Biology subjects

Joppe, M.

Publications and source records attributed to Joppe, M..

4 recordsLinked to original sources

bioRxiv2021

Directed biosynthesis of fluorinated polyketides

Modification of polyketides with fluorine offers a promising approach to develop new pharmaceuticals. While synthetic chemical methods for site-specific incorporation of fluorine in complex molecules have improved in recent years, approaches for the direct biosynthetic fluorination of natural compounds are still rare. Herein, we present a broadly applicable approach for site-specific, biocatalytic derivatization of polyketides with fluorine. Specifically, we exchanged the native acyltransferase domain (AT) of a polyketide synthase (PKS), which acts as the gatekeeper for selection of extender units, with an evolutionarily related but substrate tolerant domain from metazoan type I fatty acid synthase (FAS). The resulting PKS/FAS hybrid can utilize fluoromalonyl coenzyme A and fluoromethylmalonyl coenzyme A for polyketide chain extension, introducing fluorine or fluoro-methyl disubstitutions in polyketide scaffolds. Addition of a fluorine atom is often a decisive factor toward developing superior properties in next-generation antibiotics, including the macrolide solithromycin. We demonstrate the feasibility of our approach in the semisynthesis of a fluorinated derivative of the macrolide antibiotic YC-17.

biochemistry↗

bioRxiv2019

The resolution revolution in cryoEM requires new sample preparation procedures: A rapid pipeline to high resolution maps of yeast FAS

Single-particle electron cryo-microscopy (cryoEM) has undergone a \"resolution revolution\" that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions or sample preparation methods can result in disassembly or denaturation. The structure of the 2.6 MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. We report a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, our new protocol has yielded a 3.1 [A] map of yeast FAS from 15,000 automatically picked particles within a day. The high map quality enabled us to build a complete atomic model of an intact fungal FAS.

biochemistry↗

bioRxiv2018

The deadly touch: protein denaturation at the water-air interface and how to prevent it

Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in unsupported films of aqueous solution, which can result in partial or complete denaturation of the protein. We investigated the structure of yeast fatty acid synthase at the air-water interface by electron cryo-tomography and single-particle image processing. Around 90% of complexes adsorbed to the air-water interface are partly denatured. We show that the unfolded regions are those facing the air-water interface. Denaturation by contact with air may happen at any stage of specimen preparation. Denaturation at the air-water interface is completely avoided when the complex is plunge-frozen on a substrate of hydrophilized graphene.

biophysics↗

bioRxiv2018

Molecular mechanisms in fungal fatty acid synthase (FAS) assembly

The fungal fatty acid synthase (fFAS) multienzyme is a barrel-shaped 2.6 MDa complex comprising six times eight catalytic domains. Upon barrel-formation, up to several hundred kDa large polypeptides intertwine to bury about 170,000 [A]2 of protein surface. Functional, regulatory and structural data as well as evolutionary aspects of fFAS have been elucidated during the last decades. Notwithstanding a profound knowledge of this protein family, the biogenesis of the elaborate structure remained elusive. Remarkably, experimental data have recently demonstrated that fFAS self-assembles without the assistance of specific factors. Considering the infinitesimal probability that the barrel-shaped complex forms simply by domains approaching in the correct orientation, we were interested in understanding the sequence of events that have to orchestrate fFAS assembly. Here, we show that fFAS attains its quaternary structure along a pathway of successive domain-domain interactions, which is strongly related to the evolutionary development of this protein family. The knowledge on fFAS assembly may pave the way towards antifungal therapy, and further develops fFAS as biofactory in technological applications.

molecular biology↗