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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 ram...
Citations
... Mishra et al. reported that the mechanism of action of DGAT Type 2B in Moretierella ramanniana var. angulispora involves a catalytic triad composed of conserved Cys112, His238 and Asn276 [43]. By comparison of two DGAT2 gene homologues of an oleaginous yeast, five AA variations are related to the 2D and 3D structure of CtDGAT2a and CtDGAT2b leading to the different enzyme activity [22]. ...
Triacylglycerols is the major storage lipid in most crop seeds. As the key enzyme catalyzing the final step of triacylglycerols biosynthesis, the activity of diacylglycerol acyltransferaseis directly related to oil content. It has been shown that certain amino acids are very important for enzyme activity, one amino acid variation will greatly change the enzyme activity. In this study, we identified three amino acid point mutations that affect the Arachis hypogaea diacylglycerol acyltransferase 2 enzyme activity, T107M, K251R and L316P. According to the three amino acid variations, three single-nucleotide-mutant sequences of Arachis hypogaea diacylglycerol acyltransferase 2a were constructed and transformed into yeast strain H1246 for function verification. Results showed that T107M and K251R could change the fatty acid content and composition of the transformed yeast strains, whereas L316P led to the loss of enzyme activity. By analyzing the 2D and 3D structures of the three variants, we found that the changes of spatial structure of T107M, K251R and L316P caused the changes of the enzyme activity. Our study could provide a theoretical basis for changing the enzyme activity of DGAT by genetic engineering, and provide a new idea for increasing the oil content of the crops.
... The secondary structures of the two CtDGAT2 isozymes were predicted using phyre2 webserver (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id = index). Three-dimensional structure of CtDGAT2a and CtDGAT2b were developed using MODELLER 8v2 taking the crystal structure of glyceraldehyde-3-phosphate dehydrogenase from Methanocaldococcus jannaschii DSM 2661 (PDB ID: 3RHD) as the template showing sequence identity of .35% with both isozymes revealed by PSI-BLAST [29]. Energy minimization of the resulting model was carried out by Insight II program (version 2000.1, ...
Diacylglycerol acyltransferase (DGAT) activity is an essential enzymatic step in the formation of neutral lipid i.e., triacylglycerol in all living cells capable of accumulating storage lipid. Previously, we characterized an oleaginous yeast Candida tropicalis SY005 that yields storage lipid up to 58% under a specific nitrogen-stress condition, when the DGAT-specific transcript is drastically up-regulated. Here we report the identification, differential expression and function of two DGAT2 gene homologues- CtDGAT2a and CtDGAT2b of this C. tropicalis. Two protein isoforms are unique with respect to the presence of five additional stretches of amino acids, besides possessing three highly conserved motifs known in other reported DGAT2 enzymes. Moreover, the CtDGAT2a and CtDGAT2b are characteristically different in amino acid sequences and predicted protein structures. The CtDGAT2b isozyme was found to be catalytically 12.5% more efficient than CtDGAT2a for triacylglycerol production in a heterologous yeast system i.e., Saccharomyces cerevisiae quadruple mutant strain H1246 that is inherently defective in neutral lipid biosynthesis. The CtDGAT2b activity rescued the growth of transformed S. cerevisiae mutant cells, which are usually non-viable in the medium containing free fatty acids by incorporating them into triacylglycerol, and displayed preferential specificity towards saturated acyl species as substrate. Furthermore, we document that the efficiency of triacylglycerol production by CtDGAT2b is differentially affected by deletion, insertion or replacement of amino acids in five regions exclusively present in two CtDGAT2 isozymes. Taken together, our study characterizes two structurally novel DGAT2 isozymes, which are accountable for the enhanced production of storage lipid enriched with saturated fatty acids inherently in C. tropicalis SY005 strain as well as in transformed S. cerevisiae neutral lipid-deficient mutant cells. These two genes certainly will be useful for further investigation on the novel structure-function relationship of DGAT repertoire, and also in metabolic engineering for the enhanced production of lipid feedstock in other organisms.
... angulispora utilizes a broad range of molecular species of both diacylglycerol and acyl-CoA as substrates (Kamisaka et al., 1997). Molecular studies on DGAT for M. ramanniana have been reported (Lardizabal et al., 2001;Mishra et al., 2009). Storage lipids can be mobilized upon the requirement of cells, especially during starvation, meiosis and sporulation. ...
Polyunsaturated fatty acids (PUFAs) are functional lipids that have been widely incorporated into several industrial sectors.
Apart from animal- and plant-derived origins, oleaginous fungi belonging to Mucorales have been identified as promising alternatives
for production of n-3 and n-6 PUFAs. It was found, in Mucorales fungi, that ATP:citrate lyase, acetyl-CoA carboxylase and malic enzyme trigger lipid
overproduction, and biosynthesis of PUFA requires membrane-bound desaturases with fatty acyl substrate specificities. Accumulation
of PUFAs in the cells is associated not only with the desaturation system, but it is also tightly bound with acyltransferases
that facilitate the distribution of newly synthesized PUFA to individual lipid structures. Several physical parameters, such
as temperature, aeration, and nutrient regimes, greatly affect either the lipid content or fatty acid composition among different
Mucorales species. Conclusive evidence showed that the PUFA production yield of the fungi depends on the environmental control
of “oleaginous” enzymes, and on the transcriptional expression of the desaturase genes. These valuable studies provide perspectives
with biological rationale for microbial production of economically important lipids.
Keywordspolyunsaturated fatty acids–oleaginous fungi–fatty acid desaturation–lipid accumulation–gene expression
