Difference between revisions of "Part:BBa I718002"
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* Which species is the enzyme from? | * Which species is the enzyme from? | ||
Acinetobacter calcoaceticus ADP1 (Gram negative bacterium) | Acinetobacter calcoaceticus ADP1 (Gram negative bacterium) | ||
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* The four reactions where DAG is involved: | * The four reactions where DAG is involved: | ||
| − | - ATP + a 1,2-diacylglycerol = ADP + an L-phosphatidate | + | - ATP + a 1,2-diacylglycerol = ADP + an L-phosphatidate <br> |
| − | - a 1,2-diacylglycerol + CDP-choline -> a - phosphatidylcholine + CMP | + | - a 1,2-diacylglycerol + CDP-choline -> a - phosphatidylcholine + CMP<br> |
| − | - an L-1-phosphatidyl-ethanolamine + KDO2-lipid IVA = a 1,2-diacylglycerol + phosphatidylethanolamine-KDO2-lipidA | + | - an L-1-phosphatidyl-ethanolamine + KDO2-lipid IVA = a 1,2-diacylglycerol + phosphatidylethanolamine-KDO2-lipidA <br> |
| − | - an MDO-O-glucose + an L-1-phosphatidyl-glycerol = a 1,2-diacylglycerol + an MDO-6-(glycerophospho)-D-glucose | + | - 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? | ||
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* 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). | + | The molecular weight is about 53 kDa, the enzyme probably acts as homodimer (Stoevken, 2005).<br> |
| − | The highest activity was obtained at 40 - 45 °C (Stoevken, 2005) | + | The highest activity was obtained at 40 - 45 °C (Stoevken, 2005).<br> |
| − | The WS reaction follows a Michaelis - Menten kinetic but the DGAT reaction fit neither Michaelis - Menten neither cooperative enzyme kinetics (Stoevken, 2005). | + | The WS reaction follows a Michaelis - Menten kinetic but the DGAT reaction fit neither Michaelis - Menten neither cooperative enzyme kinetics (Stoevken, 2005).<br> |
| − | Palmitoyl-CoA is accepted with highest specificity (Stoevken, 2005). | + | Palmitoyl-CoA is accepted with highest specificity (Stoevken, 2005).<br> |
| − | 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). | + | 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> |
Revision as of 14:55, 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 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).