scholarly journals Analysis and Engineering of Substrate Shuttling by the Acyl Carrier Protein (ACP) in Fatty Acid Synthases (FASs)

2018 ◽  
Author(s):  
Emanuele Rossini ◽  
Jan Gajewski ◽  
Maja Klaus ◽  
Gerhard Hummer ◽  
Martin Grininger
Keyword(s):  
Fatty Acid ◽  
Active Sites ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Reaction Rates ◽  
Biofuel Production ◽  
Type I ◽  
Single Interface ◽  
Microbial Biofuel ◽  
The Impact

ABSTRACTIn the large enzyme complexes of natural biosynthetic pathways, molecules are assembled like in a factory. Carrier domains shuttle substrates and intermediates as covalently attached cargo within the enzyme complex between active sites. The physical confinement of the reaction increases reaction rates and hinders pathway branching. Alternating interactions of substrate-loaded carrier domains with different catalytic domains modulate the chemical environment. In this study, we aim at assessing the impact of domain-domain interactions (DDIs) on the reaction progress of a multienzyme type I fatty acid synthase (FAS) in quantitative terms. We modulate DDIs by single interface mutations, and read out the impact on substrate shuttling by recording fatty acid (FA) chain length product spectra and FAS activities. Our data show that even single interface point mutations can severely affect FA synthesis. With molecular dynamics simulations and modeling, we relate the mutation effects to specific alterations in the molecular interaction networks and domain-domain binding energetics. Some of the presented mutations induce the synthesis of short-chain FAs. These compounds are important commodity products and potent precursors for microbial biofuel production.

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 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

scholarly journals Preferential synthesis of very long chain polyunsaturated fatty acids in eutreptiella sp. (eugelnozoa) revealed by chromatographic and transcriptomic analyses

2020 ◽  
Author(s):  
Rita C. Kuo ◽  
Huan Zhang ◽  
James D. Stuart ◽  
Anthony A. Provatas ◽  
Linda Hannick ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Polyunsaturated Fatty Acids ◽  
Carbon Fixation ◽  
Acyl Carrier Protein ◽  
Lipid Production ◽  
Fatty Acid Desaturase ◽  
Biofuel Production ◽  
Type I ◽  
Long Chain

AbstractAlgal lipids are important fuel storage molecules in algae and a currency for energy transfer in the marine food chain as well as materials for biofuel production, but their production and regulation are not well understood in many species including the common coastal phytoplankton Eutreptiella spp. Here, using gas chromatography-tandem mass spectrometry (GC/MS/MS), we discovered 24 types of fatty acids (FAs) in Eutreptiella sp. with a relatively high proportion of long chain unsaturated FAs. The abundances of C16, C18 and saturated FAs decreased when phosphate in the culture medium was depleted. Among the 24 FAs, docosahexaenoic acid (22:6) and eicosapentaenoic acid (20:5) were the most abundant, suggesting that Eutreptiella sp. preferentially invests in the synthesis of very long chain polyunsaturated fatty acids (VLCPFA). Further transcriptomic analysis revealed that Eutreptiella sp. likely synthesizes VLCPFA via Δ8 pathway and uses type I and II fatty acid synthases. Using RT-qPCR, we found that some of the lipid production genes, such as β-ketoacyl-ACP reductase, fatty acid desaturase, acetyl-CoA carboxylase, acyl carrier protein, Δ8 desaturase, and Acyl-ACP thioesterase, were more actively expressed during light period. Besides, two carbon-fixation genes were more highly expressed in the high lipid illuminated cultures, suggesting a linkage between photosynthesis and lipid production.

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 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 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 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.

scholarly journals Structural insight into Pichia pastoris fatty acid synthase

2021 ◽  
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

SummaryType 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 Structural comparison of Acinetobacter baumannii β-ketoacyl-acyl carrier protein reductases in fatty acid and aryl polyene biosynthesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Woo Cheol Lee ◽  
Sungjae Choi ◽  
Ahjin Jang ◽  
Kkabi Son ◽  
Yangmee Kim
Keyword(s):  
Fatty Acid ◽  
Acinetobacter Baumannii ◽  
Gene Cluster ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Gene Clusters ◽  
Carrier Protein ◽  
Aryl Group ◽  
Visible Absorption

AbstractSome Gram-negative bacteria harbor lipids with aryl polyene (APE) moieties. Biosynthesis gene clusters (BGCs) for APE biosynthesis exhibit striking similarities with fatty acid synthase (FAS) genes. Despite their broad distribution among pathogenic and symbiotic bacteria, the detailed roles of the metabolic products of APE gene clusters are unclear. Here, we determined the crystal structures of the β-ketoacyl-acyl carrier protein (ACP) reductase ApeQ produced by an APE gene cluster from clinically isolated virulent Acinetobacter baumannii in two states (bound and unbound to NADPH). An in vitro visible absorption spectrum assay of the APE polyene moiety revealed that the β-ketoacyl-ACP reductase FabG from the A. baumannii FAS gene cluster cannot be substituted for ApeQ in APE biosynthesis. Comparison with the FabG structure exhibited distinct surface electrostatic potential profiles for ApeQ, suggesting a positively charged arginine patch as the cognate ACP-binding site. Binding modeling for the aryl group predicted that Leu185 (Phe183 in FabG) in ApeQ is responsible for 4-benzoyl moiety recognition. Isothermal titration and arginine patch mutagenesis experiments corroborated these results. These structure–function insights of a unique reductase in the APE BGC in comparison with FAS provide new directions for elucidating host–pathogen interaction mechanisms and novel antibiotics discovery.

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