A Fluorescence‐Based Assay for Quantitative Analysis of Phospholipid:Diacylglycerol Acyltransferase Activity

Falarz, Lucas J.; Xu, Yang; Singer, Stacy D.; Chen, Guanqun

doi:10.1002/lipd.12190

Cited by 3 publications

(2 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Using artificial acyl acceptors, such as di-6:0-DAG, DGAT assays do not necessarily reflect the in planta DGAT specificity with natural acyl acceptors. Whether this is the case also for PDAT assays (Falarz et al, 2019) remains to be established. The substrate specificities of C. sativa DGATs and PDAT presented here corroborate well the changes in TAG species found in seeds of C. sativa with downregulated DGAT1 and upregulated PDAT expression, respectively (Marmon et al, 2017).…”

Section: Properties Of C Sativa Pdctmentioning

confidence: 99%

Acyltransferases Regulate Oil Quality in Camelina sativa Through Both Acyl Donor and Acyl Acceptor Specificities

et al. 2020

View full text Add to dashboard Cite

Camelina sativa is an emerging biotechnology oil crop. However, more information is needed regarding its innate lipid enzyme specificities. We have therefore characterized several triacylglycerol (TAG) producing enzymes by measuring in vitro substrate specificities using different combinations of acyl-acceptors (diacylglycerol, DAG) and donors. Specifically, C. sativa acyl-CoA:diacylglycerol acyltransferase (DGAT) 1 and 2 (which both use acyl-CoA as acyl donor) and phospholipid:diacylglycerol acyltransferase (PDAT, with phosphatidylcoline as acyl donor) were studied. The results show that the DGAT1 and DGAT2 specificities are complementary, with DGAT2 exhibiting a high specificity for acyl acceptors containing only polyunsaturated fatty acids (FAs), whereas DGAT1 prefers acyl donors with saturated and monounsaturated FAs. Furthermore, the combination of substrates that resulted in the highest activity for DGAT2, but very low activity for DGAT1, corresponds to TAG species previously shown to increase in C. sativa seeds with downregulated DGAT1. Similarly, the combinations of substrates that gave the highest PDAT1 activity were also those that produce the two TAG species (54:7 and 54:8 TAG) with the highest increase in PDAT overexpressing C. sativa seeds. Thus, the in vitro data correlate well with the changes in the overall fatty acid profile and TAG species in C. sativa seeds with altered DGAT1 and PDAT activity. Additionally, in vitro studies of C. sativa phosphatidycholine:diacylglycerol cholinephosphotransferase (PDCT), another activity involved in TAG biosynthesis, revealed that PDCT accepts substrates with different desaturation levels. Furthermore, PDCT was unable to use DAG with ricineoleyl groups, and the presence of this substrate also inhibited PDCT from using other DAG-moieties. This gives insights relating to previous in vivo studies regarding this enzyme.

show abstract

Section: Properties Of C Sativa Pdctmentioning

confidence: 99%

Acyltransferases Regulate Oil Quality in Camelina sativa Through Both Acyl Donor and Acyl Acceptor Specificities

et al. 2020

View full text Add to dashboard Cite

show abstract

“…Site-directed mutagenesis of AtPDAT1 was carried out using overlap extension PCR (Heckman and Pease, 2007) with the primers listed in Table S9. Heterologous protein expression and in vitro PDAT activity assays were conducted following the procedure described in our previous study (Falarz et al, 2019). In brief, the wild-type AtP-DAT1, two mutant AtPDAT1 and LacZ negative control were cloned into the pYES2.1/V5-His TOPO vector (Invitrogen, now Thermo-Fisher Scientific, https://www.thermofisher.com), respectively, according to the manufacturer's manual.…”

Section: Site-directed Mutagenesis Of Atpdat1 Heterologous Protein Ementioning

confidence: 99%

Characterization of the diversification of phospholipid:diacylglycerol acyltransferases in the green lineage

Falarz

Caldo

et al. 2020

The Plant Journal

Self Cite

View full text Add to dashboard Cite

SUMMARY Triacylglycerols have important physiological roles in photosynthetic organisms, and are widely used as food, feed and industrial materials in our daily life. Phospholipid:diacylglycerol acyltransferase (PDAT) is the pivotal enzyme catalyzing the acyl‐CoA‐independent biosynthesis of triacylglycerols, which is unique in plants, algae and fungi, but not in animals, and has essential functions in plant and algal growth, development and stress responses. Currently, this enzyme has yet to be examined in an evolutionary context at the level of the green lineage. Some fundamental questions remain unanswered, such as how PDATs evolved in photosynthetic organisms and whether the evolution of terrestrial plant PDATs from a lineage of charophyte green algae diverges in enzyme function. As such, we used molecular evolutionary analysis and biochemical assays to address these questions. Our results indicated that PDAT underwent divergent evolution in the green lineage: PDATs exist in a wide range of plants and algae, but not in cyanobacteria. Although PDATs exhibit the conservation of several features, phylogenetic and selection‐pressure analyses revealed that overall they evolved to be highly divergent, driven by different selection constraints. Positive selection, as one major driving force, may have resulted in enzymes with a higher functional importance in land plants than green algae. Further structural and mutagenesis analyses demonstrated that some amino acid sites under positive selection are critically important to PDAT structure and function, and may be central in lecithin:cholesterol acyltransferase family enzymes in general.

show abstract

A new method for quantifying the enzyme activity of DGKs

Barbernitz,

Raben

2024

Advances in Biological Regulation

View full text Add to dashboard Cite

A Fluorescence‐Based Assay for Quantitative Analysis of Phospholipid:Diacylglycerol Acyltransferase Activity

Cited by 3 publications

References 31 publications

Acyltransferases Regulate Oil Quality in Camelina sativa Through Both Acyl Donor and Acyl Acceptor Specificities

Acyltransferases Regulate Oil Quality in Camelina sativa Through Both Acyl Donor and Acyl Acceptor Specificities

Characterization of the diversification of phospholipid:diacylglycerol acyltransferases in the green lineage

A new method for quantifying the enzyme activity of DGKs

Contact Info

Product

Resources

About