Detailed characterization of the substrate specificity of mouse wax synthase

• Autorzy: Miklaszewska M. , Kawinski A. , Banas A.
• Języki publikacji: EN

Abstrakty

EN

 Wax synthases are membrane-associated enzymes catalysing the esterification reaction between fatty acyl-CoA and a long chain fatty alcohol. In living organisms, wax esters function as storage materials or provide protection against harmful environmental influences. In industry, they are used as ingredients for the production of lubricants, pharmaceuticals, and cosmetics. Currently the biological sources of wax esters are limited to jojoba oil. In order to establish a large-scale production of desired wax esters in transgenic high-yielding oilseed plants, enzymes involved in wax esters synthesis from different biological resources should be characterized in detail taking into consideration their substrate specificity. Therefore, this study aims at determining the substrate specificity of one of such enzymes - the mouse wax synthase. The gene encoding this enzyme was expressed heterologously in Saccharomyces cerevisiae. In the in vitro assays (using microsomal fraction from transgenic yeast), we evaluated the preferences of mouse wax synthase towards a set of combinations of 11 acyl-CoAs with 17 fatty alcohols. The highest activity was observed for 14:0-CoA, 12:0-CoA, and 16:0-CoA in combination with medium chain alcohols (up to 5.2, 3.4, and 3.3 nmol wax esters/min/mg microsomal protein, respectively). Unsaturated alcohols longer than 18°C were better utilized by the enzyme in comparison to the saturated ones. Combinations of all tested alcohols with 20:0-CoA, 22:1-CoA, or Ric-CoA were poorly utilized by the enzyme, and conjugated acyl-CoAs were not utilized at all. Apart from the wax synthase activity, mouse wax synthase also exhibited a very low acyl-CoA:diacylglycerol acyltransferase activity. However, it displayed neither acyl-CoA:monoacylglycerol acyltransferase, nor acyl-CoA:sterol acyltransferase activity.

Słowa kluczowe

EN

substrate specificity; mouse; mice; wax ester; synthase; lipid; fatty alcohol

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 2013
• Tom: 60
• Numer: 2
• Opis fizyczny: p.209-215,fig.,ref.

Twórcy

autor

Miklaszewska M.

• Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdansk, Poland

autor

Kawinski A.

• Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdansk, Poland

autor

Banas A.

• Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdansk, Poland

Bibliografia

• Banas A, Dahlqvist A, Ståhl U, Lenman M, Stymne S (2000) The involvement of phospholipid:diacylglycerol acyltransferases in triacylglycerol production. Biochem Soc Trans 28: 703-705. 
• Biester EM, Hellenbrand J, Gruber J, Hamberg M, Frentzen M (2012) Identification of avian wax synthases. BMC Biochem 13:4, doi: 10.1186/1471-2091-13-4. 
• Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911-917. 
• Cheng J B, Russell D W (2004) Mammalian wax biosynthesis. II. Expression cloning of wax synthase cDNAs encoding a member of the acyltransferase enzyme family. J Biol Chem 279: 37798-37807. 
• Dahlqvist A, Ståhl U, Lenman M, Banaś A, Lee M, Sandager L, Ronne H, Stymne S (2000) Phospholipid:diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. Proc Natl Acad Sci USA 97: 6487-6492. 
• Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350: 87-96. 
• Heilmann M, Iven T, Ahmann K, Hornung E, Stymne S, Feussner I (2012) Production of wax esters in plant seed oils by oleosomal cotargeting of biosynthetic enzymes. J Lipid Res 53: 2153-2161. 
• Holtzapple E, Schmidt-Dannert C (2007) Biosynthesis of isoprenoid wax ester in Marinobacter hydrocarbonoclasticus DSM 8798: identification and characterization of isoprenoid coenzyme A synthetase and wax ester synthases. J Bacteriol 189: 3804-3812. 
• Kalscheuer R, Steinbüchel A (2003) A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J Biol Chem 278: 8075-8082. 
• Klypina N, Hanson S (2008) Arabidopsis thaliana wax synthase gene homologues show diverse expression patterns that suggest a specialized role for these genes in reproductive organs. Plant Sci 175: 312-320.
• Lardizabal K D, Metz JG, Sakamoto T, Hutton W C, Pollard M R, Lassner M W (2000) Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic Arabidopsis. Plant Physiol 122: 645-655. 
• Lassner MW, Lardizabal K, Metz JG (1999) Producing wax esters in transgenic plants by expression of genes derived from jojoba. Perspectives on new crops and new uses Janick J ed, pp 220-224. ASHS Press, Alexandria, VA.
• Li F, Wu X, Lam P, Bird D, Zheng H, Samuels L, Jetter R, Kunst L (2008) Identification of the wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase WSD1 required for stem wax ester biosynthesis in Arabidopsis. Plant Physiology 148: 97-107. 
• Lu C, Clemente TE, Cahoon E (2010) Camelina sativa as a platform for the production of oils with enhanced lubricant properties. 19th International Symposium on Plant Lipids. Cairns, Australia.
• Miwa TK (1971) Jojoba oil wax esters and derived fatty acids and alcohols - Gas chromatographic analyses. J Am Oil Chem Soc 48: 259-264.
• Sánchez M, Nicholls DG, Brindley DN (1973) The relationship between palmitoyl-coenzyme A synthetase activity and esterification of sn-glycerol 3-phosphate in rat liver mitochondria. Biochem J 132: 697-706. 
• Sandager L, Gustavsson MH, Stahl U, Dahlqvist A, Wiberg E, Banas A, Lenman M, Ronne H, Stymne S (2002) Storage lipid synthesis is nonessential in yeast. J Biol Chem 277: 6478-6482. 
• Shi S, Valle-Rodríguez JO, Khoomrung S, Siewers V, Nielsen V (2012) Functional expression and characterization of five wax ester synthases in Saccharomyces cerevisiae and their utility for biodiesel production. Biotechnology for Biofuels 5: 7 (Open Access;  http://www.biotechnologyforbiofuels.com/content/5/1/7).
• Ståhl U, Carlsson A, Lenman M, Dahlqvist A, Huang B, Banas W, Banas A, Stymne S (2004) Cloning and functional characterization of a phospholipid:diacylglycerol acyltransferase from Arabidopsis. Plant Physiol 135: 1324-1335. 
• Stöveken T, Kalscheuer R, Malkus U, Reichelt R, Steinbüchel A (2005) The wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase from Acinetobacter sp. strain ADP1: characterization of a novel type of acyltransferase. J Bacteriol 187: 1369-1376. 
• Teerawanichpan P, Qiu X (2010) Fatty acyl-CoA reductase and wax synthase from Euglena gracilis in the biosynthesis of medium-chain wax esters. Lipids 45: 263-273.