Alteration of the Fatty Acid Substrate Specificity of Lysophosphatidate Acyltransferase by Site-Directed Mutagenesis

Morand, Larry Z.; Patil, Sanjay A.; Quasney, Mary E.; German, J. Bruce

doi:10.1006/bbrc.1998.8218

Cited by 8 publications

(5 citation statements)

References 25 publications

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“…The mutant studies have increased our understanding of the regulation of lipid biosynthesis [30,31]. The isolation of nonlethal mutants in LPA-AT and DAG-AT confirms the assumption that the segregated pools of lipids are generated in different regions of the cell by different isoforms of the enzymes and are used for different functions.…”

Section: Discussionmentioning

confidence: 61%

“…The JC201 strain of E. coli contains a temperaturesensitive lesion in LPA-AT [34], and a similar mutant was also isolated from S. cere isiae [35]. Alteration of this enzyme in the mutant is detrimental as it is involved in membrane lipid synthesis [30,34]. On the other hand, mutation in LPA-AT from Nesseria meningitidis [36] and the plastidial LPA-AT [37] did not cause any significant changes in the glycerolipid profile, indicating the presence of multiple pathways for PA biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

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Alteration in the cytosolic triacylglycerol biosynthetic machinery leads to decreased cell growth and triacylglycerol synthesis in oleaginous yeast

Gangar

Raychaudhuri

Rajasekharan

2002

Biochemical Journal

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Altered nutrient content (levels of glucose) caused a drastic reduction in cell growth and triacylglycerol (TAG) production in the wild-type (WT) Rhodotorula glutinis. This was due to the decreased level of synthesis of TAG biosynthetic enzymes, reflected by a reduction in enzyme activity. A similar observation was made in the case of non-lethal mutants of TAG-deficient oleaginous yeast, namely TAG1 and TAG2, which were generated by ethyl methane sulphonate mutagenesis. Metabolic labelling of TAG-deficient cells with [(14)C]acetate, [(32)P]orthophosphate and [(14)C]mevalonate showed a negligible TAG formation with minimal alterations in phospholipid and sterol compositions. Assays on the activities of cytosolic TAG biosynthetic enzymes revealed that lysophosphatidic acid and diacylglycerol acyltransferases (ATs) were defective in TAG1 and TAG2 respectively. The activity of membrane-bound isoforms of TAG biosynthetic enzymes remains unaltered in the mutants. Analysis of cytosolic TAG biosynthetic enzymes by immunoblotting and immunoprecipitation indicated that the defective ATs were a part of the TAG biosynthetic multienzyme complex. Quantitatively, the cytosolic lysophosphatidic acid-AT was comparable between TAG1 and the WT. However, diacylglycerol-AT was relatively less in TAG2 than the WT. These results demonstrated that either by decreasing the nutrient content or mutating the enzymes of the soluble TAG biosynthetic pathway, TAG production was decreased with concomitant reduction in the cell growth.

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Section: Discussionmentioning

confidence: 61%

Section: Discussionmentioning

confidence: 99%

Alteration in the cytosolic triacylglycerol biosynthetic machinery leads to decreased cell growth and triacylglycerol synthesis in oleaginous yeast

Gangar

Raychaudhuri

Rajasekharan

2002

Biochemical Journal

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“…The identification of a substrate‐specific DGAT followed by its over‐expression in the system of interest should assist in the incorporation of the respective fatty acid into TAG. Another alternative for improving the accumulation of unusual fatty acids could be the modification of the active site through methods such as site‐directed mutagenesis (Liu et al ., ; Morand et al ., ) and hence modify or improve the substrate specificity of acyltransferases. For example, the EgDGAT1 exhibited some specificity for C14:0, but preferred C12:0 and C16:0.…”

Section: Discussionmentioning

confidence: 99%

A reconfigured Kennedy pathway which promotes efficient accumulation of medium‐chain fatty acids in leaf oils

Reynolds

Taylor²,

Cullerne

et al. 2017

Plant Biotechnology Journal

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SummaryMedium‐chain fatty acids (MCFA, C6‐14 fatty acids) are an ideal feedstock for biodiesel and broader oleochemicals. In recent decades, several studies have used transgenic engineering to produce MCFA in seeds oils, although these modifications result in unbalance membrane lipid profiles that impair oil yields and agronomic performance. Given the ability to engineer nonseed organs to produce oils, we have previously demonstrated that MCFA profiles can be produced in leaves, but this also results in unbalanced membrane lipid profiles and undesirable chlorosis and cell death. Here we demonstrate that the introduction of a diacylglycerol acyltransferase from oil palm, EgDGAT1, was necessary to channel nascent MCFA directly into leaf oils and therefore bypassing MCFA residing in membrane lipids. This pathway resulted in increased flux towards MCFA rich leaf oils, reduced MCFA in leaf membrane lipids and, crucially, the alleviation of chlorosis. Deep sequencing of African oil palm (Elaeis guineensis) and coconut palm (Cocos nucifera) generated candidate genes of interest, which were then tested for their ability to improve oil accumulation. Thioesterases were explored for the production of lauric acid (C12:0) and myristic (C14:0). The thioesterases from Umbellularia californica and Cinnamomum camphora produced a total of 52% C12:0 and 40% C14:0, respectively, in transient leaf assays. This study demonstrated that the introduction of a complete acyl‐CoA‐dependent pathway for the synthesis of MFCA‐rich oils avoided disturbing membrane homoeostasis and cell death phenotypes. This study outlines a transgenic strategy for the engineering of biomass crops with high levels of MCFA rich leaf oils.

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“…A mutation (Q to L) located in the 44 th amino acid of yeast LPAAT has been implicated in altering its substrate specificity, which led to utilization of C 26 -CoA and growth even without sphinogolipid biosynthesis (10). A temperature sensitive E. coli LPAAT mutation (17) has been mapped to the 39 th amino acid with a change from Gly to Glu (18). Changing the Thr122 of E. coli LPAAT to Ala increases the substrate specificity of the enzyme for oleoyl (C18:1)-and linoleyl (C18:3)-CoA, whereas changing this Thr to Leu increases the substrate specificity for lignoceroyl (C24:0)-CoA (18).…”

Section: Conserved Motifs In Lpaat Sequencesmentioning

confidence: 99%

The structure and functions of human lysophosphatidic acid acyltransferases

Leung¹

2001

Front Biosci

128

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Lysophosphatidic acid (LPA) and phosphatidic acid (PA) are two phospholipids involved in signal transduction and in lipid biosynthesis in cells. LPA acyltransferase (LPAAT), also known as 1-acyl sn-glycerol-3-phosphate acyltransferase (1-AGPAT) (EC 2.3.1.51), catalyzes the conversion of LPA to PA. Two human isoforms of LPAAT, designated as LPAAT-alpha (AGPAT1) and LPAAT-beta (AGPAT2), have been extensively characterized. These two proteins contain extensive sequence similarities to microbial, plant and animal LPAAT sequences. LPAAT-alpha mRNA is uniformly expressed throughout most tissues with the highest level found in skeletal muscle; whereas LPAAT-beta is differentially expressed, with the highest level found in heart and liver, and negligible level in brain and placenta. The LPAAT-alpha gene is located on chromosome 6p21.3, an area within the class III region of the major hiscompatibility complex (MHC) and the LPAAT-beta gene is mapped to chromosome 9q34.3. Enhanced transcription of LPAAT-beta is suggested for neoplasm of the female genital tract. Additionally, ectopic LPAAT expression in certain cytokine-responsive cell lines can effect amplification of cellular signaling processes, such as those leading to enhancement of synthesis of tumor necrosis factor-alpha and interleukin-6 from cells following stimulation with interleukin-1beta; this suggests that the LPAAT genes represent candidates for affecting the development of certain cancers or inflammation-associated diseases.

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Alteration of the Fatty Acid Substrate Specificity of Lysophosphatidate Acyltransferase by Site-Directed Mutagenesis

Cited by 8 publications

References 25 publications

Alteration in the cytosolic triacylglycerol biosynthetic machinery leads to decreased cell growth and triacylglycerol synthesis in oleaginous yeast

Alteration in the cytosolic triacylglycerol biosynthetic machinery leads to decreased cell growth and triacylglycerol synthesis in oleaginous yeast

A reconfigured Kennedy pathway which promotes efficient accumulation of medium‐chain fatty acids in leaf oils

The structure and functions of human lysophosphatidic acid acyltransferases

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