OVEREXPRESSION AND PURIFICATION OF THE SOLUBLE POLYHYDROXYALKANOATE SYNTHASE FROM ALCALIGENES-EUTROPHUS - EVIDENCE FOR A REQUIRED POSTTRANSLATIONAL MODIFICATION FOR CATALYTIC ACTIVITY

Polyhydroxyalkanoate (PHA) synthase has been expressed in Escherichia coli by reengineering the 5'-end of the wild-type (wt) gene and subseq uent transformation of this gene into protease-deficient E. coli UT560 0 (ompT(-)). Induction with IPTG results in soluble PHA synthase, whic h is similar to 5% of the total protein. The soluble synthase has been purified to > 90% homogeneity using FPLC chromatography on hydroxylap atite and Q-Sepharose and has a specific activity of 5 mu mol min(-1) mg(-1). The molecular weight of the PHA products is similar to 10(6) D a based on PlGel chromatography and calibration using polystyrene mole cular weight markers. The synthase in the absence of substrate appears to exist in both monomeric and dimeric forms. Incubation of the synth ase with an excess of substrate converts it into a form that is now ex tractable into CHCl3 and sediments on sucrose density ultracentrifugat ion with PHA. Studies in which the ratio of substrate, 3-D-hydroxybuty rylCoA, to synthase is varied suggest that during polymerization the e longation process occurs at a rate much faster than during the initiat ion process. A mechanistic model has been proposed for the polymerizat ion process [Griebel, R., Smith, Z., & Merrick, J. (1968) Biochemistry 7, 3676-3681 in which two cysteines are required for catalysis. This model is based on the well-characterized enzymes involved in fatty aci d biosynthesis. To test this model, several site-directed mutants of s ynthase, selected based on sequence conservation among synthases, have been prepared. The C459S mutant has activity similar to 90% that of t he wt protein, while th C319S and C319A synthases possess <0.01% the a ctivity of the wt protein. CD and antibody studies suggest that the mu tant proteins are properly folded. The detection of only a single esse ntial cysteine by mutagenesis and the requirement for posttranslationa l modification by phosphopantetheine to provide a second thiol in many enzymes utilizing coenzyme A thiol ester substrates made us consider the possibility that posttranslational modification was required for s ynthase activity as well. This hypothesis was confirmed when the plasm id containing PHA synthase (pKAS4) was transformed into E. coli SJ16, requiring beta-alanine for growth. Growth of SJ16/pKAS4 on [H-3]-beta- alanine followed by Coomassie staining of the protein and autoradiogra phy revealed that PHA synthase is overexpressed and that beta-alanine is incorporated into the protein. These results suggest PHA synthase i s posttranslationally modified by phosphopantetheine. Mutagenesis stud ies and detection of phosphopantetheine suggest that the mechanistic m odel of Griebel et al. (1968) in which two thiols are required for cat alysis is a reseasonable staring point for the examination of the mech anism of the polymerization process.