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Probing the penicillin sidechain selectivity of recombinant deacetoxycephalosporin C synthase

  • A. Dubus
  • M.D. Lloyd
  • H.-J. Lee
  • C.J. Schofield
  • J.E. Baldwin
  • J.-M. Frère

Abstract.

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.

Key words. Cephem antibiotic biosynthesis; enzyme inactivation; iron(II); 2-oxoglutarate-dependent oxygenase; spectrophotometric assay. 

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Copyright information

© Birkhäuser Verlag, 2001

Authors and Affiliations

  • A. Dubus
    • 1
  • M.D. Lloyd
    • 2
  • H.-J. Lee
    • 2
  • C.J. Schofield
    • 2
  • J.E. Baldwin
    • 2
  • J.-M. Frère
    • 1
  1. 1.Centre d'Ingéniere des Protéines, Université de Liège, Institut de Chimie B6, 4000 Liège (Belgium), Fax + 32 4 366 3364, e-mail: a.dubus@ulg.ac.be BE
  2. 2.The Oxford Centre for Molecular Sciences and The Dyson Perrins Laboratory, South Parks Road, Oxford OX1 3QY (UK)GB

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