Oxidative C-H activation for C-C bond formation: Characterization of EasC/EasE and comparison to CnsA/CnsD

A crucial step in ergot alkaloid biosynthesis is the carbon-carbon (C-C) bond formation. In synthetic organic chemistry, this step remains still challenging and therefore, time consuming and expensive. Hence, special emphasis has been placed on the field of enzyme catalyzed C-C bond formation in the last two decades. To investigate this ring closure mechanism, EasE – a flavin adenine dinucleotide (FAD) dependent homologue of the berberine bridge enzyme-like (BBE) enzyme - and EasC – a heme dependent catalase - originating from Aspergillus japonicus should be produced and investigated. Further, CnsA (BBE-like) and CnsD (catalase), which represent a similar model in Penicillium expansum, will be investigated.
For a long time, the interactions of EasC and/or EasE to form chanoclavine I from N-Me-DMAT as well as the interplay between CnsA and/or CnsD to form aurantioclavine from DMAT, were unclear. Just recently, this mystery has been solved1,2. Interestingly, the respective homologues from Aspergillus japonicus and Penicillium expansum catalyze different reactions2, which makes further investigations all the more important.
Heterologous expression of His-tagged easC and cnsD in E. coli BL21-CodonPlus(DE3)-RIL and E. coli BL21 Star(DE3) lead to soluble and active proteins. However, gene expression, purification and storage of the concentrated proteins should further be optimized in terms of stability and activity for subsequent protein crystallization. The flavoproteins EasE and CnsA turned out to be challenging to produce in various expression systems. However, soluble flavoproteins could be produced in the TNT® T7 Insect Cell Extract Protein Expression System (Promega). To get considerable amounts of soluble protein, expression of His-tagged easE and cnsA will be screened in a cell based Sf21 insect cell expression system in order to investigate these highly interesting proteins and their exceptional function in alkaloid formation.