Class III Polyhydroxybutyrate Synthase:  Involvement in Chain Termination and Reinitiation

Abstract

Polyhydroxybutyrate (PHB) synthase catalyzes the polymerization of (R)-3-hydroxybutyrylCoA (CoA ) coenzyme A) into high molecular weight PHB. Recombinant wild-type (wt) class III synthase from Allochromatium Vinosum (PhaCPhaE Av ), antibodies to this synthase and to PHB, and [ 14 C]-hydroxybutyryl-CoA (HB-CoA) have been used to detect oligomeric hydroxybutyrate (HB) units covalently bound to the synthase using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Although a distribution of products is typically observed, short (HB) n -bound synthases (designated species I) are most prevalent at low substrate to enzyme (S/E) ratios. Species I is similar to (HB) n -PhaC Av (n ) 3-10 at minimum) recently identified using D302A-PhaCPhaE Av (Tian, J., Sinskey, A. J., and Stubbe, J. (2005) Biochemistry 44, 1495-1503). Species I is shown to be an intermediate in the elongation process of PHB synthesis in vitro. The reaction catalyzed by the wt synthase in vitro was further studied under two sets of conditions: at high (70000) and low (<200) S/E ratios. At high S/E ratios, kinetic analysis of the reaction of HB-CoA with the wt synthase monitored using antibodies to PhaCPhaE Av and Western blotting revealed the disappearance of PhaC Av at early time points and its reappearance as the molecular weight of the PHB approached 1.8 MDa. At low S/E ratios, species I was observed to increase with time after complete consumption of all of the HB-CoA. The results from studies under both sets of conditions suggest that an inherent property of the synthase is chain termination and reinitiation.Polyhydroxyalkanoate (PHA 1 ) synthases catalyze the polymerization of (R)-3-hydroxyalkanoate-CoA into high molecular weight polyoxoesters under nutrient-limited conditions in the presence of a carbon source (1-4). PHA synthases are representative of enzymes involved in nontemplate-driven polymerization processes in which a watersoluble substrate is transformed into a water-insoluble granule or inclusion without a genetically determined template. The thermoplastic or elastomeric properties of PHAs (depending on the substrate) have provided the impetus for many research groups to study the mechanism and the regulation of the polymerization process, as well as the substrate specificity of the synthase. The long-range goal is to make materials with novel properties in an economically competitive fashion with oil-based polymers (5).The classification of PHA synthases, based on substrate specificity and subunit composition, has been described in detail in several reviews (6, 7). Our laboratory has focused on the class I and class III synthases that use (R)-3-hydroxybutyryl-CoA (HB-CoA) to form polyhydroxybutyrate (PHB). The class I synthase from the prototypical Wautersia eutropha (PhaC We ) has a subunit molecular weight of 64 kDa (8). The class III synthase from the prototypical Allochromatium Vinosum is composed of two subunits, designated PhaE Av (MW ≈ 40 kDa) and PhaC Av (MW ≈ 39 kDa) (9). PhaC Av contains the active si...

Mechanistic Insight with HBCH2CoA as a Probe to Polyhydroxybutyrate (PHB) Synthases

“…8,43 Thus, even at very low substrate to enzyme ratios, a small amount of protein is modified with high molecular weight PHB polymer, while most of the protein remains unmodified. 43 In order to overcome this problem, we developed a method to uniformly load PhaC, so that the elongation process could be examined. 38 Specifically, an artificial primer, [ 3 H]-saturated trimeric-CoA ([ 3 H]-sTCoA, 16 in Scheme 3), is used to acylate the enzyme, which can then be incubated with 1 .…”

Structure of the Catalytic Domain of the Class I Polyhydroxybutyrate Synthase from Cupriavidus necator

“…PhaC is thought to catalyze termination itself, although a phasin protein, PhaP, has been implicated in promoting the termination event (25,45,46). Proposals for the mechanism of termination include hydrolysis by a base-activated water molecule; transfer of the PHB chain to a nucleophilic residue on PhaC followed by hydrolysis; or chain transfer to an exogenous thiol-or hydroxyl-containing molecule (22,(47)(48)(49). Following chain termination and release of the polymer, it has been shown that the class III PhaC remains loaded with 3-10 HB units, suggesting that chain termination occurs at a site distant from the active site (47).…”

“…Proposals for the mechanism of termination include hydrolysis by a base-activated water molecule; transfer of the PHB chain to a nucleophilic residue on PhaC followed by hydrolysis; or chain transfer to an exogenous thiol-or hydroxyl-containing molecule (22,(47)(48)(49). Following chain termination and release of the polymer, it has been shown that the class III PhaC remains loaded with 3-10 HB units, suggesting that chain termination occurs at a site distant from the active site (47). Although similar studies have not been performed on the class I synthase, termination at a similarly positioned site may be generally applicable to all classes of PhaC.…”

Growth and Localization of Polyhydroxybutyrate Granules in Ralstonia eutropha

“…The three biosynthetic genes phaABC are together on an operon whose expression is relatively constant during PHB production (27,32). Chain elongation by PhaC is more rapid than chain initiation (16,49), and chain termination occurs via a poorly understood mechanism once a remarkably uniform molecular mass (polydispersity of 1.2) of approximately 10 6 Da is reached (48,49). PHB sequesters the repressing transcription factor PhaR to derepress production of PhaP1, a protein from the enigmatic phasin family (38,53,57), of which there are multiple paralogs within the R. eutropha genome (4,22,35,36).…”