Structure of a cephalosporin synthase.

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

Methods for isolation of marine-derived endophytic fungi and their bioactive secondary products

Marine-derived fungi have been shown in recent years to produce a plethora of new bioactive secondary metabolites, some of them featuring new carbon frameworks hitherto unprecedented in nature. These compounds are of interest as new lead structures for medicine as well as for plant protection. The aim of this protocol is to give a detailed description of methods useful for the isolation and cultivation of fungi associated with various marine organisms (sponges, algae and mangrove plants) for the extraction, characterization and structure elucidation of biologically active secondary metabolites produced by these marine-derived endophytic fungi, and for the preliminary evaluation of their pharmacological properties based on rapid 'in house' screening systems. Some results exemplifying the positive outcomes of the protocol are given at the end. From sampling in marine environment to completion of the structure elucidation and bioactivity screening, a period of at least 3 months has to be scheduled

Deconstructing the Chlamydial Cell Wall

The evolutionary separated Gram-negative Chlamydiales show a biphasic life cycle and replicate exclusively within eukaryotic host cells. Members of the genus Chlamydia are responsible for many acute and chronic diseases in humans, and Chlamydia-related bacteria are emerging pathogens. We revisit past efforts to detect cell wall material in Chlamydia and Chlamydia-related bacteria in the context of recent breakthroughs in elucidating the underlying cellular and molecular mechanisms of the chlamydial cell wall biosynthesis. In this review, we also discuss the role of cell wall biosynthesis in chlamydial FtsZ-independent cell division and immune modulation. In the past, penicillin susceptibility of an invisible wall was referred to as the "chlamydial anomaly." In light of new mechanistic insights, chlamydiae may now emerge as model systems to understand how a minimal and modified cell wall biosynthetic machine supports bacterial cell division and how cell wall-targeting beta-lactam antibiotics can also act bacteriostatically rather than bactericidal. On the heels of these discussions, we also delve into the effects of other cell wall antibiotics in individual chlamydial lineages