A Novel High-Throughput Assay Reveals That the Temperature Induced Increases in Transphosphatidylation of Phospholipase D Are Dependent on the Alcohol Acceptor Concentration

Overview

Journal Biomolecules

Publisher MDPI

Specialties Biochemistry
Molecular Biology

Date 2022 May 28

PMID 35625563

Authors

Hengzhang Yang

Rudiger Woscholski

Affiliations

Soon will be listed here.

Abstract

Phospholipase D reacts with alcohols or water, transphosphatidylating or hydrolysing lipids such as phosphatidylcholine, generating phosphatidylalcohols or phosphatidic acid, respectively. The enzyme has been employed in many applications making use of the transphosphatidylation reaction and the enzyme's tolerance for organic solvents in order to synthesize natural and artificial phospholipids. Yet, its catalytic properties with respect to the transphosphatidylation reaction are not well understood. Here, we introduce a novel high-throughput assay, making use of 96-well plates, that employs Fluorescamine for the detection of transphosphatidylated amino alcohols. This assay allowed to monitor the and at different temperatures, revealing that the former will be elevated by the temperature, while the latter is increased by a combination of both temperature and alcohol acceptor concentration being elevated, suggesting that increase in temperature may open up a new binding site for the alcohol acceptor.

Citing Articles

NAD Promotes Superovulation of Huaxi Cattle Through Regulation of Cumulus Cell Proliferation and Oocyte Maturation.

Wang S, Liu M, Di A, Jiang X, Wu J, Zhang J Int J Mol Sci. 2025; 26(5).

PMID: 40076893 PMC: 11900452. DOI: 10.3390/ijms26052276.

Imidazole Headgroup Phospholipid Shows Asymmetric Distribution in Vesicles and Zinc-Dependent Esterase Activity.

Sachet-Fernandez G, Hindley J, Ces O, Woscholski R Biomolecules. 2024; 14(11).

PMID: 39595540 PMC: 11592132. DOI: 10.3390/biom14111363.

References

Takeda N, Akiyama Y . Rapid determination of sulphonamides in milk using liquid chromatographic separation and fluorescamine derivatization. J Chromatogr. 1992; 607(1):31-5. DOI: 10.1016/0021-9673(92)87051-9. View

Shuto S, Itoh H, Sakai A, Nakagami K, Imamura S, Matsuda A . Nucleosides and nucleotides--CXXXVII. Antitumor phospholipids with 5-fluorouridine as a cytotoxic polar-head: synthesis of 5'-phosphatidyl-5-fluorouridines by phospholipase D-catalyzed transphosphatidylation. Bioorg Med Chem. 1995; 3(3):235-43. DOI: 10.1016/0968-0896(95)00003-y. View

Gotor V, Flitsch S . Increasing the diversity of biocatalytic reactions. Curr Opin Chem Biol. 2011; 15(2):185-6. DOI: 10.1016/j.cbpa.2011.02.001. View

Ashby J, Duan Y, Ligans E, Tamsi M, Zhong W . High-throughput profiling of nanoparticle-protein interactions by fluorescamine labeling. Anal Chem. 2015; 87(4):2213-9. DOI: 10.1021/ac5036814. View

Hosokawa M, Shimatani T, Kanada T, Inoue Y, Takahashi K . Conversion to docosahexaenoic acid-containing phosphatidylserine from squid skin lecithin by phospholipase D-mediated transphosphatidylation. J Agric Food Chem. 2000; 48(10):4550-4. DOI: 10.1021/jf991186s. View

Yu C, Liu S, Panagia V . The transphosphatidylation activity of phospholipase D. Mol Cell Biochem. 1996; 157(1-2):101-5. DOI: 10.1007/BF00227886. View

Evans C, Ridella J . An evaluation of fluorometric proteinase assays which employ fluorescamine. Anal Biochem. 1984; 142(2):411-20. DOI: 10.1016/0003-2697(84)90485-8. View

Selvy P, Lavieri R, Lindsley C, Brown H . Phospholipase D: enzymology, functionality, and chemical modulation. Chem Rev. 2011; 111(10):6064-119. PMC: 3233269. DOI: 10.1021/cr200296t. View

Brennan J, Tillitt D . Development of a dual luciferase activity and fluorescamine protein assay adapted to a 384 micro-well plate format: Reducing variability in human luciferase transactivation cell lines aimed at endocrine active substances. Toxicol In Vitro. 2017; 47:18-25. DOI: 10.1016/j.tiv.2017.10.030. View

10.

Martin S, DeBlanc R, Hergenrother P . Determination of the substrate specificity of the phospholipase D from Streptomyces chromofuscus via an inorganic phosphate quantitation assay. Anal Biochem. 2000; 278(2):106-10. DOI: 10.1006/abio.1999.4420. View

11.

Dittrich N, Ulbrich-Hofmann R . Transphosphatidylation by immobilized phospholipase D in aqueous media. Biotechnol Appl Biochem. 2001; 34(3):189-94. DOI: 10.1042/ba20010032. View

12.

Damnjanovic J, Iwasaki Y . Phospholipase D as a catalyst: application in phospholipid synthesis, molecular structure and protein engineering. J Biosci Bioeng. 2013; 116(3):271-80. DOI: 10.1016/j.jbiosc.2013.03.008. View

13.

Baykov A, Evtushenko O, Avaeva S . A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay. Anal Biochem. 1988; 171(2):266-70. DOI: 10.1016/0003-2697(88)90484-8. View

14.

Carrea G, DArrigo P, Piergianni V, Roncaglio S, Secundo F, Servi S . Purification and properties of two phospholipases D from Streptomyces sp. Biochim Biophys Acta. 1995; 1255(3):273-9. DOI: 10.1016/0005-2760(94)00241-p. View

15.

Geladopoulos T, Sotiroudis T, Evangelopoulos A . A malachite green colorimetric assay for protein phosphatase activity. Anal Biochem. 1991; 192(1):112-6. DOI: 10.1016/0003-2697(91)90194-x. View

16.

Yang H, Roberts M . Phosphohydrolase and transphosphatidylation reactions of two Streptomyces phospholipase D enzymes: covalent versus noncovalent catalysis. Protein Sci. 2003; 12(9):2087-98. PMC: 2324005. DOI: 10.1110/ps.03192503. View

17.

Aurich I, Hirche F, Ulbrich-Hofmann R . The determination of phospholipase D activity in emulsion systems. Anal Biochem. 1999; 268(2):337-42. DOI: 10.1006/abio.1998.3072. View

18.

Truppo M . Biocatalysis in the Pharmaceutical Industry: The Need for Speed. ACS Med Chem Lett. 2017; 8(5):476-480. PMC: 5430392. DOI: 10.1021/acsmedchemlett.7b00114. View

19.

Sato R, Itabashi Y, Suzuki A, Hatanaka T, Kuksis A . Effect of temperature on the stereoselectivity of phospholipase D toward glycerol in the transphosphatidylation of phosphatidylcholine to phosphatidylglycerol. Lipids. 2005; 39(10):1019-23. DOI: 10.1007/s11745-004-1325-0. View

20.

Omar M, Hammad M, Salman B, Derayea S . Highly sensitive spectrofluorimetric method for determination of doxazosin through derivatization with fluorescamine; Application to content uniformity testing. Spectrochim Acta A Mol Biomol Spectrosc. 2015; 157:55-60. DOI: 10.1016/j.saa.2015.12.012. View