Molecular mechanisms underlying catalytic activity of delta 6 desaturase from Glossomastix chrysoplasta and Thalassiosira pseudonana

Shi, Haisu; 粟, 史海; Wu, Rina; 娜, 乌日; Zheng, Yan; Yue, Xiqing; 庆, 岳喜

doi:10.1194/jlr.m079806

Cited by 14 publications

(13 citation statements)

References 22 publications

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“…GcFADS12 possessed typical characteristics of other FADS12 family members, including three histidine‐rich conserved motifs (HXXHHK, HWXVXXTXLQH and HLVHH) and six transmembrane domains (Fig. ), and these observations were in agreement with our previous reports on Δ6 fatty acid desaturase . The amino acid sequence of GcFADS12 was highly similar to the one that S. Casaregola submitted to the GenBank (protein id: CDO51572.1), which was in agreement with the observation from our phylogenetic analysis of FADS12 (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…LB agar plates for plasmid construction in Escherichia coli Top 10 and SC‐U synthetic minimal medium for gene expression in S. cerevisiae were used as described previously . G. candidum was grown on Yeast Peptone Dextrose medium (containing 10 g·L −1 yeast extract, 10 g·L −1 peptone, 10 g·L −1 glucose and 20 g·L −1 agar) at 28 °C.…”

Section: Methodsmentioning

confidence: 99%

“…In EFA biosynthesis, Δ12 fatty acid desaturase (FADS12), Δ15 fatty acid desaturase (FADS15) and Δ6 fatty acid desaturase (FADS6) play a key role in regulating the level of EFAs. In a previous study, the molecular mechanism, substrate specificity and catalytic activity for FADS6 were analyzed and applied to PUFA synthesis . FADS12 is a key bifunctional membrane‐bound desaturase that converts oleic acid (OA, 18:1 Δ9 ) to linoleic acid (LA, 18:2 Δ9,12 ) and LA to α‐linolenic acid (ALA, 18:3 Δ9,12,15 ) by introducing a double bond between the carbons 12 and 13 from the carboxyl end of the substrate in the biosynthesis of EFAs .…”

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confidence: 99%

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Δ12 fatty acid desaturase gene from Geotrichum candidum in cheese: molecular cloning and functional characterization

Luo

Shi

et al. 2018

FEBS Open Bio

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Soft cheese with white rind lacks essential fatty acids ( EFA s), and as a result its long‐term consumption may lead to various kinds of cardiovascular and cerebrovascular diseases, such as hyperlipidemia, hypertension, and atherosclerosis. Geotrichum candidum is a dimorphic yeast that plays an important role in the ripening of mold cheese. A gene coding for Δ12 fatty acid desaturase, a critical bifunctional enzyme desaturating oleic acid ( OA ) and linoleic acid ( LA ) to produce LA and α‐linolenic acid ( ALA ), respectively, was isolated from G. candidum , and then cloned and heterologously expressed in Saccharomyces cerevisiae . This gene, named Gc FADS 12 , had an open reading frame of 1257 bp and codes for a protein of 419 amino acids with a predicted molecular mass of 47.5 kDa. Characterization showed that Gc FADS 12 had the ability to convert OA to LA and LA to ALA , and the conversion rates for OA and LA were 20.40 ± 0.66% and 6.40 ± 0.57%, respectively. We also found that the protein product of Gc FADS 12 catalyzes the conversion of the intermediate product ( LA ) to ALA by addition of OA as the sole substrate. The catalytic activity of Gc FADS 12 on OA and LA was unaffected by fatty acid concentrations. Kinetic analysis revealed that Gc FADS 12 had stronger affinity for the OA than for the LA substrate. This study offers a solid basis for improving the production of EFA s by G. candidum in cheese.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Δ12 fatty acid desaturase gene from Geotrichum candidum in cheese: molecular cloning and functional characterization

Luo

Shi

et al. 2018

FEBS Open Bio

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“…Then, the expression level of each chimeric desaturase in Pichia pastoris was determined by western blotting analysis as described previously ( Shi et al, 2015); each sample loading volume was adjusted to the same level according to the western blot results to determine desaturase activity of each chimera. Chimeric desaturase activity were analyzed by gas chromatography (GC) as described previously ( Shi et al, 2016; Shi et al, 2018b).…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, it is important to study differences in the mechanisms underlying differential FADS12 activity in various species. At present, research on the catalytic mechanism of fatty acid desaturase is still in its infancy (Lee et al, 2016), except that the three-dimensional structure of FADS9 was analyzed and found to be mushroom-shaped (Bai, 2015; Wang, 2015) and the molecular mechanisms underlying FADS2 substrate specificity ( Shi et al, 2015) and catalytic activity ( Shi et al, 2018a) were analyzed in our previous study. However, studies on fatty acid desaturase in dominant strains of cheese have not yet been undertaken.…”

Section: Introductionmentioning

confidence: 99%

Both Determinants of Allosteric and Active Sites Responsible for Catalytic Activity of Delta 12 Fatty Acid Desaturase by Domain Swapping

Shi

Tian

et al. 2019

Preprint

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Cheese lacks essential fatty acids (EFAs). Delta 12 fatty acid desaturase (FADS12) is a critical enzyme required for EFA biosynthesis in fermentation of the predominant strains of cheese. Previously, we identified the FADS12 gene and characterized its function for the first time in Geotrichum candidum, a dominant strain used to manufacture soft cheese with white rind. In this study, we analyzed the molecular mechanism of FADS12 function by swapping domains from Mortierella alpina and G. candidum that had, respectively, high and low oleic acid conversion rates. The results revealed three regions that are essential to this process, including regions from the end of the second transmembrane domain to the beginning of the third transmembrane domain, from the end of the third transmembrane domain to the beginning of the fourth transmembrane domain, and from the 30-amino acid from the end of the sixth transmembrane domain to the C-terminal end region. Based on our domain swapping analyses, nine pairs of amino acids including H112, S118, H156, Q161, K301, R306, E307, A309 and S323 in MaFADS12 (K123, A129, N167, M172, T302, D307, I308, E310 and D324 in GcFADS12) were identified as having a significantly effect on FADS12 catalytic efficiency, and linoleic acid and its analogues (12,13-cyclopropenoid fatty acid) were found to inhibit the catalytic activity of FADS12 and related recombinant enzymes. Furthermore, the molecular mechanism of FADS12 inhibition was analyzed. The results revealed two allosteric domains, including one domain from the N-terminal region to the beginning of the first transmembrane domain and another from the 31st amino acid from the end of the sixth transmembrane domain to the C terminus. Y4 and F398 amino acid residues from MaFADS12 and eight pairs of amino acids including G56, L60, L344, G10, Q13, S24, K326 and L344 in MaFADS12 (while Y66, F70, F345, F20, Y23, Y34, F327 and F345 in GcFADS12) played a pivotal role in FADS12 inhibition. Finally, we found that both allosteric and active sites were responsible for the catalytic activity of FADS12 at various temperatures, pH, and times. This study offers a solid theoretical basis to develop preconditioning methods to increase the rate at which GcFADS12 converts oleic and linoleic acids to produce higher levels of EFAs in cheese.

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Location of the photosynthetic carbon metabolism in microcompartments and separated phases in microalgal cells

Launay,

Avilan,

Gérard

et al. 2023

FEBS Letters

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Carbon acquisition, assimilation and storage in eukaryotic microalgae and cyanobacteria occur in multiple compartments that have been characterised by the location of the enzymes involved in these functions. These compartments can be delimited by bilayer membranes, such as the chloroplast, the lumen, the peroxisome, the mitochondria or monolayer membranes, such as lipid droplets or plastoglobules. They can also originate from liquid–liquid phase separation such as the pyrenoid. Multiple exchanges exist between the intracellular microcompartments, and these are reviewed for the CO2 concentration mechanism, the Calvin–Benson–Bassham cycle, the lipid metabolism and the cellular energetic balance. Progress in microscopy and spectroscopic methods opens new perspectives to characterise the molecular consequences of the location of the proteins involved, including intrinsically disordered proteins.

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Molecular mechanisms underlying catalytic activity of delta 6 desaturase from Glossomastix chrysoplasta and Thalassiosira pseudonana

Cited by 14 publications

References 22 publications

Δ12 fatty acid desaturase gene from Geotrichum candidum in cheese: molecular cloning and functional characterization

Δ12 fatty acid desaturase gene from Geotrichum candidum in cheese: molecular cloning and functional characterization

Both Determinants of Allosteric and Active Sites Responsible for Catalytic Activity of Delta 12 Fatty Acid Desaturase by Domain Swapping

Location of the photosynthetic carbon metabolism in microcompartments and separated phases in microalgal cells

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