ABSTRACT
Most small GTPases actuate their functions on subcellular membranes, which are increasingly seen as integral components of small GTPase signalling. In this review, we used the highly studied regulation of Arf GTPases by their GEFs to categorize the molecular principles of membrane contributions to small GTPase signalling, which have been highlighted by integrated structural biology combining in vitro reconstitutions in artificial membranes and high-resolution structures. As an illustration of how this framework can be harnessed to better understand the cooperation between small GTPases, their regulators and membranes, we applied it to the activation of the small GTPase Rac1 by DOCK-ELMO, identifying novel contributions of membranes to Rac1 activation. We propose that these structure-based principles should be considered when interrogating the mechanisms whereby small GTPase systems ensure spatial and temporal control of cellular signalling on membranes.
Subject(s)
Monomeric GTP-Binding Proteins , Signal Transduction , Cell Membrane , Membranes, ArtificialABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Prenylated indole diketopiperazine (DKP) alkaloids are important bioactive molecules or their precursors. In the context of synthetic biology, efficient means for their biological production would increase their chemical diversification and the discovery of novel bioactive compounds. Here, we prove the suitability of the Escherichia coli chassis for the production of prenylated indole DKP alkaloids. We used enzyme combinations not found in nature by co-expressing bacterial cyclodipeptide synthases (CDPSs) that assemble the DKP ring and fungal prenyltransferases (PTs) that transfer the allylic moiety from the dimethylallyl diphosphate (DMAPP) to the indole ring of tryptophanyl-containing cyclodipeptides. Of the 11 tested combinations, seven resulted in the production of eight different prenylated indole DKP alkaloids as determined by LC-MS/MS and NMR characterization. Two were previously undescribed. Engineering E. coli by introducing a hybrid mevalonate pathway for increasing intracellular DMAPP levels improved prenylated indole DKP alkaloid production. Purified product yields of 2-26 mg/L per culture were obtained from culture supernatants. Our study paves the way for the bioproduction of novel prenylated indole DKP alkaloids in a tractable chassis that can exploit the cyclodipeptide diversity achievable with CDPSs and the numerous described PT activities.
