Difference between revisions of "Part:BBa I718002"
| Line 1: | Line 1: | ||
| − | * Which species is the enzyme from? | + | * '''Which species is the enzyme from?''' |
Acinetobacter calcoaceticus ADP1 (Gram negative bacterium) | Acinetobacter calcoaceticus ADP1 (Gram negative bacterium) | ||
| − | * What reaction does it catalyze? | + | * '''What reaction does it catalyze?''' |
diacylglycerol + acyl-coA -> triglyceride + coA-SH | 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) | 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? | + | * '''Does it work in E. coli?''' |
Yes. The authors of the papers (references) have shown that it works already in vitro after purification. | Yes. The authors of the papers (references) have shown that it works already in vitro after purification. | ||
| − | * Does it require anything? | + | * '''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. | 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: | + | * '''The four reactions where DAG is involved:''' |
- ATP + a 1,2-diacylglycerol = ADP + an L-phosphatidate <br> | - ATP + a 1,2-diacylglycerol = ADP + an L-phosphatidate <br> | ||
- a 1,2-diacylglycerol + CDP-choline -> a - phosphatidylcholine + CMP<br> | - a 1,2-diacylglycerol + CDP-choline -> a - phosphatidylcholine + CMP<br> | ||
| Line 19: | Line 19: | ||
- an MDO-O-glucose + an L-1-phosphatidyl-glycerol = a 1,2-diacylglycerol + an MDO-6-(glycerophospho)-D-glucose <br> | - an MDO-O-glucose + an L-1-phosphatidyl-glycerol = a 1,2-diacylglycerol + an MDO-6-(glycerophospho)-D-glucose <br> | ||
| − | * Another reactions catalyzed by DGAT? | + | * '''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. | 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: | + | * '''In vitro characterization of WS/DGAT:''' |
The molecular weight is about 53 kDa, the enzyme probably acts as homodimer (Stoevken, 2005).<br> | The molecular weight is about 53 kDa, the enzyme probably acts as homodimer (Stoevken, 2005).<br> | ||
Revision as of 14:59, 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).
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).
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)