RadH: A Versatile Halogenase for Integration into Synthetic Pathways.

Flavin-dependent halogenases are useful enzymes for providing halogenated molecules with improved biological activity, or intermediates for synthetic derivatization. We demonstrate how the fungal halogenase RadH can be used to regioselectively halogenate a range of bioactive aromatic scaffolds. Site-directed mutagenesis of RadH was used to identify catalytic residues and provide insight into the mechanism of fungal halogenases. A high-throughput fluorescence screen was also developed, which enabled a RadH mutant to be evolved with improved properties. Finally we demonstrate how biosynthetic genes from fungi, bacteria, and plants can be combined to encode a new pathway to generate a novel chlorinated coumarin "non-natural" product in E. coli.

Structure and biocatalytic scope of thermophilic flavin-dependent halogenase and flavin reductase enzymes.

Flavin-dependent halogenase (Fl-Hal) enzymes have been shown to halogenate a range of synthetic as well as natural aromatic compounds. The exquisite regioselectively of Fl-Hal enzymes can provide halogenated building blocks which are inaccessible using standard halogenation chemistries. Consequently, Fl-Hal are potentially useful biocatalysts for the chemoenzymatic synthesis of pharmaceuticals and other valuable products, which are derived from haloaromatic precursors. However, the application of Fl-Hal enzymes, in vitro, has been hampered by their poor catalytic activity and lack of stability. To overcome these issues, we identified a thermophilic tryptophan halogenase (Th-Hal), which has significantly improved catalytic activity and stability, compared with other Fl-Hal characterised to date. When used in combination with a thermostable flavin reductase, Th-Hal can efficiently halogenate a number of aromatic substrates. X-ray crystal structures of Th-Hal, and the reductase partner (Th-Fre), provide insights into the factors that contribute to enzyme stability, which could guide the discovery and engineering of more robust and productive halogenase biocatalysts.

A fitness assay for comparing RNAi effects across multiple C. elegans genotypes.

BACKGROUND: RNAi technology by feeding of E. coli containing dsRNA in C. elegans has significantly contributed to further our understanding of many different fields, including genetics, molecular biology, developmental biology and functional genomics. Most of this research has been carried out in a single genotype or genetic background. However, RNAi effects in one genotype do not reveal the allelic effects that segregate in natural populations and contribute to phenotypic variation. RESULTS: Here we present a method that allows for rapidly comparing RNAi effects among diverse genotypes at an improved high throughput rate. It is based on assessing the fitness of a population of worms by measuring the rate at which E. coli is consumed. Critically, we demonstrate the analytical power of this method by QTL mapping the loss of RNAi sensitivity (in the germline) in a recombinant inbred population derived from a cross between Bristol and a natural isolate from Hawaii. Hawaii has lost RNAi sensitivity in the germline. We found that polymorphisms in ppw-1 contribute to this loss of RNAi sensitivity, but that other loci are also likely to be important. CONCLUSIONS: In summary, we have established a fast method that improves the throughput of RNAi in liquid, that generates quantitative data, that is easy to implement in most laboratories, and importantly that enables QTL mapping using RNAi.

Characterization of imine reductases in reductive amination for the exploration of structure-activity relationships.

Imine reductases (IREDs) have shown great potential as catalysts for the asymmetric synthesis of industrially relevant chiral amines, but a limited understanding of sequence activity relationships makes rational engineering challenging. Here, we describe the characterization of 80 putative and 15 previously described IREDs across 10 different transformations and confirm that reductive amination catalysis is not limited to any particular subgroup or sequence motif. Furthermore, we have identified another dehydrogenase subgroup with chemoselectivity for imine reduction. Enantioselectivities were determined for the reduction of the model substrate 2-phenylpiperideine, and the effect of changing the reaction conditions was also studied for the reductive aminations of 1-indanone, acetophenone, and 4-methoxyphenylacetone. We have performed sequence-structure analysis to help explain clusters in activity across a phylogenetic tree and to inform rational engineering, which, in one case, has conferred a change in chemoselectivity that had not been previously observed.

An RNAi-based dimorphic genetic screen identified the double bromodomain protein BET-1 as a sumo-dependent attenuator of RAS-mediated signalling.

Attenuation of RAS/RAF/MAPK signalling is essential to prevent hyperactivation of this oncogenic pathway. In C. elegans, the sumoylation pathway and a combination of histone tail modifications regulate gene expression to attenuate the LET-60 (RAS) signalling pathway. We hypothesised that a number of chromatin regulators are likely to depend on sumoylation to attenuate the pathway. To reveal these, we designed an RNAi-based dimorphic genetic screen that selects candidates based on their ability to act as enhancers of a sumo mutant phenotype, such interactions would suggest that the candidates may be physically associated with sumoylation. We found 16 enhancers, one of which BET-1, is a conserved double bromodomain containing protein. We further characterised BET-1 and showed that it can physically associate with SMO-1 and UBC-9, and that it can be sumoylated in vitro within the second bromodomain at lysine 252. Previous work has shown that BET-1 can bind acetyl-lysines on histone tails to influence gene expression. In conclusion, our screening approach has identified BET-1 as a Sumo-dependent attenuator of LET-60-mediated signalling and our characterisation suggests that BET-1 can be sumoylated.