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A molecular model for diacylglycerol acyltransferase from Mortierella ramanniana var. angulispora.

ABSTRACT: Acyl CoA diacylglycerol acyltransferase (DGAT, EC 2.3.120) is recognized as a key player of cellular diacylglycerol metabolism. It catalyzes the terminal, yet the committed step in triacylglycerol synthesis using diacylglycerol and fatty acyl CoA as substrates. The protein sequence of diacylglycerol acyltransferse (DGAT) Type 2B in Moretierella ramanniana var. angulispora (Protein_ID = AAK84180.1) was retrieved from GenBank. However, a structure is not yet available for this sequence. The 3D structure of DGAT Type 2B was modeled using a template structure (PDB ID: 1K30) obtained from Protein databank (PDB) identified by searching with position specific iterative BLAST (PSI-BLAST). The template (PDB ID: 1K30) describes the structure of DGAT from Cucurbita moschata. Modeling was performed using Modeller 9v2 and protein model is hence generated. The DGAT type 2B protein model was subsequently docked with six inhibitors (sphingosine; trifluoroperazine; phosphatidic acid; lysophospatidylserine; KCl; 1, 2-diolein) using AutoDock (a molecular docking program). The binding of inhibitors to the protein model of DGAT type 2B is discussed.

SUBMITTER: Mishra S

PROVIDER: S-EPMC2732034 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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A molecular model for diacylglycerol acyltransferase from Mortierella ramanniana var. angulispora.

Mishra Sanjay S Dwivedi Surya Prakash SP Dwivedi Neeraja N Kumar Ajay A Rawat Anil A Kamisaka Yasushi Y

Bioinformation 20090628 9

Acyl CoA diacylglycerol acyltransferase (DGAT, EC 2.3.120) is recognized as a key player of cellular diacylglycerol metabolism. It catalyzes the terminal, yet the committed step in triacylglycerol synthesis using diacylglycerol and fatty acyl CoA as substrates. The protein sequence of diacylglycerol acyltransferse (DGAT) Type 2B in Moretierella ramanniana var. angulispora (Protein_ID = AAK84180.1) was retrieved from GenBank. However, a structure is not yet available for this sequence. The 3D str ...[more]

PMID: 19759814

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Project description:Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme in triacylglycerol biosynthesis in eukaryotic organisms. Triacylglycerols are important energy-storage oils in plants such as peanuts, soybeans and rape. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2) genes were cloned from the peanut cultivar 'Luhua 14' using a homologous gene sequence method and rapid amplification of cDNA ends. To understand the role of AhDGAT2 in triacylglycerol biosynthesis, two AhDGAT2 nucleotide sequences that differed by three amino acids were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli Rosetta (DE3). Following IPTG induction, the isozymes (AhDGAT2a and AhDGAT2b) were expressed as 64.5 kDa GST fusion proteins. Both AhDGAT2a and AhDGAT2b occurred in the host cell cytoplasm and inclusion bodies, with larger amounts in the inclusion bodies. Overexpression of AhDGATs depressed the host cell growth rates relative to non-transformed cells, but cells harboring empty-vector, AhDGAT2a-GST, or AhDGAT2b-GST exhibited no obvious growth rate differences. Interestingly, induction of AhDGAT2a-GST and AhDGAT2b-GST proteins increased the sizes of the host cells by 2.4-2.5 times that of the controls (post-IPTG induction). The total fatty acid (FA) levels of the AhDGAT2a-GST and AhDGAT2a-GST transformants, as well as levels of C12:0, C14:0, C16:0, C16:1, C18:1n9c and C18:3n3 FAs, increased markedly, whereas C15:0 and C21:0 levels were lower than in non-transformed cells or those containing empty-vectors. In addition, the levels of some FAs differed between the two transformant strains, indicating that the two isozymes might have different functions in peanuts. This is the first time that a full-length recombinant peanut DGAT2 has been produced in a bacterial expression system and the first analysis of its effects on the content and composition of fatty acids in E. coli. Our results indicate that AhDGAT2 is a strong candidate gene for efficient FA production in E. coli.

Project description:Diacylglycerol acyltransferases (DGATs) esterify sn-1,2-diacylglycerol with a long-chain fatty acyl-CoA, the last and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. At least 74 DGAT2 sequences from 61 organisms have been identified, but the expression of any DGAT2 as a partial or full-length protein in Escherichia coli had not been reported. The main objective of this study was to express and purify recombinant DGAT2 (rDGAT2) from E. coli for antigen production with a minor objective to compare rDGAT2 expression in yeast. A plasmid was engineered to express tung tree DGAT2 fused to maltose binding protein and poly-histidine (His) affinity tags. Immunoblotting showed that rDGAT2 was detected in the soluble, insoluble, and membrane fractions. The rDGAT2 in the soluble fraction was partially purified by amylose resin, nickel-nitrilotriacetic agarose (Ni-NTA) beads, and tandem affinity chromatography. Multiple proteins co-purified with rDGAT2. Size exclusion chromatography estimated the size of the rDGAT2-enriched fraction to be approximately eight times the monomer size. Affinity-purified rDGAT2 fractions had a yellow tint and contained fatty acids. The rDGAT2 in the insoluble fraction was partially solubilized by seven detergents with SDS being the most effective. Recombinant DGAT2 was purified to near homogeneity by SDS solubilization and Ni-NTA affinity chromatography. Mass spectrometry identified rDGAT2 as a component in the bands corresponding to the monomer and dimer forms as observed by SDS-PAGE. Protein bands with monomer and dimer sizes were also observed in the microsomal membranes of Saccharomyces cerevisiae expressing hemagglutinin-tagged DGAT2. Nonradioactive assay showed TAG synthesis activity of DGAT2 from yeast but not E. coli. The results suggest that rDGAT2 is present as monomer and dimer forms on SDS-PAGE, associated with other proteins, lipids, and membranes, and that post-translational modification of rDGAT2 may be required for its enzymatic activity and/or the E. coli protein is misfolded.

Dataset Information

A molecular model for diacylglycerol acyltransferase from Mortierella ramanniana var. angulispora.

Publications

A molecular model for diacylglycerol acyltransferase from Mortierella ramanniana var. angulispora.

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