possibility of additional modification of other nucleophilic residues cannot be excluded. Inactivation was largely prevented by the addition of substrates and ferrous iron, implying that DEPC treatment results in the modification of active-site histidines, which act as ligands for ferrous iron. CO2 offered no protection against DEPC inactivation, either in the absence or presence of substrates and/or ferrous iron.

Amino Acid Oxidoreductases↗
PubMed1995

delta-L-(alpha-aminoadipoyl)-L-cysteinyl-D-valine synthetase: the order of peptide bond formation and timing of the epimerisation reaction.

delta-L-(alpha-Aminoadipoyl)-L-cysteinyl-D-valine (ACV) synthetase catalyses the formation of the common precursor tripeptide of both the penicillin and cephalosporin antibiotics from the L-enantiomers of its constituent amino acids. Replacement of cysteine with L-O-methylserine in preparative-scale incubations led to the isolation of both L-O-methylserinyl-L-valine and L-O-methylserinyl-D-valine dipeptides. The dipeptides were characterized with the aid of authentic synthetic standards by both 1H NMR and electrospray ionization MS. A revised mechanism for ACV biosynthesis involving formation of the cysteinyl-valine peptide bond before the epimerisation of valine and subsequent condensation with the delta-carboxyl of L-alpha-aminoadipate is therefore proposed.

Amino Acid Sequence↗
PubMed1995

Crystallization and preliminary X-ray diffraction studies on recombinant isopenicillin N synthase from Aspergillus nidulans.

Recombinant Aspergillus nidulans isopenicillin N synthase was purified from an Escherichia coli expression system. The apoenzyme in the presence of saturating concentrations of MnCl2 could be crystallized by either macro- or microseeding, using the hanging drop vapor diffusion technique with polyethylene glycol 8000 as precipitant. The crystals (0.5-1.0 mm overall dimensions) diffract X-rays to at least 2.0 A resolution at synchrotrons and belong to space group P212121 with unit cell dimensions of a = 59.2 A, b = 127.0 A, and c = 139.6 A. The asymmetric unit contains one dimer, and the solvent content of the crystals is 60%. The crystals are radiation sensitive.

Aspergillus nidulans↗
PubMed1994

Crystallization and preliminary X-ray diffraction studies on a recombinant isopenicillin N synthase from Cephalosporium acremonium.

Recombinant isopenicillin N synthase from Cephalosporium acremonium was expressed in Escherichia coli and the protein was purified. After nearly 5000 crystallization trials, the apo enzyme was crystallized by the hanging drop vapour diffusion technique, using polyethylene glycol and lithium sulphate as precipitants. Two crystal forms have been obtained with either octahedral or elongated prismatic habits. The larger octahedral crystals (0.1 mm over-all dimensions) belong to space group I4 with unit cell dimensions of a = b = 124.7 A, c = 156.9 A, and diffract X-rays to about 3.5 A resolution at synchrotrons. The crystallographic asymmetric unit contains a dimer.

Acremonium↗
PubMed1994

Substrate specificity of L-delta-(alpha-aminoadipoyl)-L-cysteinyl-D-valine synthetase from Cephalosporium acremonium: demonstration of the structure of several unnatural tripeptide products.

Potential substrates for L-delta-(alpha-aminoadipoyl)-L-(cysteinyl)-D-valine (ACV) synthetase were initially identified using both the amino-acid-dependent ATP<-->pyrophosphate exchange reaction catalysed by the enzyme and the incorporation of 14C-radiolabelled cysteine and valine into potential peptide products. S-Carboxymethylcysteine was an effective substitute for alpha-aminoadipate and both allylglycine and vinylglycine could substitute for cysteine, indicating that the thiol group of cysteine is not essential for peptide formation. L-allo-Isoleucine but not L-isoleucine substituted effectively for valine. The structures of the presumed peptide products derived from these amino acids were confirmed by combined use of electrospray-ionization m.s. (e.s.m.s.) and 1H n.m.r. These results clearly indicate that, in common with other peptide synthetases, but in contrast with ribosomal peptide synthesis, ACV synthetase has a relatively broad substrate specificity.

2-Aminoadipic Acid↗
PubMed1992

The role of temperature and nutritional status in flight initiation by Triatoma infestans.

Flight initiation in Triatoma infestans is associated with low nutritional status and increases with rising temperature; it appears to be largely independent of bug age and sex. A predictive model for the probability of flight initiation was constructed based on weight:length ratios of the bugs and maximum ambient temperature, both of which can be ascertained in the field. The model accurately predicted the proportion of bugs initiating flight in > 85% of the groups used in our study. The predictive equation was found to give significant fits with two independent data sets. From our results it might be expected that flight would be rare during colder (< 20 degrees C) months but that 5-10% of the normal population of an infested house would fly on any given night during the hotter months when temperatures approach 30 degrees C. If bug nutritional status falls significantly, this proportion could be expected to rise to 30%.

Animal Nutritional Physiological Phenomena↗
PubMed1992

Dispersive flight by Triatoma infestans under natural climatic conditions in Argentina.

Male and female T. infestans were released on two consecutive nights in the salinas of Cordoba Province, Argentina, when air temperatures during the flying period averaged 28.5 degrees C. 136 males (43% of the total released) and 170 females (57%) flew on the first night, and 6 males (18%) and 7 females (27%) on the second. Of these, we recovered 23 males and 14 females within 100 m, and a further female within 200 m, all from the first night's release. The remainder appear to have flown more than 100 m and possibly more than 200 m. In support of this conclusion 4 male and 5 female bugs were recovered in an abandoned brick house 550 m from the release point. Since the proportion of bugs apparently flying more than 200 m is considerably greater than that deduced from previous experiments at lower temperatures (Lehane & Schofield, 1981) it is possible that temperature influences not only the proportion of bugs flying but also the distance flown. Flight appears to be affected by wind speed. On the first night, when there was negligible wind, 43% of male and 57% of female bugs flew. On the second night, with winds gusting at 4-5 m/s, only 18% of the male and 27% of the female bugs flew.

Animals↗
PubMed1991

Isolation and partial characterisation of ACV synthetase from Cephalosporium acremonium and Streptomyces clavuligerus. Evidence for the presence of phosphopantothenate in ACV synthetase.

delta-(L-alpha-Aminoadipoyl)-L-cysteinyl-D-valine (ACV) synthetase was isolated and partially characterised from Cephalosporium acremonium CO728 and Streptomyces clavuligerus. The purification procedure resulted in a 745- and 277-fold increase in specific enzyme activity, respectively. Both enzymes had similar apparent molecular masses of ca. 300 kdaltons by SDS-polyacrylamide electrophoresis, under reducing and denaturing conditions, and in excess of 600 kdaltons in the native state by gel filtration. Attempts to obtain an N-terminal amino acid sequence of ACV synthetase from C. acremonium were unsuccessful, hence internal amino acid sequence data were obtained after tryptic digestion of the protein. Phosphopantothenic acid was shown to be associated with the enzyme from both sources, which suggests the possible involvement of pantothenate as a 'swinging arm' in the formation of the tripeptide ACV.

Acremonium↗
PubMed1991

Vector population responses to control interventions.

In 1981, Kenyan authorities attempted eradication of tsetse (Glossina pallidipes) from the Lambwe valley, by sequential aerial spraying of endosulfan. Fly populations were reduced by over 99.9% in the main habitats, but recovered to their original levels within one year. In 1986, an Argentine research team attempted local elimination of domestic Triatominae (Triatoma infestans) in an area of Santiago del Estero. House infestation rates were reduced to an apparent zero, but recovered to pre-control levels in two years. In Sri Lanka, a combination of mosquito control with active case detection and treatment reduced malaria incidence to just 17 cases in 1964. Interruption of the programme then saw case incidence return to over 500,000 by 1969. These three examples-from African trypanosomiasis, South American Chagas disease and malaria-all illustrate the same process. Vector populations, infestation rates, and rates of disease transmission, can be reduced. The real problem is not in achieving the initial reduction, but in subsequently either driving the disease to extinction or maintaining it below acceptable levels. And this problem takes us beyond the simple analysis of interventions, into the complex realms of population behaviour set in its political, social and economic context. It seems that we have been too rarely concerned to plan and implement the long term surveillance and selective interventions that are required to translate initial gains into real success. In biological terms, we tend to ignore the complexity and robustness of vector populations and disease transmission cycles, especially their varied capacity to recover from interference. Moreover, in socio-political terms, a reduced problem-even one representing potential for future catastrophe-tends to claim less priority than present problems, even though premature redeployment of resources may abnegate gains already achieved. The problem is strongly compounded by methods of economic analysis where the benefits of avoiding future problems are strongly discounted in favour of short-term temporary gains. This paper seeks to open discussion on these long-term planning issues.

Animals↗
PubMed1990

Molecular weight analysis of isopenicillin N synthase by electrospray mass spectrometry.

The use of electrospray mass spectrometry as a tool in analytical biochemistry was illustrated by determination of the molecular weights of wildtype and recombinant isopenicillin N synthase (IPNS). The molecular weight of recombinant IPNS produced using an expression system which generated soluble protein was found to be between 38,364 and 38,376 Da, ca 60 mass units higher than that of the wildtype material, consistent with the presence of an additional N-terminal glycine in the former. Observed molecular weights were all ca 70 Da higher than that calculated from sequence information, consistent with the complexion of a partially hydrated iron atom to the enzyme during analysis.

Escherichia coli↗
PubMed1990

Acyl coenzyme A: 6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum and Aspergillus nidulans.

A study of the final stages of the biosynthesis of the penicillins in Penicillium chrysogenum has revealed two types of enzyme. One hydrolyses phenoxymethyl penicillin to 6-aminopenicillanic acid (6-APA). The other, also obtained from Aspergillus nidulans, transfers a phenylacetyl group from phenylacetyl CoA to 6-APA. The acyltransferase, purified to apparent homogeneity, had a molecular mass of 40 kDa. It also catalyses the conversion of isopenicillin N (IPN) to benzylpenicillin (Pen G) and hydrolyses IPN to 6-APA. In the presence of SDS it dissociates, with loss of activity, into fragments of ca 30 and 10.5 kDa, but activity is regained when these fragments recombine in the absence of SDS.

Acyl Coenzyme A↗
PubMed1990

High level expression in Escherichia coli of a fungal gene under the control of strong promoters.

A recent report (Patino et al., (1989) FEMS Microbiol. Lett. 58, 139-144) described the low level expression, in Escherichia coli, of the Isopenicillin N Synthase (IPNS) gene from Cephalosporium acremonium under the control of strong promoters. We report here our work on the expression of the IPNS gene. Plasmids containing the IPNS gene under the control of the trp or trc promoters directed synthesis of high levels of active IPNS in E. coli. Constitutive and inductive high level IPNS expression systems have been developed. Importantly, the expression vectors do not encode beta-lactamase so IPNS activity can be determined directly by biological assays. Analysis by nmr verified that the IPNS produced from these expression systems catalysed the conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (LLD-ACV) to isopenicillin N in high yield.

Acremonium↗