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 paper 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)

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).