Part:BBa_I718002

• 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. We showed it works in vivo in LB medium with or without supplementation in sodium oleate (see experience).

• 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 bacterium.

• 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).

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

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).

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)