High-Speed Atomic Force Microscopy Visualization of the Dynamics of the Multienzyme Fatty Acid Synthase

Benning, Friederike; Sakiyama, Yusuke; Mazur, Adam; Bukhari, Habib S. T.; Lim, Roderick Y. H.; Maier, Timm

doi:10.1021/acsnano.7b04216

Cited by 23 publications

(30 citation statements)

References 43 publications

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“…based on single-molecule fluorescence. Most recently, it has also been demonstrated that high-speed atomic force microscopy is able to resolve large-scale conformational changes in mFAS at a spatial and temporal resolution permitting detailed analysis of the transitions of single mFAS molecules between different conformational states 41 . However, further work will be required to apply this technique for a comprehensive analysis of mFAS structural dynamics.…”

Section: Metazoan Fatty Acid Synthasementioning

confidence: 99%

The architectures of iterative type I PKS and FAS

2018

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Covering: up to mid of 2018 Type I fatty acid synthases (FASs) are giant multienzymes catalyzing all steps of the biosynthesis of fatty acids from acetyl- and malonyl-CoA by iterative precursor extension. Two strikingly different architectures of FAS evolved in yeast (as well as in other fungi and some bacteria) and metazoans. Yeast-type FAS (yFAS) assembles into a barrel-shaped structure of more than 2 MDa molecular weight. Catalytic domains of yFAS are embedded in an extensive scaffolding matrix and arranged around two enclosed reaction chambers. Metazoan FAS (mFAS) is a 540 kDa X-shaped dimer, with lateral reaction clefts, minimal scaffolding and pronounced conformational variability. All naturally occurring yFAS are strictly specialized for the production of saturated fatty acids. The yFAS architecture is not used for the biosynthesis of any other secondary metabolite. On the contrary, mFAS is related at the domain organization level to major classes of polyketide synthases (PKSs). PKSs produce a variety of complex and potent secondary metabolites; they either act iteratively (iPKS), or are linked via directed substrate transfer into modular assembly lines (modPKSs). Here, we review the architectures of yFAS, mFAS, and iPKSs. We rationalize the evolution of the yFAS assembly, and provide examples for re-engineering of yFAS. Recent studies have provided novel insights into the organization of iPKS. A hybrid crystallographic model of a mycocerosic acid synthase-like Pks5 yielded a comprehensive visualization of the organization and dynamics of fully-reducing iPKS. Deconstruction experiments, structural and functional studies of specialized enzymatic domains, such as the product template (PT) and the starter-unit acyltransferase (SAT) domain have revealed functional principles of non-reducing iterative PKS (NR-PKSs). Most recently, a six-domain loading region of an NR-PKS has been visualized at high-resolution together with cryo-EM studies of a trapped loading intermediate. Altogether, these data reveal the related, yet divergent architectures of mFAS, iPKS and also modPKSs. The new insights highlight extensive dynamics, and conformational coupling as key features of mFAS and iPKS and are an important step towards collection of a comprehensive series of snapshots of PKS action.

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Section: Metazoan Fatty Acid Synthasementioning

confidence: 99%

The architectures of iterative type I PKS and FAS

2018

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“…( C ) Conformational dynamics of animal FAS. Swinging and swivelling motions around the flexible hinge region have been observed by single particle EM and high-speed atomic force microscopy 8 , 15 . Full rotation of the condensing wing by 180° was further confirmed by mutagenesis studies 49 .…”

Section: Introductionmentioning

confidence: 99%

“…in an early fluorescence resonance energy transfer (FRET) study on chicken liver FAS 13 , 14 . More detailed insights into the conformational versatility of animal FAS were finally given by a recent negative stain electron microscopy (EM) study on rat FAS, high-speed atomic force microscopy on insect FAS and by computational modelling with porcine FAS data 7 , 8 , 15 . These studies revealed large conformational changes within the enzyme with complete relative rotational and swinging freedom between the condensing and processing wing (Fig.…”

Section: Introductionmentioning

confidence: 99%

Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression

Heil

Rittner

Goebel

et al. 2018

Sci Rep

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The access to information on the dynamic behaviour of large proteins is usually hindered as spectroscopic methods require the site-specific attachment of biophysical probes. A powerful emerging tool to tackle this issue is amber codon suppression. Till date, its application on large and complex multidomain proteins of MDa size has not been reported. Herein, we systematically investigate the feasibility to introduce different non-canonical amino acids into a 540 kDa homodimeric fatty acid synthase type I by genetic code expansion with subsequent fluorescent labelling. Our approach relies on a microplate-based reporter assay of low complexity using a GFP fusion protein to quickly screen for sufficient suppression conditions. Once identified, these findings were successfully utilized to upscale both the expression scale and the protein size to full-length constructs. These fluorescently labelled samples of fatty acid synthase were subjected to initial biophysical experiments, including HPLC analysis, activity assays and fluorescence spectroscopy. Successful introduction of such probes into a molecular machine such as fatty acid synthases may pave the way to understand the conformational variability, which is a primary intrinsic property required for efficient interplay of all catalytic functionalities, and to engineer them.

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“…C) Conformational dynamics of animal FAS. Swinging and swivelling motions around the flexible hinge region have been observed by single particle EM and high-speed atomic force microscopy 8,15 . Full rotation of the condensing wing by 180° was further confirmed by mutagenesis studies 50 .…”

Section: Introductionmentioning

confidence: 99%

Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression

Heil

Rittner

Goebel

et al. 2018

Preprint

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17Amber codon suppression is a powerful tool to site-specifically modify proteins to 18 generate novel biophysical probes. Yet, its application on large and complex 19 multidomain proteins is challenging, leading to difficulties during structural and 20 conformational characterization using spectroscopic methods. The animal fatty acid 21 synthase type I is a 540 kDa homodimer displaying large conformational variability. 22As the key enzyme of de novo fatty acid synthesis, it attracts interest in the fields of 23 obesity, diabetes and cancer treatment. Substrates and intermediates remain 24 covalently bound to the enzyme during biosynthesis and are shuttled to all catalytic 25 domains by the acyl carrier protein domain. Thus, conformational variability of animal 26 FAS is an essential aspect for fatty acid biosynthesis. We investigate this 27 multidomain protein as a model system for probing amber codon suppression by 28 genetic encoding of non-canonical amino acids. The systematic approach relies on a 29 microplate-based reporter assay of low complexity, that was used for quick screening 30 of suppression conditions. Furthermore, the applicability of the reporter assay is 31 demonstrated by successful upscaling to both full-length constructs and increased 32 expression scale. The obtained fluorescent probes of murine FAS type I could be 33 subjected readily to a conformational analysis using single-molecule fluorescence 34 resonance energy transfer. 35All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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High-Speed Atomic Force Microscopy Visualization of the Dynamics of the Multienzyme Fatty Acid Synthase

Cited by 23 publications

References 43 publications

The architectures of iterative type I PKS and FAS

The architectures of iterative type I PKS and FAS

Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression

Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression

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