Dataset Information

Intrinsic disorder in the regulatory N-terminal domain of diacylglycerol acyltransferase 1 from Brassica napus.

ABSTRACT: Proteins with multifunctional regulatory domains often demonstrate structural plasticity or protein disorder, allowing the binding of multiple regulatory factors and post-translational modifications. While the importance of protein disorder is clear, it also poses a challenge for in vitro characterization. Here, we report protein intrinsic disorder in a plant molecular system, which despite its prevalence is less studied. We present a detailed biophysical characterization of the entire cytoplasmic N-terminal domain of Brassica napus diacylglycerol acyltransferase, (DGAT1), which includes an inhibitory module and allosteric binding sites. Our results demonstrate that the monomeric N-terminal domain can be stabilized for biophysical characterization and is largely intrinsically disordered in solution. This domain interacts with allosteric modulators of DGAT1, CoA and oleoyl-CoA, at micromolar concentrations. While solution scattering studies indicate conformational heterogeneity in the N-terminal domain of DGAT1, there is a small gain of secondary structure induced by ligand binding.

SUBMITTER: Panigrahi R

PROVIDER: S-EPMC6232145 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Intrinsic disorder in the regulatory N-terminal domain of diacylglycerol acyltransferase 1 from Brassica napus.

Panigrahi Rashmi R Matsui Tsutomu T Song Andrew H AH Caldo Kristian Mark P KMP Young Howard S HS Weselake Randall J RJ Lemieux M Joanne MJ

Scientific reports 20181112 1

Proteins with multifunctional regulatory domains often demonstrate structural plasticity or protein disorder, allowing the binding of multiple regulatory factors and post-translational modifications. While the importance of protein disorder is clear, it also poses a challenge for in vitro characterization. Here, we report protein intrinsic disorder in a plant molecular system, which despite its prevalence is less studied. We present a detailed biophysical characterization of the entire cytoplasm ...[more]

PMID: 30420764

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Project description:BackgroundBrassica napus is one of the most important oilseed crops, and can supply considerable amounts of edible oil as well as provide raw materials for the production of biodiesel in the biotechnology industry. Lysophosphatidic acid acyltransferase (LPAT), a key enzyme in the Kennedy pathway, catalyses fatty acid chains into 3-phosphoglycerate and promotes further production of oil in the form of triacylglycerol. However, because B. napus is an allotetraploid with two subgenomes, the precise genes which involved in oil production remain unclear due to the intractability of efficiently knocking out all copies with high genetic redundancy. Therefore, a robust gene editing technology is necessary for gene function analysis.ResultsAn efficient gene editing technology was developed for the allotetraploid plant B. napus using the CRISPR-Cas9 system. Previous studies showed poor results in either on-target or off-target activity in B. napus. In the present study, four single-gRNAs and two multi-gRNAs were deliberately designed from the conserved coding regions of BnLPAT2 which has seven homologous genes, and BnLPAT5, which has four homologous genes. The mutation frequency was found to range from 17 to 68%, while no mutation was observed in the putative off-target sites. The seeds of the Bnlpat2/Bnlpat5 mutant were wizened and showed enlarged oil bodies, disrupted distribution of protein bodies and increased accumulation of starch in mature seeds. The oil content decreased, with an average decrease of 32% for Bnlpat2 lines and 29% for Bnlpat5 lines in single-gRNA knockout lines, and a decline of 24% for Bnlpat2 mutant lines (i.e., g123) and 39% for Bnlpat2/Bnlpat5 double mutant lines (i.e., g134) in multi-gRNA knockout lines.ConclusionsSeven BnLPAT2 homologous genes and four BnLPAT5 homologous genes were cleaved completely using the CRISPR-Cas9 system, which indicated that it is effective for editing all homologous genes in allotetraploid rapeseed, despite the relatively low sequence identities of both gene families. The size of the oil bodies increased significantly while the oil content decreased, confirming that BnLPAT2 and BnLPAT5 play a role in oil biosynthesis. The present study lays a foundation for further oil production improvement in oilseed crop species.

Dataset Information

Intrinsic disorder in the regulatory N-terminal domain of diacylglycerol acyltransferase 1 from Brassica napus.

Publications

Intrinsic disorder in the regulatory N-terminal domain of diacylglycerol acyltransferase 1 from Brassica napus.

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