title: Overexpression and purification of the soluble polyhydroxyalkanoate synthase from Alcaligenes eutrophus: evidence for a required posttranslational modification for catalytic activity.
abstract: Polyhydroxyalkanoate (PHA) synthase has been expressed in Escherichia coli by reengineering the 5'-end of the wild-type (wt) gene and subsequent transformation of this gene into protease-deficient E. coli UT5600 (ompT-). Induction with IPTG results in soluble PHA synthase, which is approximately 5% of the total protein. The soluble synthase has been purified to > 90% homogeneity using FPLC chromatography on hydroxylapatite and Q-Sepharose and has a specific activity of 5 mumol min-1 mg-1. The molecular weight of the PHA product is approximately 10(6) Da based on PlGel chromatography and calibration using polystyrene molecular weight markers. The synthase in the absence of substrate appears to exist in both monomeric and dimeric forms. Incubation of the synthase with an excess of substrate converts it into a form that is now extractable into CHCl3 and sediments on sucrose density ultracentrifugation with PHA. Studies in which the ratio of substrate, 3-D-hydroxybutyrylCoA, to synthase is varied suggest that during polymerization the elongation process occurs at a rate much faster than during the initiation process. A mechanistic model has been proposed for the polymerization 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 acid biosynthesis. To test this model, several site-directed mutants of synthase, selected based on sequence conservation among synthases, have been prepared. The C459S mutant has activity approximately 90% that of the wt protein, while the C319S and C319A synthases possess < 0.01% the activity of the wt protein. CD and antibody studies suggest that the mutant proteins are properly folded. The detection of only a single essential cysteine by mutagenesis and the requirement for posttranslational 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 synthase activity as well. This hypothesis was confirmed when the plasmid containing PHA synthase (pKAS4) was transformed into E. coli SJ16, requiring beta-alanine for growth. Growth of SJ16/pKAS4 on [3H]-beta-alanine followed by Coomassie staining of the protein and autoradiography revealed that PHA synthase is overexpressed and that beta-alanine is incorporated into the protein. These results suggest PHA synthase is posttranslationally modified by phosphopantetheine.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge 02139-4307.
authors.forename: T U
authors.initials: TU
authors.lastname: Gerngross
authors.forename: K D
authors.initials: KD
authors.lastname: Snell
authors.forename: O P
authors.initials: OP
authors.lastname: Peoples
authors.forename: A J
authors.initials: AJ
authors.lastname: Sinskey
authors.forename: E
authors.initials: E
authors.lastname: Csuhai
authors.forename: S
authors.initials: S
authors.lastname: Masamune
authors.forename: J
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D000217:Acyltransferases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D000421:Alcaligenes / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010944:Plants / Q000201:enzymology; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase