STRUCTURE OF A CEPHALOSPORIN SYNTHASE DETERMINED FROM MEROHEDRALLY TWINNED CRYSTALS

K. Valegardl, A.C. Terwisscha van Scheltinga2, M.D Lloyd3, T. Hara1, S. Ramaswamy2, A. Perrakis4, A. Thompson4, H.J. Lee3, J.E. Baldwin3, C.J. Schofield3, J. Hajdul, I. Andersson2

lDepartment of Biochemistry, Uppsala University, Box 576, S-75123 Uppsala, Sweden
2Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, Box 590, S-75124 Uppsala, Sweden
3Oxford Centre for Molecular Sciences and Dyson Perrins Laboratory, Oxford University, South Parks Road, Oxford OXI 3QY, U.K.
4 EMBL Grenoble, c/o ILL, Avenue des Martyrs, B.P.156, F-38042 Grenoble Cedex 9, France (5) ESRF, B. P. 220, F-38042 Grenoble Cedex, France

 

The key step in cephalosporin biosynthsesis is the expansion of the flve-membered ring of the penicillin nucleus into the six-membered ring of the cephalosporin nucleus [1,2). In prokaryotes this step is catalysed by the Fe(II)-dependent enzyme deacetoxycephalosporin C synthase, DAOCS. The enzyme uses dioxygen-derived ferryl intermediate in catalysis. The ferryl form of DAOCS is produced by the oxidative splitting of the cosubstrate, 2- oxoglutarate. This route of controlled ferryl formation and reaction is common to many mononuclear ferrous enzymes [3].

We present the first crystal structure of a 2-oxoacid dependent oxygenase. Structures for apo- DAOCS, the enzyme complexed with Fe(II), and with Fe(II) and 2-oxoglutarate are described at 1.3,1.5 and 1.5 A resolutions, respectively. Based on these structures we propose a mechanism for intermediate. the formation of the transient ferryl intermediate.

All crystals used were merohedral twins. This specific type of crystal twinning is caused by a rotation involving a symmetry element of the lattice, which causes the reciprocal lattices of the twins to coincide completely. The twinning ratio (deflned as the volume fraction of the smaller crystal in the twin) varies from crystal to crystal, from a neglible degree to almost equal fractions of the two individual crystals in the twin. In order to be able to use these data for MIR structure solution, a strategy was developed to determine the degree of twinning for each individual data set, and correct for that.

  1. Baldwin, J.E., Schofield, C. J. ( 1992) In The Chemistry of ß -lactams (Ed. Page, M.I.) Blackie, London, pp.l-78.
  2. Yoshida, M., Konomi, T., Kohsaka, M., Baldwin, J.E., Herchen, S., Singh, P., Hunt, N.A., Demain, A.L. ( 1978) Proc. Natl. Acad. Sci. USA, 75, 6253-6257.
  3. Hegg, E.L., Que, L. ( 1997) Eur. J. Biochem., 250, 625- 629.