scholarly journals Quantification and Discovery of Acyl-ACPs by LC-MS/MS

2019 ◽  
Author(s):  
Jeong-Won Nam ◽  
Lauren M. Jenkins ◽  
Jia Li ◽  
Bradley S. Evans ◽  
Jan G. Jaworski ◽  
...  

ABSTRACTAcyl carrier proteins (ACPs) are the scaffolds for fatty acid biosynthesis in living systems, rendering them essential to a comprehensive understanding of lipid metabolism; however, accurate quantitative methods to assess individual acyl-ACPs do not exist. A robust method was developed to quantify acyl-ACPs at picogram levels. Acyl-ACP elongation intermediates (3-hydroxyacyl-ACPs and 2, 3-trans-enoyl-ACPs), and unexpected medium chain (C10:1, C14:1) and polyunsaturated long chain acyl-ACPs (C16:3) were also identified, indicating the sensitivity of the method and that descriptions of lipid metabolism and ACP function are incomplete. Such ACPs are likely important to medium chain lipid production for fuels and highlight poorly understood lipid remodeling events in the chloroplast. The approach is broadly applicable to Type II FAS systems found in plants, bacteria, and mitochondria of animal and fungal systems because it uses a strategy that capitalizes on a highly conserved Asp-Ser-Leu-Asp (DSLD) amino acid sequence in ACPs to which acyl groups are attached. This allows for sensitive quantification using LC-MS/MS with de novo generated standards and an isotopic dilution strategy and will fill a gap in understanding, providing insights through quantitative exploration of fatty acid biosynthesis processes for optimal biofuels, renewable feed stocks, and medical studies in health and disease.

Open Biology ◽  
2017 ◽  
Vol 7 (2) ◽  
pp. 160277 ◽  
Author(s):  
Matías Cabruja ◽  
Sonia Mondino ◽  
Yi Ting Tsai ◽  
Julia Lara ◽  
Hugo Gramajo ◽  
...  

Unlike most bacteria, mycobacteria rely on the multi-domain enzyme eukaryote-like fatty acid synthase I (FAS I) to make fatty acids de novo. These metabolites are precursors of the biosynthesis of most of the lipids present both in the complex mycobacteria cell wall and in the storage lipids inside the cell. In order to study the role of the type I FAS system in Mycobacterium lipid metabolism in vivo , we constructed a conditional mutant in the fas-acpS operon of Mycobacterium smegmatis and analysed in detail the impact of reduced de novo fatty acid biosynthesis on the global architecture of the cell envelope. As expected, the mutant exhibited growth defect in the non-permissive condition that correlated well with the lower expression of fas-acpS and the concomitant reduction of FAS I, confirming that FAS I is essential for survival. The reduction observed in FAS I provoked an accumulation of its substrates, acetyl-CoA and malonyl-CoA, and a strong reduction of C 12 to C 18 acyl-CoAs, but not of long-chain acyl-CoAs (C 19 to C 24 ). The most intriguing result was the ability of the mutant to keep synthesizing mycolic acids when fatty acid biosynthesis was impaired. A detailed comparative lipidomic analysis showed that although reduced FAS I levels had a strong impact on fatty acid and phospholipid biosynthesis, mycolic acids were still being synthesized in the mutant, although with a different relative species distribution. However, when triacylglycerol degradation was inhibited, mycolic acid biosynthesis was significantly reduced, suggesting that storage lipids could be an intracellular reservoir of fatty acids for the biosynthesis of complex lipids in mycobacteria. Understanding the interaction between FAS I and the metabolic pathways that rely on FAS I products is a key step to better understand how lipid homeostasis is regulated in this microorganism and how this regulation could play a role during infection in pathogenic mycobacteria.


1991 ◽  
Vol 81 (2) ◽  
pp. 251-255
Author(s):  
Manfred Focke ◽  
Andrea Feld ◽  
Hartmut K. Lichtenthaler

1992 ◽  
Vol 47 (5-6) ◽  
pp. 382-386 ◽  
Author(s):  
Bernd List ◽  
Andrea Golz ◽  
Wilhelm Boland ◽  
Hartmut K. Lichtenthaler

The antibiotic cerulenin was shown to be a potent dose-dependent inhibitor of de novo fattyacid biosynthesis in intact isolated chloroplasts of different plants (measured as [14C]acetate incorporation into the total fatty-acid fraction). Various chemical derivatives of cerulenin were synthesized and tested in the chloroplast assay-system of oat, spinach and pea. Modifications of the hydrocarbon chain of cerulenin (e.g. tetrahydro-cerulenin and its short-chain cis-2,3-epoxy-4-oxoheptanamide derivative) decreased the inhibitory activity of cerulenin, whereas variations of the epoxy-oxo-amide structural element led to a complete loss of inhibition potency. The results indicate that the naturally occurring antibiotic cerulenin is the most active specific inhibitor of de novo fatty-acid biosynthesis, but the formation of the hydroxylactam ring seems to be an essential requirement for the inhibitory activity. Those structural analogues of cerulenin, which can no longer form a hydroxylactam ring, do not possess any inhibitory capacity.


1963 ◽  
Vol 41 (1) ◽  
pp. 1267-1274
Author(s):  
Peter F. Hall ◽  
Edward E. Nishizawa ◽  
Kristen B. Eik-Nes

The fatty acids palmitic, palmitoleic, stearic, and oleic have been isolated from rabbit testis and evidence for the synthesis of palmitic and stearic acids de novo from acetate-1-C14is presented. ICSH did not produce demonstrable stimulation of the synthesis of these acids in vitro although the hormone stimulated the production of testosterone-C14by the same tissue. Adrenal tissue was shown to contain palmitic, stearic, and oleic acids, and ACTH did not increase the incorporation of acetate-1-C14into a fatty acid fraction extracted following incubation of adrenal tissue in the presence of this substrate. Fatty acid biosynthesis, therefore, is probably not influenced by the mechanisms by which tropic hormones increase steroid formation.


1990 ◽  
Vol 45 (5) ◽  
pp. 518-520 ◽  
Author(s):  
Manfred Focke ◽  
Andrea Feld ◽  
Hartmut K. Lichtenthaler

Thiolactomycin was shown to be a potent inhibitor of de novo fatty acid biosynthesis in intact isolated chloroplasts (measured as [14C]acetate incorporation into total fatty acids). In our attempt to further localize the inhibition site we confirmed the inhibition with a fatty acid synthetase preparation, measuring the incorporation of [14C]malonyl-CoA into total fatty acids. From the two proposed enzymic targets of the fatty acid synthetase by thiolactomycin we could exclude the acetyl-CoA: ACP transacetylase. It appears that the inhibition by thiolactomycin occurs on the level of the condensing enzymes, i.e. the 3-oxoacyl-ACP synthases. We also demonstrated that the two starting enzymes of de novo fatty acid biosynthesis, the acetyl-CoA synthetase and the acetyl-CoA carboxylase, are not affected by thiolactomycin.


2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Regiane Kawasaki ◽  
Rafael A. Baraúna ◽  
Artur Silva ◽  
Marta S. P. Carepo ◽  
Rui Oliveira ◽  
...  

Exiguobacterium antarcticumB7 is extremophile Gram-positive bacteria able to survive in cold environments. A key factor to understanding cold adaptation processes is related to the modification of fatty acids composing the cell membranes of psychrotrophic bacteria. In our study we show thein silicoreconstruction of the fatty acid biosynthesis pathway ofE. antarcticumB7. To build the stoichiometric model, a semiautomatic procedure was applied, which integrates genome information using KEGG and RAST/SEED. Constraint-based methods, namely, Flux Balance Analysis (FBA) and elementary modes (EM), were applied. FBA was implemented in the sense of hexadecenoic acid production maximization. To evaluate the influence of the gene expression in the fluxome analysis, FBA was also calculated using thelog2⁡FCvalues obtained in the transcriptome analysis at 0°C and 37°C. The fatty acid biosynthesis pathway showed a total of 13 elementary flux modes, four of which showed routes for the production of hexadecenoic acid. The reconstructed pathway demonstrated the capacity ofE. antarcticumB7 tode novoproduce fatty acid molecules. Under the influence of the transcriptome, the fluxome was altered, promoting the production of short-chain fatty acids. The calculated models contribute to better understanding of the bacterial adaptation at cold environments.


2020 ◽  
Author(s):  
Michael Burkart ◽  
Thomas Bartholow ◽  
Terra Sztain ◽  
Ashay Patel ◽  
D Lee ◽  
...  

Abstract Fatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB in Escherichia coli. This work reveals the molecular basis of six discrete binding events responsible for E. coli FAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways. ONE SENTENCE SUMMARY: Through a combination of structural and computational analysis, a comparative evaluation of protein-protein interactions in de novo fatty acid biosynthesis in E. coli is performed.


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