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Structural determinant of functionality in acyl lipid desaturases.

ABSTRACT: Little is known about the structure-function relationship of membrane-bound lipid desaturases. Using a domain-swapping strategy, we found that the N terminus (comprising the two first transmembrane segments) region of Bacillus cereus DesA desaturase improves Bacillus subtilis Des activity. In addition, the replacement of the first two transmembrane domains from Bacillus licheniformis inactive open reading frame (ORF) BL02692 with the corresponding domain from DesA was sufficient to resurrect this enzyme. Unexpectedly, we were able to restore the activity of ORF BL02692 with a single substitution (Cys40Tyr) of a cysteine localized in the first transmembrane domain close to the lipid-water interface. Substitution of eight residues (Gly90, Trp104, Lys172, His228, Pro257, Leu275, Tyr282, and Leu284) by site-directed mutagenesis produced inactive variants of DesA. Homology modeling of DesA revealed that His228 is part of the metal binding center, together with the canonical His boxes. Trp104 shapes the hydrophobic tunnel, whereas Gly90 and Lys172 are probably involved in substrate binding/recognition. Pro257, Leu275, Tyr282, and Leu284 might be relevant for the structural arrangement of the active site or interaction with electron donors. This study reveals the role of the N-terminal region of ?5 phospholipid desaturases and the individual residues necessary for the activity of this class of enzymes.

SUBMITTER: Sastre DE

PROVIDER: S-EPMC6168307 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Structural determinant of functionality in acyl lipid desaturases.

Sastre Diego E DE Saita Emilio E Uttaro Antonio D AD de Mendoza Diego D Altabe Silvia G SG

Journal of lipid research 20180807 10

Little is known about the structure-function relationship of membrane-bound lipid desaturases. Using a domain-swapping strategy, we found that the N terminus (comprising the two first transmembrane segments) region of Bacillus cereus DesA desaturase improves Bacillus subtilis Des activity. In addition, the replacement of the first two transmembrane domains from Bacillus licheniformis inactive open reading frame (ORF) BL02692 with the corresponding domain from DesA was suffic ...[more]

PMID: 30087203

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Project description:Background:Dietary omega-3 (n-3), long chain (LC-,???20 carbons), polyunsaturated fatty acids (PUFAs) derived largely from marine animal sources protect against inflammatory processes and enhance brain development and function. With the depletion of natural stocks of marine animal sources and an increasing demand for n-3 LC-PUFAs, alternative, sustainable supplies are urgently needed. As a result, n-3 18-carbon and LC-PUFAs are being generated from plant or algal sources, either by engineering new biosynthetic pathways or by augmenting existing systems. Results:We utilized an engineered plasmid encoding two cyanobacterial acyl-lipid desaturases (DesB and DesD, encoding ?15 and ?6 desaturases, respectively) and "vesicle-inducing protein in plastids" (Vipp1) to induce production of stearidonic acid (SDA, 18:4 n-3) at high levels in three strains of cyanobacteria (10, 17 and 27% of total lipids in Anabaena sp. PCC7120, Synechococcus sp. PCC7002, and Leptolyngbya sp. strain BL0902, respectively). Lipidomic analysis revealed that in addition to SDA, the rare anti-inflammatory n-3 LC-PUFA eicosatetraenoic acid (ETA, 20:4 n-3) was synthesized in these engineered strains, and?~?99% of SDA and ETA was complexed to bioavailable monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) species. Importantly, novel molecular species containing alpha-linolenic acid (ALA), SDA and/or ETA in both acyl positions of MGDG and DGDG were observed in the engineered Leptolyngbya and Synechococcus strains, suggesting that these could provide a rich source of anti-inflammatory molecules. Conclusions:Overall, this technology utilizes solar energy, consumes carbon dioxide, and produces large amounts of nutritionally important n-3 PUFAs and LC-PUFAs. Importantly, it can generate previously undescribed, highly bioavailable, anti-inflammatory galactosyl lipids. This technology could therefore be transformative in protecting ocean fisheries and augmenting the nutritional quality of human and animal food products.

Project description:BACKGROUND:With declining wild fish populations, farmed salmon has gained popularity as a source for healthy long-chain highly unsaturated fatty acids (LC-HUFA). However, the introduction of plant oil in farmed salmon feeds has reduced the content of these beneficial LC-HUFA. The synthetic capability for LC-HUFAs depends upon the dietary precursor fatty acids and the genetic potential, thus there is a need for in-depth understanding of LC-HUFA synthetic genes and their interactions with other genes involved in lipid metabolism. Several key genes of LC-HUFA synthesis in salmon belong to the fatty acid desaturases 2 (fads2) family. The present study applied whole transcriptome analysis on two CRISPR-mutated salmon strains (crispants), 1) ?6abc/5Mt with mutations in ?5fads2, ?6fads2-a, ?6fads2-b and ?6fads2-c genes, and 2) ?6bcMt with mutations in ?6fads2-b and ?6fads2-c genes. Our purpose is to evaluate the genetic effect fads2 mutations have on other lipid metabolism pathways in fish, as well as to investigate mosaicism in a commercial species with a very long embryonal period. RESULTS:Both ?6abc/5Mt and ?6bcMt crispants demonstrated high percentage of indels within all intended target genes, though different indel types and percentage were observed between individuals. The ?6abc/5Mt fish displayed several disruptive indels which resulted in over 100 differentially expressed genes (DEGs) enriched in lipid metabolism pathways in liver. This includes up-regulation of srebp1 genes which are known key transcription regulators of lipid metabolism as well as a number of down-stream genes involved in fatty acid de-novo synthesis, fatty acid ?-oxidation and lipogenesis. Both elovl5 and elovl2 genes were not changed, suggesting that the genes were not targeted by Srebp1. The mutation of ?6bcMt surprisingly resulted in over 3000 DEGs which were enriched in factors encoding genes involved in mRNA regulation and stability. CONCLUSIONS:CRISPR-Cas9 can efficiently mutate multiple fads2 genes simultaneously in salmon. The results of the present study have provided new information on the transcriptional regulations of lipid metabolism genes after reduction of LC-HUFA synthesis pathways in salmon.

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Structural determinant of functionality in acyl lipid desaturases.

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Structural determinant of functionality in acyl lipid desaturases.

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