Publications

• Kinetic and crystallographic studies on deacetoxycephalosporin C synthase (DAOCS).

  Lee H.J., Lloyd M.D., Harlos K., Clifton I.J., Baldwin J.E., Schofield C.J.

  Deacetoxycephalosporin C synthase (DAOCS) is an iron(II) and 2-oxoglutarate-dependent oxygenase that catalyzes the conversion of penicillin N to deacetoxycephalosporin C, the committed step in the biosynthesis of cephalosporin antibiotics. The crystal structure of DAOCS revealed that the C terminu ... >> More

  Deacetoxycephalosporin C synthase (DAOCS) is an iron(II) and 2-oxoglutarate-dependent oxygenase that catalyzes the conversion of penicillin N to deacetoxycephalosporin C, the committed step in the biosynthesis of cephalosporin antibiotics. The crystal structure of DAOCS revealed that the C terminus of one molecule is inserted into the active site of its neighbor in a cyclical fashion within a trimeric unit. This arrangement has hindered the generation of crystalline enzyme-substrate complexes. Therefore, we constructed a series of DAOCS mutants with modified C termini. Oxidation of 2-oxoglutarate was significantly uncoupled from oxidation of the penicillin substrate in certain truncated mutants. The extent of uncoupling varied with the number of residues deleted and the penicillin substrate used. Crystal structures were determined for the DeltaR306 mutant complexed with iron(II) and 2-oxoglutarate (to 2.10 A) and the DeltaR306A mutant complexed with iron(II), succinate and unhydrated carbon dioxide (to 1.96 A). The latter may mimic a product complex, and supports proposals for a metal-bound CO(2) intermediate during catalysis. << Less

  J Mol Biol 308:937-948(2001) [PubMed] [EuropePMC]

• Purification and properties of deacetoxycephalosporin C synthase from recombinant Escherichia coli and its comparison with the native enzyme purified from Streptomyces clavuligerus.

  Dotzlaf J.E., Yeh W.K.

  A putatively rate-limiting synthase (expandase) of Streptomyces clavuligerus was stabilized in vitro and purified 46-fold from cell-free extracts; a major enriched protein with a Mr of 35,000 was further purified by electrophoretic elution. Based on a 22-residue amino-terminal sequence of the prot ... >> More

  A putatively rate-limiting synthase (expandase) of Streptomyces clavuligerus was stabilized in vitro and purified 46-fold from cell-free extracts; a major enriched protein with a Mr of 35,000 was further purified by electrophoretic elution. Based on a 22-residue amino-terminal sequence of the protein, the synthase gene of S. clavuligerus was cloned and expressed in Escherichia coli (Kovacevic, S., Weigel, B.J., Tobin, M.B., Ingolia, T.D., and Miller, J. R. (1989) J. Bacteriol. 171, 754-760). The synthase protein was detected mainly from granules of recombinant E. coli. The recombinant synthase was solubilized from the granules by urea, and for the first time a highly active synthase was purified to near homogeneity. The synthase was a monomer with a Mr of 34,600 and exhibited two isoelectric points of 6.1 and 5.3. Its catalytic activity required alpha-ketoglutarate, Fe2+, and O2, was stimulated by dithiothreitol or ascorbate but not by ATP, and was optimal at pH 7.0 in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer and at 36 degrees C. The Fe2+ requirement was specific, and at least one sulfhydryl group in the purified enzyme was apparently essential for the ring expansion. The Km values of the enzyme for penicillin N and alpha-ketoglutarate were 29 and 18 microM, respectively, and the Ka for Fe2+ was 8 microM. The recombinant synthase was indistinguishable from the native synthase of S. clavuligerus by those biochemical properties. In addition to the enzymic ring expansion of penicillin N to deacetoxycephalosporin C, the recombinant synthase catalyzed a novel hydroxylation of 3-exomethylenecephalosporin C to deacetylcephalosporin C. << Less

  J Biol Chem 264:10219-10227(1989) [PubMed] [EuropePMC]

• Isolation of deacetoxycephalosporin C from fermentation broths of Penicillium chrysogenum transformants: construction of a new fungal biosynthetic pathway.

  Cantwell C., Beckmann R., Whiteman P., Queener S.W., Abraham E.P.

  Deacetoxycephalosporin C (DAOC), a precursor of cephalosporins excreted by Cephalosporium and Streptomyces species, has been produced in Penicillium chrysogenum transformed with DNA containing a hybrid penicillin N expandase gene (cefEh) and a hybrid isopenicillin N epimerase gene (cefDh). DAOC fr ... >> More

  Deacetoxycephalosporin C (DAOC), a precursor of cephalosporins excreted by Cephalosporium and Streptomyces species, has been produced in Penicillium chrysogenum transformed with DNA containing a hybrid penicillin N expandase gene (cefEh) and a hybrid isopenicillin N epimerase gene (cefDh). DAOC from a P. chrysogenum transformant was identified by ultraviolet light (UV), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) and mass spectrum analyses. P. chrysogenum transformed with DNA containing cefEh without cefDh did not produce DAOC. Untransformed P. chrysogenum produced penicillin V (phenoxymethylpenicillin) but not DAOC. Transformants also produced penicillin V but, in general, less than untransformed P. chrysogenum. The cefEh and cefDh genes were constructed by replacing the open reading frame (ORF) of cloned P. chrysogenum pcbC and penDE genes with the ORF of the Streptomyces clavuligerus expandase gene, cefE, and the ORF of the Streptomyces lipmanii epimerase gene, cefD, respectively. Analyses of representative transformants suggested that production of DAOC occurred via cefEh and cefDh genes stably integrated in the P. chrysogenum genome. DNA from untransformed P. chrysogenum did not hybridize to cefE or cefD gene probes. << Less

  Proc Biol Sci 248:283-289(1992) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.