Abstract

Deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus catalyses the oxidative ring expansion of the penicillin nucleus into the nucleus of cephalosporins. The reaction requires dioxygen and 2-oxoglutarate as co-substrates to create a reactive iron-oxygen intermediate from a ferrous iron in the active site. The active enzyme is monomeric in solution. The structure of DAOCS was determined earlier from merohedrally twinned crystals where the last four C-terminal residues (308-311) of one molecule penetrate the active site of a neighbouring molecule, creating a cyclic trimeric structure in the crystal. Shortening the polypeptide chain from the C terminus by more than four residues diminishes activity. Here, we describe a new crystal form of DAOCS in which trimer formation is broken and the C-terminal arm is free. These crystals show no signs of twinning, and were obtained from DAOCS labelled with an N-terminal His-tag. The modified DAOCS is catalytically active. The free C-terminal arm protrudes into the solvent, and the C-terminal domain (residues 268299) is rotated by about 16degrees towards the active site. The last 12 residues (300-311) are disordered. Structures for various enzyme-substrate and enzyme-product complexes in the new crystal form confirm overlapping binding sites for penicillin and 2-oxoglutarate. The results support the notion that 2-oxoglutarate and dioxygen need to react first to produce an oxidizing iron species, followed by reaction with the penicillin substrate. The position of the penicillin nucleus is topologically similar in the two crystal forms, but the penicillin side-chain in the new non-twinned crystals overlaps with the position of residues 304-306 of the C-terminal arm in the twinned crystals. An analysis of the interactions between the C-terminal region and residues in the active site indicates that DAOCS could also accept polypeptide chains as ligands, and these could bind near the iron. (C) 2004 Elsevier Ltd. All rights reserved

Published in

Journal of Molecular Biology
2004, Volume: 343, number: 1, pages: 157-171
Publisher: ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD

SLU Authors

• Öster, Linda

  • Department of Molecular Biology, Swedish University of Agricultural Sciences
    

• Terwisscha Van Scheltinga, Anke

  • Department of Molecular Biology, Swedish University of Agricultural Sciences
    

• Valegård, Karin

  • Department of Molecular Biology, Swedish University of Agricultural Sciences
    

• Andersson, Inger

  • Department of Molecular Biology, Swedish University of Agricultural Sciences
    

Publication Identifiers

DOI: https://doi.org/10.1016/j.jmb.2004.07.049

Permanent link to this page (URI)

https://res.slu.se/id/publ/4805