ORCID Profile
0000-0003-2858-8929
Current Organisation
University of Oxford
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Publisher: Elsevier BV
Date: 09-2002
DOI: 10.1016/S0003-2697(02)00246-4
Abstract: A system has been developed for subjecting protein crystals to hyperbaric pressures of oxygen gas in order to promote enzymatic reaction. Crystals of an oxygenase or oxidase enzyme are grown anaerobically by hanging drop vapor diffusion, under crystallization conditions modified to eliminate combustible materials such as plastic coverslips and grease. The crystalline enzyme:substrate complex can then be exposed to oxygen gas at pressures up to 60 bar using a custom-built device or "bomb." In this way, reaction is initiated synchronously throughout the crystal and subsequent flash freezing allows the trapping of enzyme:product complexes in high occupancy. These complexes can then be structurally characterized by conventional monochromatic X-ray crystallography. The bomb is furnished from naval brass and lubricated with Fomblin RT15 perfluorinated polyether grease in order to ensure compatibility with the highly oxidizing environment.
Publisher: Elsevier BV
Date: 12-2013
Publisher: Elsevier BV
Date: 12-2001
DOI: 10.1016/S1074-5521(01)00090-4
Abstract: Isopenicillin N synthase (IPNS) catalyses formation of bicyclic isopenicillin N, precursor to all penicillin and cephalosporin antibiotics, from the linear tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine. IPNS is a non-haem iron(II)-dependent enzyme which utilises the full oxidising potential of molecular oxygen in catalysing the bicyclisation reaction. The reaction mechanism is believed to involve initial formation of the beta-lactam ring (via a thioaldehyde intermediate) to give an iron(IV)-oxo species, which then mediates closure of the 5-membered thiazolidine ring. Here we report experiments employing time-resolved crystallography to observe turnover of an isosteric substrate analogue designed to intercept the catalytic pathway at an early stage. Reaction in the crystalline enzyme-substrate complex was initiated by the application of high-pressure oxygen, and subsequent flash freezing allowed an oxygenated product to be trapped, bound at the iron centre. A mechanism for formation of the observed thiocarboxylate product is proposed. In the absence of its natural reaction partner (the N-H proton of the L-cysteinyl-D-valine amide bond), the proposed hydroperoxide intermediate appears to attack the putative thioaldehyde species directly. These results shed light on the events preceding beta-lactam closure in the IPNS reaction cycle, and enhance our understanding of the mechanism for reaction of the enzyme with its natural substrate.
Publisher: American Chemical Society (ACS)
Date: 31-03-2007
DOI: 10.1021/BI062314Q
Abstract: Isopenicillin N synthase (IPNS), a non-heme iron oxidase central to penicillin and cephalosporin biosynthesis, catalyzes an energetically demanding chemical transformation to produce isopenicillin N from the tripeptide delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-valine (ACV). We describe the synthesis of two cyclopropyl-containing tripeptide analogues, delta-(l-alpha-aminoadipoyl)-l-cysteinyl-beta-methyl-d-cyclopropylglycine and delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-cyclopropylglycine, designed as probes for the mechanism of IPNS. We have solved the X-ray crystal structures of these substrates in complex with IPNS and propose a revised mechanism for the IPNS-mediated turnover of these compounds. Relative to the previously determined IPNS-Fe(II)-ACV structure, key differences exist in substrate orientation and water occupancy, which allow for an explanation of the differences in reactivity of these substrates.
Publisher: Royal Society of Chemistry (RSC)
Date: 21-03-2003
DOI: 10.1039/B212270G
Abstract: Isopenicillin N synthase (IPNS) catalyses conversion of the linear tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N (IPN), the central step in biosynthesis of the beta-lactam antibiotics. The unsaturated substrate analogue delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-vinylglycine (ACvG) has previously been incubated with IPNS and single product was isolated, a 2-alpha-hydroxymethyl isopenicillin N (HMPen), formed via a monooxygenase mode of reactivity. ACvG has now been crystallised with IPNS and the structure of the anaerobic IPNS:Fe(II):ACvG complex determined to 1.15 A resolution. Furthermore, by exposing the anaerobically grown crystals to high-pressure oxygen gas, a structure corresponding to the bicyclic product HMPen has been obtained at 1.60 A resolution. In light of these and other IPNS structures, and recent developments with related dioxygenases, the [2 + 2] cycloaddition mechanism for HMPen formation from ACvG has been revised, and a stepwise radical mechanism is proposed. This revised mechanism remains consistent with the observed stereospecificity of the transformation, but fits better with apparent constraints on the coordination geometry around the active site iron atom.
Publisher: Elsevier BV
Date: 07-2002
Publisher: American Association for the Advancement of Science (AAAS)
Date: 30-08-2019
Abstract: Malaria elimination goals are constantly eroded by the challenge of emerging drug and insecticide resistance. Alam et al. have taken established drug targets—CLK protein kinases involved in regulation of RNA splicing—and investigated how inhibition of the parasite's enzymes blocks completion of its complex life cycle. They identified an inhibitor of the parasite's CLK protein kinase that was 100-fold less active against the most closely related human protein kinase and effective at clearing rodent malaria parasites. Not only does this compound halt the development of sexual stages but it also limits transmission to the mosquito vector of the parasite, a key requirement for malaria drugs. Science , this issue p. eaau1682
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Jon Elkins.