Difference between revisions of "Part:BBa I718002"
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In Acinetobacter ADP1, the enzyme is associated with lipids inclusions but also with the membrane and a minor amount in the cytoplasm (Stoevken, 2005). In recombinant E.coli, the majority of WS/DGAT was membrane-associated but to some extent also located in the cytosolic fraction (Stoevken, 2005).<br>
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In Acinetobacter ADP1, the enzyme is associated with lipids inclusions but also with the membrane and a minor amount in the cytoplasm (Stoevken, 2005). In recombinant E.coli, the majority of WS/DGAT was membrane-associated but to some extent also located in the cytosolic fraction (Stoevken, 2005).
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Palmitoyl-CoA is accepted with highest specificity (Stoevken, 2005).
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[[Image:paris_DGAT_localisation.jpg|center]]
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''Localization of WS/DGAT by immunogold TEM of Acinetobacter sp. strain ADP1. Arrows indicate the gold particles. The scale bars correspond to 500 nm. LI, lipid inclusion; CM, cytoplasm membrane; OM, outer membrane.''
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===References===
 
===References===

Revision as of 15:12, 10 July 2007

  • Which species is the enzyme from?

Acinetobacter calcoaceticus ADP1 (Gram negative bacterium).

  • What reaction does it catalyze?

diacylglycerol + acyl-coA -> triglyceride + coA-SH.

Studies employing total membrane fractions or extracts of recombinants E. coli strains revealed that this enzyme has a very broad substrate range, accepting long chain fatty alcohols and acyl-CoA esters ranging from C12 to C20 as well as monoacylglycerol as substrates (Kalscheuer, 2004)

  • Does it work in E. coli?

Yes. The authors of the papers (references) have shown that it works already in vitro after purification.

  • Does it require anything?

Yes. DAG is already a compound of the phospholipid metabolism. E. coli has a Long Chain Fatty Acid (LCFA) transporter, FadL. Adding oleate in the LB medium (5mM) is probably needed.

  • The four reactions where DAG is involved:

- ATP + a 1,2-diacylglycerol = ADP + an L-phosphatidate
- a 1,2-diacylglycerol + CDP-choline -> a - phosphatidylcholine + CMP
- an L-1-phosphatidyl-ethanolamine + KDO2-lipid IVA = a 1,2-diacylglycerol + phosphatidylethanolamine-KDO2-lipidA
- an MDO-O-glucose + an L-1-phosphatidyl-glycerol = a 1,2-diacylglycerol + an MDO-6-(glycerophospho)-D-glucose

  • Another reactions catalyzed by DGAT?

Yes. DGAT is also an acyl-CoA fatty alcohol acyltransferase (wax ester synthase, WS) catalyzing the final condensation of acyl-CoA and fatty alcohol. Knowing that E.coli does not produce fatty alcohol, this reaction is probably not avaible in this bacteria.

  • In vitro characterization of WS/DGAT:

The molecular weight is about 53 kDa, the enzyme probably acts as homodimer (Stoevken, 2005).

The highest activity was obtained at 40 - 45 °C (Stoevken, 2005).

WS activity (filled squares) and DGAT activity (filled triangles) from two independent experiments with error bars


The WS reaction follows a Michaelis - Menten kinetic but the DGAT reaction fit neither Michaelis - Menten neither cooperative enzyme kinetics (Stoevken, 2005).

WS activity (filled squares) and DGAT activity (filled triangles) from two independent experiments with error bars


Palmitoyl-CoA is accepted with highest specificity (Stoevken, 2005).

Paris DGAT lipid.jpg


In Acinetobacter ADP1, the enzyme is associated with lipids inclusions but also with the membrane and a minor amount in the cytoplasm (Stoevken, 2005). In recombinant E.coli, the majority of WS/DGAT was membrane-associated but to some extent also located in the cytosolic fraction (Stoevken, 2005). Palmitoyl-CoA is accepted with highest specificity (Stoevken, 2005).

Paris DGAT localisation.jpg

Localization of WS/DGAT by immunogold TEM of Acinetobacter sp. strain ADP1. Arrows indicate the gold particles. The scale bars correspond to 500 nm. LI, lipid inclusion; CM, cytoplasm membrane; OM, outer membrane.

References

Kalscheuer,R. and Steinbuchel,A. A Novel Bifunctional Wax Ester Synthase/Acyl-CoA:Diacylglycerol Acyltransferase Mediates Wax Ester and Triacylglycerol Biosynthesis in Acinetobacter calcoaceticus ADP1. J. Biol. Chem. 278 (10), 8075-8082 (2003).

Stoveken T, Kalscheuer R, Malkus U, Reichelt R, Steinbuchel A. The wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase from Acinetobacter sp. strain ADP1: characterization of a novel type of acyltransferase. J Bacteriol. ;187(4):1369-76 (2005)