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 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 Purification and Biochemical Characterization of theMycobacterium tuberculosisβ-Ketoacyl-acyl Carrier Protein Synthases KasA and KasB

2001 ◽  
Vol 276 (50) ◽  
pp. 47029-47037 ◽  
Author(s):  
Merrill L. Schaeffer ◽  
Gautam Agnihotri ◽  
Craig Volker ◽  
Howard Kallender ◽  
Patrick J. Brennan ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Mycolic Acid ◽  
Carrier Protein ◽  
Mycolic Acids ◽  
E Coli ◽  
Long Chain

Mycolic acids are vital components of theMycobacterium tuberculosiscell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents. Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performsde novofatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors. In this study, we present the initial characterization of purified KasA and KasB, two β-ketoacyl-ACP synthase (KAS) enzymes of theM. tuberculosisFASII system. KasA and KasB were expressed inE. coliand purified by affinity chromatography. Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP. Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons. Activity of KasA and KasB increased with use ofM. tuberculosisAcpM, suggesting that structural differences between AcpM andE. coliACP may affect their recognition by the enzymes. Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin. These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery.

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 Dual-localized enzymatic components that constitute the mitochondrial and plastidial fatty acid synthase systems

2020 ◽  
Author(s):  
Xin Guan ◽  
Yozo Okazaki ◽  
Rwisdom Zhang ◽  
Kazuki Saito ◽  
Basil J Nikolau
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Acyl Chain ◽  
Type Ii ◽  
Dual Localization ◽  
Genes Encoding ◽  
Enoyl Acp Reductase ◽  
Identification And Characterization

ABSTRACTWe report the identification and characterization of genes encoding three enzymes that are shared between the mitochondrial and plastidial-localized Type II fatty acid synthase systems (mtFAS and ptFAS, respectively). Two of these enzymes, β-ketoacyl-ACP reductase (pt/mtKR) and enoyl-ACP reductase (pt/mtER) catalyze two of the reactions that constitute the core, 4-reaction cycle of the FAS system, which iteratively elongate the acyl-chain by 2-carbon atoms per cycle. The third enzyme, malonyl-CoA:ACP transacylase (pt/mtMCAT) catalyzes the reaction that loads the mtFAS system with substrate, by malonylating the phosphopantetheinyl cofactor of acyl carrier protein (ACP). GFP-transgenic experiments determined the dual localization of these enzymes, which were validated by the characterization of mutant alleles, which were transgenically rescued by transgenes that were singularly retargeted to either plastids or mitochondria. The singular retargeting of these proteins to plastids rescued the embryo-lethality associated with disruption of the essential ptFAS system, but these rescued plants display phenotypes typical of the lack of mtFAS function. Specifically, these phenotypes include reduced lipoylation of the H subunit of the glycine decarboxylase complex, the hyperaccumulation of glycine, and reduced growth; all these traits are reversible by growing these plants in an elevated CO2 atmosphere, which suppresses mtFAS-associated, photorespiration-dependent chemotypes.

scholarly journals Biophysical Characterization of Acyl Carrier Protein Domains from a Polyunsaturated Fatty Acid Synthase

2012 ◽  
Vol 26 (S1) ◽  
Author(s):  
Uldaeliz Trujillo Rodriguez ◽  
Delise Oyola-Robles ◽  
Stefan Arold ◽  
Fernando Alves De Melo ◽  
John E. Ladbury ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Polyunsaturated Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Protein Domains ◽  
Carrier Protein ◽  
Biophysical Characterization
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