Engineering Arabidopsis long-chain acyl-CoA synthetase 9 variants with enhanced enzyme activity

2019 ◽  
Vol 476 (1) ◽  
pp. 151-164 ◽  
Author(s):  
Yang Xu ◽  
Kristian Mark P. Caldo ◽  
Roman Holic ◽  
Elzbieta Mietkiewska ◽  
Jocelyn Ozga ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Fatty Acid ◽  
Amino Acid ◽  
Amino Acid Residue ◽  
Acid Site ◽  
Amino Acid Site ◽  
Acyl Donor ◽  
Evolutionary Analysis ◽  
Chain Acyl ◽  
Long Chain

Abstract Long-chain acyl-CoA synthetase (LACS, EC 6.2.1.3) catalyzes the ATP-dependent activation of free fatty acid to form acyl-CoA, which, in turn, serves as the major acyl donor for various lipid metabolic pathways. Increasing the size of acyl-CoA pool by enhancing LACS activity appears to be a useful approach to improve the production and modify the composition of fatty acid-derived compounds, such as triacylglycerol. In the present study, we aimed to improve the enzyme activity of Arabidopsis thaliana LACS9 (AtLACS9) by introducing random mutations into its cDNA using error-prone PCR. Two AtLACS9 variants containing multiple amino acid residue substitutions were identified with enhanced enzyme activity. To explore the effect of each amino acid residue substitution, single-site mutants were generated and the amino acid substitutions C207F and D238E were found to be primarily responsible for the increased activity of the two variants. Furthermore, evolutionary analysis revealed that the beneficial amino acid site C207 is conserved among LACS9 from plant eudicots, whereas the other beneficial amino acid site D238 might be under positive selection. Together, our results provide valuable information for the production of LACS variants for applications in the metabolic engineering of lipid biosynthesis in oleaginous organisms.

Substrate preferences of long-chain acyl-CoA synthetase and diacylglycerol acyltransferase contribute to enrichment of flax seed oil with α-linolenic acid

2018 ◽  
Vol 475 (8) ◽  
pp. 1473-1489 ◽  
Author(s):  
Yang Xu ◽  
Roman Holic ◽  
Darren Li ◽  
Xue Pan ◽  
Elzbieta Mietkiewska ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Linolenic Acid ◽  
Seed Oil ◽  
Diacylglycerol Acyltransferase ◽  
Acyl Donor ◽  
Flax Seed ◽  
Chain Acyl ◽  
Long Chain

Seed oil from flax (Linum usitatissimum) is enriched in α-linolenic acid (ALA; 18:3Δ9cis,12cis,15cis), but the biochemical processes underlying the enrichment of flax seed oil with this polyunsaturated fatty acid are not fully elucidated. Here, a potential process involving the catalytic actions of long-chain acyl-CoA synthetase (LACS) and diacylglycerol acyltransferase (DGAT) is proposed for ALA enrichment in triacylglycerol (TAG). LACS catalyzes the ATP-dependent activation of free fatty acid to form acyl-CoA, which in turn may serve as an acyl-donor in the DGAT-catalyzed reaction leading to TAG. To test this hypothesis, flax LACS and DGAT cDNAs were functionally expressed in Saccharomyces cerevisiae strains to probe their possible involvement in the enrichment of TAG with ALA. Among the identified flax LACSs, LuLACS8A exhibited significantly enhanced specificity for ALA over oleic acid (18:1Δ9cis) or linoleic acid (18:2Δ9cis,12cis). Enhanced α-linolenoyl-CoA specificity was also observed in the enzymatic assay of flax DGAT2 (LuDGAT2-3), which displayed ∼20 times increased preference toward α-linolenoyl-CoA over oleoyl-CoA. Moreover, when LuLACS8A and LuDGAT2-3 were co-expressed in yeast, both in vitro and in vivo experiments indicated that the ALA-containing TAG enrichment process was operative between LuLACS8A- and LuDGAT2-3-catalyzed reactions. Overall, the results support the hypothesis that the cooperation between the reactions catalyzed by LACS8 and DGAT2 may represent a route to enrich ALA production in the flax seed oil.

scholarly journals Long-chain acyl-CoA synthetases and fatty acid channeling

2007 ◽  
Vol 2 (4) ◽  
pp. 465-476 ◽  
Author(s):  
Douglas G Mashek ◽  
Lei O Li ◽  
Rosalind A Coleman
Keyword(s):  
Fatty Acid ◽  
Chain Acyl ◽  
Long Chain

scholarly journals Recent Advances in the Pathophysiology of Fatty Acid Oxidation Defects: Secondary Alterations of Bioenergetics and Mitochondrial Calcium Homeostasis Caused by the Accumulating Fatty Acids

2020 ◽  
Vol 11 ◽  
Author(s):  
Alexandre Umpierrez Amaral ◽  
Moacir Wajner
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Calcium Homeostasis ◽  
Acid Oxidation ◽  
Cultured Fibroblasts ◽  
Medium Chain ◽  
Chain Acyl ◽  
Energy Deprivation ◽  
Long Chain

Deficiencies of medium-chain acyl-CoA dehydrogenase, mitochondrial trifunctional protein, isolated long-chain 3-hydroxyacyl-CoA dehydrogenase, and very long-chain acyl-CoA dehydrogenase activities are considered the most frequent fatty acid oxidation defects (FAOD). They are biochemically characterized by the accumulation of medium-chain, long-chain hydroxyl, and long-chain fatty acids and derivatives, respectively, in tissues and biological fluids of the affected patients. Clinical manifestations commonly include hypoglycemia, cardiomyopathy, and recurrent rhabdomyolysis. Although the pathogenesis of these diseases is still poorly understood, energy deprivation secondary to blockage of fatty acid degradation seems to play an important role. However, recent evidence indicates that the predominant fatty acids accumulating in these disorders disrupt mitochondrial functions and are involved in their pathophysiology, possibly explaining the lactic acidosis, mitochondrial morphological alterations, and altered mitochondrial biochemical parameters found in tissues and cultured fibroblasts from some affected patients and also in animal models of these diseases. In this review, we will update the present knowledge on disturbances of mitochondrial bioenergetics, calcium homeostasis, uncoupling of oxidative phosphorylation, and mitochondrial permeability transition induction provoked by the major fatty acids accumulating in prevalent FAOD. It is emphasized that further in vivo studies carried out in tissues from affected patients and from animal genetic models of these disorders are necessary to confirm the present evidence mostly achieved from in vitro experiments.

scholarly journals Electrospray Tandem Mass Spectrometry for Analysis of Acylcarnitines in Dried Postmortem Blood Specimens Collected at Autopsy from Infants with Unexplained Cause of Death

2001 ◽  
Vol 47 (7) ◽  
pp. 1166-1182 ◽  
Author(s):  
Donald H Chace ◽  
James C DiPerna ◽  
Brenda L Mitchell ◽  
Bethany Sgroi ◽  
Lindsay F Hofman ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Fatty Acid ◽  
Amino Acid ◽  
Tandem Mass Spectrometry ◽  
Metabolic Disorders ◽  
Tandem Mass ◽  
Postmortem Blood ◽  
Blood Specimens ◽  
Medical Examiners ◽  
Long Chain

Abstract Background: Deaths from inherited metabolic disorders may remain undiagnosed after postmortem examination and may be classified as sudden infant death syndrome. Tandem mass spectrometry (MS/MS) may reveal disorders of fatty acid oxidation in deaths of previously unknown cause. Methods: We obtained filter-paper blood from 7058 infants from United States and Canadian Medical Examiners. Acylcarnitine and amino acid profiles were obtained by MS/MS. Specialized interpretation was used to evaluate profiles for disorders of fatty acid, organic acid, and amino acid metabolism. The analyses of postmortem blood specimens were compared with the analyses of bile specimens, newborn blood specimens, and specimens obtained from older infants at risk for metabolic disorders. Results: Results on 66 specimens suggested diagnoses of metabolic disorders. The most frequently detected disorders were medium-chain and very-long-chain acyl-CoA dehydrogenase deficiencies (23 and 9 cases, respectively), glutaric acidemia type I and II deficiencies (3 and 8 cases, respectively), carnitine palmitoyl transferase type II/translocase deficiencies (6 cases), severe carnitine deficiency (4 cases), isovaleric acidemia/2-methylbutyryl-CoA dehydrogenase deficiencies (4 cases), and long-chain hydroxyacyl-CoA dehydrogenase/trifunctional protein deficiencies (4 cases). Conclusions: Postmortem metabolic screening can explain deaths in infants and children and provide estimates of the number of infant deaths attributable to inborn errors of metabolism. MS/MS is cost-effective for analysis of postmortem specimens and should be considered for routine use by Medical Examiners and pathologists in unexpected/unknown infant and child death.

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