%0 Journal Article
%T Overexpression and characterization of medium-chain-length polyhydroxyalkanoate granule bound polymerases from Pseudomonas putida GPo1
%A Qun Ren
%A Guy de Roo
%A Bernard Witholt
%A Manfred Zinn
%A Linda Th£¿ny-Meyer
%J Microbial Cell Factories
%D 2009
%I BioMed Central
%R 10.1186/1475-2859-8-60
%X PHA polymerase 1 (PhaC1) and PHA polymerase 2 (PhaC2) from P. putida GPo1 were overexpressed in the PHA-negative host P. putida GPp104 and purified from isolated PHA granules. Only minor activity (two to three orders of magnitude lower than that of the granule bound proteins) could be recovered when the enzymes were purified to homogeneity. Therefore, kinetic properties and substrate ranges were determined for the granule bound polymerases. The polymerases differed significantly with respect to their association with PHA granules, enzyme kinetics and substrate specificity. PhaC2 appeared to bind PHA granules more tightly than PhaC1. When R-3-hydroxyoctanoic acid was used as substrate, the granule-bound PhaC1 exhibited a Km of 125 (¡À 35) ¦ÌM and a Vmax of 40.8 (¡À 6.2) U/mg PhaC1, while a Km of 37 (¡À 10) ¦ÌM and a Vmax of 2.7 (¡À 0.7) U/mg PhaC2 could be derived for the granule-bound PhaC2. Granule-bound PhaC1 showed a strong preference for medium chain length (mcl-) 3-hydroxyacly-CoAs, with highest affinity towards 3-hydroxydecanoyl-CoA (40 U/mg PhaC1). Granule-bound PhaC2 demonstrated a far broader specificity ranging from short chain length up to long chain length substrates. Activity increased with increasing chain length with a maximum activity for 3-hydroxyacyl-CoAs containing 12 or more C-atoms.The kinetic properties and substrate ranges were determined for both granule bound polymerases. Evidence was provided for the first time that two PHA polymerases exhibited significant differences in granule release and in vitro activity profiles, suggesting that there are substantial functional differences between granule bound PhaC1 and PhaC2.Many bacteria are able to accumulate polyhydroxyalkanoates (PHA) in the cytoplasm as storage compounds for energy and carbon [1]. The key enzymes for PHA biosynthesis are PHA polymerases. These enzymes catalyze the covalent linkage of 3-hydroxyacyl coenzyme A (CoA) thioesters by transesterification with concomitant release of CoA. PHA
%U http://www.microbialcellfactories.com/content/8/1/60