In vitro activities of granule-bound poly[(R)-3-hydroxyalkanoate]polymerase C1 of Pseudomonas oleovorans--development of an activity test for medium-chain-length-poly(3-hydroxyalkanoate) polymerases.
A newly developed in vitro activity assay for medium-chain-length (mcl)-poly(3-hydroxyalkanoate) polymerases is described. Polymerase C1 of Pseudomonas oleovorans GPo1 attached to isolated granules was used as model enzyme. A direct correlation was found between (R)-3-hydroxyoctanoylCoA depletion and poly(3-hydroxyalkanoate) synthesis due to polymerase C1 activity. Highest activities of 1.13 U/mg granule bound protein and highest specific activities of 2.3 U/mg polymerase C1 were determined towards (RS)-3-hydroxyoctanoylCoA. A first determination of a Km value for mcl poly(3-hydroxyalkanoate) polymerases was performed leading to an estimated Km of 0.16 (+/-0.1) mM for granule bound polymerase C1 with (R)-3-hydroxyoctanoylCoA as substrate. Polymerase C1 showed no activity towards (RS)-3-hydroxybutyrylCoA and a specific activity of 0.28 U/mg polymerase C1 for (R)-3-hydroxyvalerylCoA. (R)-3-HydroxyoctanoylCoA and a mixture of (RS)-3-hydroxyoctanoylCoA were both depleted for more than 75% by granule-bound polymerase C1, suggesting a non-rate-limiting epimerase activity attached to poly(3-hydroxyalkanoate) granules isolated from Pseudomonas putida GPp104[pGEc405]. Whereas no relationship was found between the activity of granule-bound polymerase C1 and poly(3-hydroxyalkanoate) content of the granules, higher activities were measured when a higher substrate concentration or more enzyme was present in the in vitro activity assay.
Kraak
M N
MN
Institute of Biotechnology, ETH Hönggerberg, Zürich, Switzerland.
Kessler
B
B
Witholt
B
B
D000217:Acyltransferases / Q000378:metabolism*; D001426:Bacterial Proteins; D011549:Pseudomonas / Q000201:enzymology* / Q000254:growth & development
D001426:Bacterial Proteins; D000217:Acyltransferases; C067032:poly(3-hydroxyalkenoate)polymerase