Studies on the active site of deacetoxycephalosporin C synthase

The Fe(II) and 2-oxogolutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25% of total soluble protein in Escherichia coli and purified by an efficient l arge-scale procedure. Purified protein catalysed the conversions of penicil lins N and G to deacetoxycephems. Gel filtration and Light scattering studi es showed that in solution monomeric apo-DAOCS is in equilibrium with a tri meric form from which it crystallizes. DAOCS was crystallized +/-Fe(II) and /or 2-oxooglutarate using the hanging drop method. Crystals diffracted to b eyond 1.3 Angstrom resolution and belonged to the R3 space group (unit cell dimensions: a = b = 106.4 Angstrom, c = 71.2 Angstrom; alpha = beta = 90 d egrees gamma = 120 degrees (in the hexagonal setting)). Despite the structu re revealing that Met180 is located close to the reactive oxidizing centre of DAOCS, there was no functional difference between the wild-type and sele nomethionine derivatives. X-ray absorption spectroscopic studies in solutio n generally supported the iron co-ordination chemistry defined by the cryst al structures. The Fe K-edge positions of 7121.2 and 7121.4 eV for DAOCS al one and with 2-oxoglutarate were both consistent with the presence of Fe(II ). For Fe(II) in DAOCS the best fit to the Extended X-ray Absorption Fine S tructure (EXAFS) associated with the Fe K-edge was found with two His imida zolate groups at 1.96 Angstrom, three nitrogen or oxygen atoms at 2.11 Angs trom and one other light atom at 2.04 Angstrom. For the Fe(II) in the DAOCS -2-oxoglutarate complex the EXAFS spectrum was successfully interpreted by backscattering from two His residues (Fe-nr at 1.99 Angstrom), a bidentate O,O-co-ordinated 2-oxoglutarate with Fe-O distances of 2.08 Angstrom, anoth er O atom at 2.08 Angstrom and one at 2.03 Angstrom. Analysis of the X-ray crystal structural data suggests a binding mode for the penicillin N substr ate and possible roles for the C terminus in stabilising the enzyme and ord ering the reaction mechanism. (C) 1999 Academic Press.