No abstract

Penicillins and cephalosporins are among the most widely used therapeutic agents. These antibiotics are produced from fermentation-derived materials as their chemical synthesis is not commercially viable. Unconventional steps in their biosynthesis are catalysed by Fe(II)-dependent oxidases/oxygenases; isopenicillin N synthase (IPNS) creates in one step the bicyclic nucleus of penicillins, and deacetoxycephalosporin C synthase (DAOCS) catalyses the expansion of the penicillin nucleus into the nucleus of cephalosporins. Both enzymes use dioxygen-derived ferryl intermediates in catalysis but, in contrast to IPNS, the ferryl form of DAOCS is produced by the oxidative splitting of a co-substrate, 2-oxoglutarate (alpha-ketoglutarate). This route of controlled ferryl formation and reaction is common to many mononuclear ferrous enzymes, which participate in a broader range of reactions than their well-characterized counterparts, the haem enzymes. Here we report the first crystal structure of a 2-oxoacid-dependent oxygenase. High-resolution structures for apo-DAOCS, the enzyme complexed with Fe(II), and with Fe(II) and 2-oxoglutarate, were obtained from merohedrally twinned crystals. Using a model based on these structures, we propose a mechanism for ferryl formation.

The Fe(II) and 2-oxoglutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25 % of total soluble protein in Escherichia coli and puri®ed by an ef®cient large-scale procedure. Puri®ed protein catalysed the conversions of penicillins N and G to deacetoxycephems. Gel ®ltration and light scattering studies showed that in solution monomeric apo-DAOCS is in equilibrium with a trimeric form from which it crystallizes. DAOCS was crystallized AEFe(II) and/or 2-oxoglutarate using the hanging drop method. Crystals diffracted to beyond 1.3 A Ê resolution and belonged to the R3 space group (unit cell dimensions: a b 106.4 A Ê , c 71.2 A Ê ; a b 90 , g 120 (in the hexagonal setting)). Despite the structure revealing that Met180 is located close to the reactive oxidizing centre of DAOCS, there was no functional difference between the wild-type and selenomethionine derivatives. X-ray absorption spectroscopic studies in solution generally supported the iron co-ordination chemistry de®ned by the crystal structures. The Fe K-edge positions of 7121.2 and 7121.4 eV for DAOCS alone and with 2-oxoglutarate were both consistent with the presence of Fe(II). For Fe(II) in DAOCS the best ®t to the Extended X-ray Absorption Fine Structure (EXAFS) associated with the Fe K-edge was found with two His imidazolate groups at 1.96 A Ê , three nitrogen or oxygen atoms at 2.11 A Ê and one other light atom at 2.04 A Ê . For the Fe(II) in the DAOCS-2-oxoglutarate complex the EXAFS spectrum was successfully interpreted by backscattering from two His residues (Fe-N at 1.99 A Ê ), a bidentate O,O-co-ordinated 2-oxoglutarate with Fe-O distances of 2.08 A Ê , another O atom at 2.08 A Ê and one at 2.03 A Ê . Analysis of the X-ray crystal structural data suggests a binding mode for the penicillin N substrate and possible roles for the C terminus in stabilising the enzyme and ordering the reaction mechanism.

Plastic deformation processes in oriented polyethylene are investigated by the X-ray method, and changes in texture produced by transverse compression are interpreted in terms of twinning, slip and phase transformation. The crystal structure of the new form produced by the phase transformation is determined, and the unit cell is a monoclinic one with a=8.09, b=2.53, c=4.79 Å and β=107.9°. The phase transformation is considered to be of diffusion-less type and a transition mechanism similar to that of twinning is proposed.

Interleukin-18 (IL-18), a cytokine formerly known as interferon-gamma- (IFN-gamma-) inducing factor, has pleiotropic immunoregulatory functions, including augmentation of IFN-gamma production, Fas-mediated cytotoxicity and developmental regulation of T-lymphocyte helper type I. We determined the solution structure of IL-18 as a first step toward understanding its receptor activation mechanism. It folds into a beta-trefoil structure that resembles that of IL-1. Extensive mutagenesis revealed the presence of three sites that are important for receptor activation: two serve as binding sites for IL-18 receptor alpha (IL-18Ralpha), located at positions similar to those of IL-1 for IL-1 receptor type I (IL-1RI), whereas the third site may be involved in IL-18 receptor beta (IL-18Rbeta) binding. The structure and mutagenesis data provide a basis for understanding the IL-18-induced heterodimerization of receptor subunits, which is necessary for receptor activation.