Defining the extreme substrate specificity of <i>Euonymus alatus</i> diacylglycerol acetyltransferase, an unusual membrane-bound <i>O</i>-acyltransferase

Bansal, Sunil; Durrett, Timothy P.

doi:10.1042/bsr20160277

Cited by 14 publications

(14 citation statements)

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“…Together, these results demonstrated that EaDAcT was able to acetylate MCFA diacylglycerols (DAGs) such as 1,2-dilaurin- sn -3-glycerol and 1,2-dimyristoyl- sn -3-glycerol to generate acetyl-dilaurin and acetyl-dimyristin in camelina seed. Such activities have been shown in vitro in our previous work ( Bansal and Durrett, 2016 a ).…”

Section: Resultssupporting

confidence: 76%

Towards the synthetic design of camelina oil enriched in tailored acetyl-triacylglycerols with medium-chain fatty acids

Bansal

Kim

et al. 2018

Journal of Experimental Botany

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Section: Resultssupporting

confidence: 76%

Towards the synthetic design of camelina oil enriched in tailored acetyl-triacylglycerols with medium-chain fatty acids

Bansal

Kim

et al. 2018

Journal of Experimental Botany

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“…Instead, in Brassica napus DGAT1 this hydrophilic region has been shown to interact with long-chain acyl-CoA in a positive cooperative fashion (Weselake et al, 2006). The absence of this region therefore might explain why EaDAcT possesses a non-cooperative response to acetyl-CoA concentration (Bansal and Durrett, 2016a), whereas DGAT1 enzymes possess a positive cooperative response to oleyl-CoA (Roesler et al, 2016), though additional quantification of EaDAcT activity at lower concentrations of acetyl-CoA is needed to confirm the enzyme's non-cooperative kinetics.…”

Section: Membrane Localization and Orientation Of Eadactmentioning

confidence: 99%

“…The presence of an acetyl group instead of a longer fatty acyl group gives acetyl-TAG altered physical properties compared with regular triacylglycerols (here referred to as lcTAG), including reduced viscosity and lower melting temperatures (Durrett et al, 2010;Liu et al, 2015). The preferred substrate of EaDAcT is DAG, but it is also capable of acetylating fatty alcohols to form alkyl acetates, albeit with reduced efficiency (Bansal and Durrett, 2016a;Ding et al, 2016). This weak wax synthase activity is consistent with the enzyme's sequence similarity to the jojoba wax synthase, which distances EaDAcT from the DGAT1 enzymes with which it shares a common DAG substrate (Durrett et al, 2010;Liu et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Membrane topology and identification of key residues of EaDAcT, a plant MBOAT with unusual substrate specificity

et al. 2017

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Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) catalyzes the transfer of an acetyl group from acetyl-CoA to the sn-3 position of diacylglycerol to form 3-acetyl-1,2-diacyl-sn-glycerol (acetyl-TAG). EaDAcT belongs to a small, plant-specific subfamily of the membrane bound O-acyltransferases (MBOAT) that acylate different lipid substrates. Sucrose gradient density centrifugation revealed that EaDAcT colocalizes to the same fractions as an endoplasmic reticulum (ER)-specific marker. By mapping the membrane topology of EaDAcT, we obtained an experimentally determined topology model for a plant MBOAT. The EaDAcT model contains four transmembrane domains (TMDs), with both the N- and C-termini orientated toward the lumen of the ER. In addition, there is a large cytoplasmic loop between the first and second TMDs, with the MBOAT signature region of the protein embedded in the third TMD close to the interface between the membrane and the cytoplasm. During topology mapping, we discovered two cysteine residues (C187 and C293) located on opposite sides of the membrane that are important for enzyme activity. In order to identify additional amino acid residues important for acetyltransferase activity, we isolated and characterized acetyltransferases from other acetyl-TAG-producing plants. Among them, the acetyltransferase from Euonymus fortunei possessed the highest activity in vivo and in vitro. Mutagenesis of conserved amino acids revealed that S253, H257, D258 and V263 are essential for EaDAcT activity. Alteration of residues unique to the acetyltransferases did not alter the unique acyl donor specificity of EaDAcT, suggesting that multiple amino acids are important for substrate recognition.

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“…In a few publications dedicated to the investigation of Ac DAG, the following synonyms can be encountered: acetylated triacylglycerols (acTAG; Bansal & Durrett, ; Durrett et al, ), 1,2‐diacyl‐3‐acetates (Kleiman et al, ), α‐acetotriglycerides (Spitzer & Aichholz, ), and monoacetyldiglycerides (MADG) (Limb, Kim, Han, & Jhon, ; Yang et al, ), of which only one unambiguously indicates the sn ‐position in the Ac DAG molecule acylated by the acetic acid residue. In the glycerol framework of Ac DAG, only one sn ‐position is acetylated—the terminal one (or, α‐position); and, therefore, logically, natural Ac DAG should be a racemic mixture of sn ‐1,2(2,3)‐diacyl‐3(1)‐acetylglycerols (Fig.…”

Section: Introductionmentioning

confidence: 99%

Evidence of Strict Stereospecificity in the Structure of sn‐1,2‐Diacyl‐3‐Acetyl‐Glycerols from Euonymus maximowiczianus Seeds Using Nuclear Magnetic Resonance Spectroscopy

Sidorov

Shashkov

Solovyev

et al. 2018

Lipids

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Asymmetric, optically active sn-1,2-diacyl-3-acetyl-glycerols (AcDAG) have been known to scientists for several decades. However, to date, the problem of their structure has not been definitely resolved, which has led to a vast diversity of terms used for their designation in the literature. Using two-dimensional nuclear magnetic resonance, we have investigated AcDAG from the mature seeds of Euonymus maximowiczianus, from which we have been able to both identify a correlation of the methyl group in acetic acid residue with protons at the carbon atom at sn-3 position in the glycerol residue of the AcDAG molecule and, for the first time, demonstrate that this correlation is observed exclusively with one carbon atom at the α-position, but not with two as would have been expected in case of a racemic mixture. Moreover, results of our analysis of AcDAG isolated from the seeds of E. maximowiczianus directly confirm that diacylglycerol-3-acetyl-transferase is responsible for their biosynthesis, which reveals a strict specificity not only to acetyl-CoA as one of the substrates but also to the sn-3-position of the glycerol residue in sn-1,2-diacylglycerol during their biosynthesis.

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Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

Abstract: The membrane-bound O-acyltransferase Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) preferentially uses acetyl-CoA to acetylate sn-1,2 DAGs but other acyl-donor and acyl-acceptor substrates can be used with low efficiency.

Cited by 14 publications

References 23 publications

Towards the synthetic design of camelina oil enriched in tailored acetyl-triacylglycerols with medium-chain fatty acids

Towards the synthetic design of camelina oil enriched in tailored acetyl-triacylglycerols with medium-chain fatty acids

Membrane topology and identification of key residues of EaDAcT, a plant MBOAT with unusual substrate specificity

Evidence of Strict Stereospecificity in the Structure of sn‐1,2‐Diacyl‐3‐Acetyl‐Glycerols from Euonymus maximowiczianus Seeds Using Nuclear Magnetic Resonance Spectroscopy

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