1w2a: Difference between revisions

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 268-299) is rotated by about 16 degrees 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.

1W2A is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Streptomyces_clavuligerus Streptomyces clavuligerus] with FE and EDO as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Deacetoxycephalosporin-C_synthase Deacetoxycephalosporin-C synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.14.20.1 1.14.20.1] Structure known Active Site: AC1. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1W2A OCA].  

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