scholarly journals Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression

2018 ◽  
Author(s):  
Christina S. Heil ◽  
Alexander Rittner ◽  
Bjarne Goebel ◽  
Daniel Beyer ◽  
Martin Grininger
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Protein Domain ◽  
Type I ◽  
Reporter Assay ◽  
Multidomain Proteins ◽  
Conformational Variability ◽  
Amber Codon ◽  
Amber Codon Suppression

AbstractAmber codon suppression is a powerful tool to site-specifically modify proteins to generate novel biophysical probes. Yet, its application on large and complex multidomain proteins is challenging, leading to difficulties during structural and conformational characterization using spectroscopic methods. The animal fatty acid synthase type I is a 540 kDa homodimer displaying large conformational variability. As the key enzyme of de novo fatty acid synthesis, it attracts interest in the fields of obesity, diabetes and cancer treatment. Substrates and intermediates remain covalently bound to the enzyme during biosynthesis and are shuttled to all catalytic domains by the acyl carrier protein domain. Thus, conformational variability of animal FAS is an essential aspect for fatty acid biosynthesis. We investigate this multidomain protein as a model system for probing amber codon suppression by genetic encoding of non-canonical amino acids. The systematic approach relies on a microplate-based reporter assay of low complexity, that was used for quick screening of suppression conditions. Furthermore, the applicability of the reporter assay is demonstrated by successful upscaling to both full-length constructs and increased expression scale. The obtained fluorescent probes of murine FAS type I could be subjected readily to a conformational analysis using single-molecule fluorescence resonance energy transfer.

scholarly journals A Mammalian Type I Fatty Acid Synthase Acyl Carrier Protein Domain Does Not Sequester Acyl Chains

2007 ◽  
Vol 283 (1) ◽  
pp. 518-528 ◽  
Author(s):  
Eliza Ploskoń ◽  
Christopher J. Arthur ◽  
Simon E. Evans ◽  
Christopher Williams ◽  
John Crosby ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Protein Domain ◽  
Carrier Protein ◽  
Type I ◽  
Acyl Chains

Acyl carrier protein: structure–function relationships in a conserved multifunctional protein family

2007 ◽  
Vol 85 (6) ◽  
pp. 649-662 ◽  
Author(s):  
David M. Byers ◽  
Huansheng Gong
Keyword(s):  
Fatty Acid ◽  
Active Sites ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Divalent Cations ◽  
Structural Features ◽  
Carrier Protein ◽  
Type I ◽  
Prosthetic Group

Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid synthesis. In yeast and mammals, ACP exists as a separate domain within a large multifunctional fatty acid synthase polyprotein (type I FAS), whereas it is a small monomeric protein in bacteria and plastids (type II FAS). Bacterial ACPs are also acyl donors for synthesis of a variety of products, including endotoxin and acylated homoserine lactones involved in quorum sensing; the distinct and essential nature of these processes in growth and pathogenesis make ACP-dependent enzymes attractive antimicrobial drug targets. Additionally, ACP homologues are key components in the production of secondary metabolites such as polyketides and nonribosomal peptides. Many ACPs exhibit characteristic structural features of natively unfolded proteins in vitro, with a dynamic and flexible conformation dominated by 3 parallel α helices that enclose the thioester-linked acyl group attached to a phosphopantetheine prosthetic group. ACP conformation may also be influenced by divalent cations and interaction with partner enzymes through its “recognition” helix II, properties that are key to its ability to alternately sequester acyl groups and deliver them to the active sites of ACP-dependent enzymes. This review highlights recent progress in defining how the structural features of ACP are related to its multiple carrier roles in fatty acid metabolism.

scholarly journals Antimycobacterial action of thiolactomycin: an inhibitor of fatty acid and mycolic acid synthesis.

1996 ◽  
Vol 40 (12) ◽  
pp. 2813-2819 ◽  
Author(s):  
R A Slayden ◽  
R E Lee ◽  
J W Armour ◽  
A M Cooper ◽  
I M Orme ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Mycolic Acid ◽  
Carrier Protein ◽  
Type I ◽  
Dependent Manner ◽  
Fas Ii

Thiolactomycin (TLM) possesses in vivo antimycobacterial activity against the saprophytic strain Mycobacterium smegmatis mc2155 and the virulent strain M. tuberculosis Erdman, resulting in complete inhibition of growth on solid media at 75 and 25 micrograms/ml, respectively. Use of an in vitro murine macrophage model also demonstrated the killing of viable intracellular M. tuberculosis in a dose-dependent manner. Through the use of in vivo [1,2-14C]acetate labeling of M. smegmatis, TLM was shown to inhibit the synthesis of both fatty acids and mycolic acids. However, synthesis of the shorter-chain alpha'-mycolates of M. smegmatis was not inhibited by TLM, whereas synthesis of the characteristic longer-chain alpha-mycolates and epoxymycolates was almost completely inhibited at 75 micrograms/ml. The use of M. smegmatis cell extracts demonstrated that TLM specifically inhibited the mycobacterial acyl carrier protein-dependent type II fatty acid synthase (FAS-II) but not the multifunctional type I fatty acid synthase (FAS-I). In addition, selective inhibition of long-chain mycolate synthesis by TLM was demonstrated in a dose-response manner in purified, cell wall-containing extracts of M. smegmatis cells. The in vivo and in vitro data and knowledge of the mechanism of TLM resistance in Escherichia coli suggest that two distinct TLM targets exist in mycobacteria, the beta-ketoacyl-acyl carrier protein synthases involved in FAS-II and the elongation steps leading to the synthesis of the alpha-mycolates and oxygenated mycolates. The efficacy of TLM against M. smegmatis and M. tuberculosis provides the prospects of identifying fatty acid and mycolic acid biosynthetic genes and revealing a novel range of chemotherapeutic agents directed against M. tuberculosis.

scholarly journals Structural insight into Pichia pastoris fatty acid synthase

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Joseph S. Snowden ◽  
Jehad Alzahrani ◽  
Lee Sherry ◽  
Martin Stacey ◽  
David J. Rowlands ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Protein Production ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Expression System ◽  
Model Organism ◽  
Type I ◽  
Structural Insight ◽  
Fatty Acid Synthases ◽  
Insight Into

AbstractType I fatty acid synthases (FASs) are critical metabolic enzymes which are common targets for bioengineering in the production of biofuels and other products. Serendipitously, we identified FAS as a contaminant in a cryoEM dataset of virus-like particles (VLPs) purified from P. pastoris, an important model organism and common expression system used in protein production. From these data, we determined the structure of P. pastoris FAS to 3.1 Å resolution. While the overall organisation of the complex was typical of type I FASs, we identified several differences in both structural and enzymatic domains through comparison with the prototypical yeast FAS from S. cerevisiae. Using focussed classification, we were also able to resolve and model the mobile acyl-carrier protein (ACP) domain, which is key for function. Ultimately, the structure reported here will be a useful resource for further efforts to engineer yeast FAS for synthesis of alternate products.

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

2019 ◽  
Author(s):  
Jennifer W. Lou ◽  
Kali R. Iyer ◽  
S. M. Naimul Hasan ◽  
Leah E. Cowen ◽  
Mohammad T. Mazhab-Jafari
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Protein Translocation ◽  
Acyl Carrier Protein ◽  
Reaction Chamber ◽  
Type I ◽  
Acyl Carrier Proteins ◽  
Enoyl Reductase ◽  
Ketoacyl Synthase

ABSTRACTDuring fatty acid biosynthesis, acyl carrier proteins (ACPs) from type I fungal fatty acid synthase (FAS) shuttle substrates and intermediates within a reaction chamber that hosts multiple spatially-fixed catalytic centers. A major challenge in understanding the mechanism of ACP-mediated substrate shuttling is experimental observation of its transient interaction landscape within the reaction chamber. Here, we have shown that ACP spatial distribution is sensitive to the presence of substrates in a catalytically inhibited state, which enables high-resolution investigation of the ACP-dependent conformational transitions within the enoyl reductase (ER) reaction site. In two fungal FASs with distinct ACP localization, the shuttling domain is targeted to the ketoacyl-synthase (KS) domain and away from other catalytic centers, such as acetyl-transferase (AT) and ER domains by steric blockage of the KS active site followed by addition of substrates. These studies strongly suggest that acylation of phosphopantetheine arm of ACP may be an integral part of the substrate shuttling mechanism in type I fungal FAS.

scholarly journals Characterization of fatty acid synthase monomers restrained from reassociating by immobilization to a solid support

1993 ◽  
Vol 292 (2) ◽  
pp. 361-364 ◽  
Author(s):  
J R Petithory ◽  
S Smith
Keyword(s):  
Fatty Acid ◽  
Active Site ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Solid Support ◽  
Protein Domain ◽  
Serine Residue ◽  
Subunit Dissociation ◽  
Per Se

The controversial question as to whether the ketoreductase activity of the animal fatty acid synthase is lost on dissociation of the homodimer has been addressed by using immobilized subunits which cannot reassociate under the conditions of assay. Ketoreductase activity, assessed with the model substrate S-acetoacetyl-N-acetylcysteamine, was identical in immobilized monomers and dimers, exhibiting normal Michaelis-Menten kinetics with Km values in the millimolar range. When acetoacetyl-CoA was used as a substrate, however, biphasic kinetics were observed in the case of the dimer, with estimated Km values in the micro- and milli-molar ranges, but only the high-Km reaction was observed with the monomer. Thus when the ketoreductase activities of the monomer and dimer are assessed with acetoacetyl-CoA at concentrations sufficient to saturate only the low-Km reaction, it appears that the ketoreductase activity towards acetoacetyl-CoA is lost upon dissociation. Reduction of acetoacetyl-CoA via the low-Km pathway is CoA-dependent, indicating that acetoacetyl-CoA can react with the dimer by two mechanisms: a high-Km pathway analogous to that utilized by model substrates and a low-Km pathway in which substrate and product are transferred between acyl-CoA and acyl-enzyme forms. The results indicate that the ketoreductase activity per se is unaffected by subunit dissociation and are consistent with a model in which the transfer of substrate from CoA ester to the acyl-carrier-protein domain necessitates juxtaposition of the transferase active-site serine residue of one subunit and the phosphopantetheine moiety of the adjacent subunit.

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

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jennifer W. Lou ◽  
Kali R. Iyer ◽  
S. M. Naimul Hasan ◽  
Leah E. Cowen ◽  
Mohammad T. Mazhab-Jafari
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
Protein Translocation ◽  
Acyl Carrier Protein ◽  
Reaction Chamber ◽  
Type I ◽  
Acyl Carrier Proteins ◽  
Enoyl Reductase ◽  
Ketoacyl Synthase

Abstract During fatty acid biosynthesis, acyl carrier proteins (ACPs) from type I fungal fatty acid synthase (FAS) shuttle substrates and intermediates within a reaction chamber that hosts multiple spatially-fixed catalytic centers. A major challenge in understanding the mechanism of ACP-mediated substrate shuttling is experimental observation of its transient interaction landscape within the reaction chamber. Here, we have shown that ACP spatial distribution is sensitive to the presence of substrates in a catalytically inhibited state, which enables high-resolution investigation of the ACP-dependent conformational transitions within the enoyl reductase (ER) reaction site. In two fungal FASs with distinct ACP localization, the shuttling domain is targeted to the ketoacyl-synthase (KS) domain and away from other catalytic centers, such as acetyl-transferase (AT) and ER domains by steric blockage of the KS active site followed by addition of substrates. These studies strongly suggest that acylation of phosphopantetheine arm of ACP may be an integral part of the substrate shuttling mechanism in type I fungal FAS.

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