Trapping the Complex Molecular Machinery of Polyketide and Fatty Acid Synthases with Tunable Silylcyanohydrin Crosslinkers

Abstract

Many families of natural products are synthesized by large multidomain biological machines commonly referred to as megasynthases.W hile the advance of mechanism-based tools has opened new windows into the structural features within the protein-protein interfaces guiding carrier protein dependent enzymes,t here is an immediate need for tools that can be engaged to link co-translated domains in asite-selective manner.N ow,t he use of silylcyanohydrins is demonstrated in at wo-step,t wo-site selective crosslinking for the trapping of carrier-protein interactions within megasynthases.T his advance provides an ew tool to trap intermediate states within multimodular systems,akey step towardunderstanding the specificities within fatty acid (FAS) and polyketide (PKS) synthases.

Fluorometric Analysis of Carrier-Protein-Dependent Biosynthesis through a Conformationally Sensitive Solvatochromic Pantetheinamide Probe

“…The reaction was diluted with EtOAc (1 × 40 mL) and washed with saturated aqueous NaHCO 3 ( Proteins. Polyhistidine-tagged proteins AcpP, 33 hACP, 15 DEBS Acp4, 43 actACP C17S, 34 ΔEntB, 44 PltL, 36 and PltF 38 were expressed and purified based on previously described methods. AcpP, ΔEntB (C-terminal CP domain of EntB), PltL, and PltF were cloned in pET-22 vectors (Amp R ) with C-terminal hexahistidine tags.…”

Curiouser and curiouser: progress in understanding the programming of iterative highly-reducing polyketide synthases

“…The lack of the ACP (and its downstream thiolesterase [TE] domain) in the vFAS crystal structure was attributed to its likely conformational mobility. 24 A second structure, apparently of a FAS/PKS involved in mycocerosic acid biosynthesis in Mycobacterium smegmatis shows a highly similar domain organisation, 26 although because it lacks any kind of C -MeT domain it is a relatively poor model of the fungal hr-PKS.…”

Electron cryomicroscopy observation of acyl carrier protein translocation in type I fungal fatty acid synthase

“…In type II bacterial FAS, where ACP is only composed of the canonical lobe, substrate loading state can modulate the affinity profile of a panel of ACP mutants toward KS protein and active-site specific cross-linkers have proven to be valuable tools to trap transiently formed ACP complexes with other catalytic proteins such as ER and dehydratase (DH). [9][10][11][12] We therefore hypothesized that the loadingstate of ACP may modulate its interaction with the catalytic centers in the context of fully assembled type I fungal FAS and allow for redistribution of ACP toward a specific reaction site. Both endogenously purified enzymes are catalytically active (i.e.…”