Structural insight into Pichia pastoris fatty acid synthase

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.

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Structural insight into Pichia pastoris fatty acid synthase

10.1101/2021.01.27.428426 ◽

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.

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Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomicroscopy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0913547107 ◽

2010 ◽

Vol 107 (20) ◽

pp. 9164-9169 ◽

Cited By ~ 46

Author(s):

P. Gipson ◽

D. J. Mills ◽

R. Wouts ◽

M. Grininger ◽

J. Vonck ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthase ◽

Electron Cryomicroscopy ◽

Structural Insight ◽

Insight Into

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

Journal of Biological Chemistry ◽

10.1074/jbc.m703454200 ◽

2007 ◽

Vol 283 (1) ◽

pp. 518-528 ◽

Cited By ~ 53

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

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

Biochemistry and Cell Biology ◽

10.1139/o07-109 ◽

2007 ◽

Vol 85 (6) ◽

pp. 649-662 ◽

Cited By ~ 115

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.12.2813 ◽

1996 ◽

Vol 40 (12) ◽

pp. 2813-2819 ◽

Cited By ~ 105

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.

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Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression

10.1101/282525 ◽

2018 ◽

Cited By ~ 1

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.

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

10.1101/636167 ◽

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.

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

Scientific Reports ◽

10.1038/s41598-019-49261-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

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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Functional Characterization of an Evolutionarily Distinct Phosphopantetheinyl Transferase in the Apicomplexan Cryptosporidium parvum

Eukaryotic Cell ◽

10.1128/ec.4.7.1211-1220.2005 ◽

2005 ◽

Vol 4 (7) ◽

pp. 1211-1220 ◽

Cited By ~ 23

Author(s):

Xiaomin Cai ◽

Dustin Herschap ◽

Guan Zhu

Keyword(s):

Fatty Acid ◽

Cryptosporidium Parvum ◽

Acyl Carrier Protein ◽

Functional Characterization ◽

Type I ◽

Phosphopantetheinyl Transferase ◽

Apicomplexan Parasites ◽

Fatty Acid Synthases

ABSTRACT Recently, two types of fatty acid synthases (FASs) have been discovered from apicomplexan parasites. Although significant progress has been made in characterizing these apicomplexan FASs, virtually nothing was previously known about the activation and regulation of these enzymes. In this study, we report the discovery and characterization of two distinct types of phosphopantetheinyl transferase (PPTase) that are responsible for synthesizing holo-acyl carrier protein (ACP) from three apicomplexan parasites: surfactin production element (SFP) type in Cryptosporidium parvum (CpSFP-PPT), holo-ACP synthase (ACPS)-type in Plasmodium falciparum (PfACPS-PPT), and both SFP and ACPS types in Toxoplasma gondii (TgSFP-PPT and TgACPS-PPT). CpSFP-PPT and TgSFP-PPT are monofunctional, cytosolic, and phylogenetically related to animal PPTases. However, PfACPS-PPT and TgACPS-PPT are bifunctional (fused with a metal-dependent hydrolase), likely targeted to the apicoplast, and more closely related to proteobacterial PPTases. The function of apicomplexan PPTases has been confirmed by detailed functional analysis using recombinant CpSFP-PPT expressed from an artificially synthesized gene with codon usage optimized for Escherichia coli. The recombinant CpSFP-PPT was able to activate the ACP domains from the C. parvum type I FAS in vitro using either CoA or acetyl-CoA as a substrate, or in vivo when coexpressed in bacteria, with kinetic characteristics typical of PPTases. These observations suggest that the two types of fatty acid synthases in the Apicomplexa are activated and regulated by two evolutionarily distinct PPTases.

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