Understanding the role of active site residues in CotB2 catalysis using a cluster model

Terpene cyclases are responsible for the initial cyclization cascade in the multistep synthesis of a large number of terpenes. CotB2 is a diterpene cyclase from Streptomyces melanosporofaciens, which catalyzes the formation of cycloocta-9-en-7-ol, a precursor to the next-generation anti-inflammatory drug cyclooctatin. In this work, we present evidence for the significant role of the active site's residues in CotB2 on the reaction energetics using quantum mechanical calculations in an active site cluster model. The results revealed the significant effect of the active site residues on the relative electronic energy of the intermediates and transition state structures with respect to gas phase data. A detailed understanding of the role of the enzyme environment on the CotB2 reaction cascade can provide important information towards a biosynthetic strategy for cyclooctatin and the biomanufacturing of related terpene structures.

Current understanding and biotechnological application of the bacterial diterpene synthase CotB2

CotB2 catalyzes the first committed step in cyclooctatin biosynthesis of the soil bacterium Streptomyces melanosporofaciens. To date, CotB2 represents the best studied bacterial diterpene synthase. Its reaction mechanism has been addressed by isoptope labeling, targeted mutagenesis and theoretical computations in the gas phase, as well as full enzyme molecular dynamic simulations. By X-ray crystallography different snapshots of CotB2 from the open, inactive, to the closed, active conformation have been obtained in great detail, allowing us to draw detailed conclusions regarding the catalytic mechanism at the molecular level. Moreover, numerous alternative geranylgeranyl diphosphate cyclization products obtained by CotB2 mutagenesis have exciting applications for the sustainable production of high value bioactive substances.

The first structure of a bacterial diterpene cyclase: CotB2

Sesquiterpenes and diterpenes are a diverse class of secondary metabolites that are predominantly derived from plants and some prokaryotes. The properties of these natural products encompass antitumor, antibiotic and even insecticidal activities. Therefore, they are interesting commercial targets for the chemical and pharmaceutical industries. Owing to their structural complexity, these compounds are more efficiently accessed by metabolic engineering of microbial systems than by chemical synthesis. This work presents the first crystal structure of a bacterial diterpene cyclase, CotB2 from the soil bacteriumStreptomyces melanosporofaciens, at 1.64 Å resolution. CotB2 is a diterpene cyclase that catalyzes the cyclization of the linear geranylgeranyl diphosphate to the tricyclic cyclooctat-9-en-7-ol. The subsequent oxidation of cyclooctat-9-en-7-ol by two cytochrome P450 monooxygenases leads to bioactive cyclooctatin. Plasticity residues that decorate the active site of CotB2 have been mutated, resulting in alternative monocyclic, dicyclic and tricyclic compounds that show bioactivity. These new compounds shed new light on diterpene cyclase reaction mechanisms. Furthermore, the product of mutant CotB2W288Gproduced the new antibiotic compound (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene, which acts specifically against multidrug-resistantStaphylococcus aureus. This opens a sustainable route for the industrial-scale production of this bioactive compound.

Foliar application of elicitors alters isoflavone concentrations and other seed characteristics of field-grown soybean

Soybean [Glycine max (L.) Merr.] is a key species used by the nutraceutical industry, as it contains isoflavones that have beneficial effects on human health. A 2-yr field study was conducted in Sainte-Anne-de-Bellevue, QC, to determine the effects of foliar application of elicitor compounds on isoflavone concentrations of seeds, seed yield, and other important seed characteristics. Two soybean cultivars (AC Proteina and AC Orford) were treated with one of four elicitors: lipo-chitooligosaccharides (LCO) Bj V (C18:1 MeFuc) and Bj V (Ac, C16, MeFuc), chitosan, and actinomycetes spores (Streptomyces melanosporofaciens strain EF-76), each applied at two soybean stages of development [vegetative (V4), and early podding (R3)]. Untreated controls were also included. Total and individual isoflavone concentrations were determined by HPLC. Seed yield, 100-seed weight, seeds per pod, pods per plant and oil and crude protein concentrations were concurrently determined. Total and individual isoflavone concentrations were affected by year and cultivar, which also interacted with elicitors. Interactions indicated that plant response to elicitors varied greatly depending on cultivars and years. Compared with an untreated control, some elicitors resulted in total isoflavone concentration increases ranging between 67 and 87%. No single elicitor treatment, however, consistently increased isoflavone concentrations. There were three-way interactions (year, cultivar and elicitors) for seed yield and number of pods per plants indicating, again, substantial variation in response to elicitors. Response was much greater in 2004, where elicitors consistently increased seed yield and number of pods per plant. Elicitors did not affect crude protein and oil concentrations. The use of elicitor compounds seems promising as a means of increasing isoflavone concentrations and seed yield; evaluations in a greater number of environments are now required. Key words: Soybean, isoflavone, daidzein, genistein, glycitein, protein, oil, seed yield, chitosan, actinomycetes, lipo-chitooligosaccharides

Microscopic Examination of Chitosan–Polyphosphate Beads with Entrapped Spores of the Biocontrol Agent,Streptomyces melanosporofaciensEF-76

Spores of the biocontrol agent,Streptomyces melanosporofaciensEF-76, were entrapped by complex coacervation in beads composed of a macromolecular complex (MC) of chitosan and polyphosphate. A proportion of spores entrapped in beads survived the entrapment procedure as shown by treating spores from chitosan beads with a dye allowing the differentiation of live and dead cells. The spore-loaded chitosan beads could be digested by a chitosanase, suggesting that, once introduced in soil, the beads would be degraded to release the biocontrol agent. Spore-loaded beads were examined by optical and scanning electron microscopy because the release of the biological agent depends on the spore distribution in the chitosan beads. The microscopic examination revealed that the beads had a porous surface and contained a network of inner microfibrils. Spores were entrapped in both the chitosan microfibrils and the bead lacuna.