Chemistry with an Artificial Primer of Polyhydroxybutyrate Synthase Suggests a Mechanism for Chain Termination

Abstract

Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the conversion of 3-(R)-hydroxybutyryl CoA (HBCoA) to PHB, which is deposited as granules in the cytoplasm of microorganisms. The class I PhaC from Caulobacter crescentus (PhaCCc) is a highly soluble protein with a turnover number of 75 s–1 and no lag phase in coenzyme A (CoA) release. Studies with [1-14C]HBCoA and PhaCCc monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and autoradiography reveal that the rate of elongation is much faster than the rate of initiation. Priming with the artificial primer [3H]sTCoA and monitoring for CoA release reveal a single CoA/PhaC, suggesting that the protein is uniformly loaded and that the elongation process could be studied. Reaction of sT-PhaCCc with [1-14C]HBCoA revealed that priming with sTCoA increased the uniformity of elongation, allowing distinct polymerization species to be observed by SDS–PAGE and autoradiography. However, in the absence of HBCoA, [3H]sT-PhaC unexpectedly generates [3H]sDCoA with a rate constant of 0.017 s–1. We propose that the [3H]sDCoA forms via attack of CoA on the oxoester of the [3H]sT-PhaC chain, leaving the synthase attached to a single HB unit. Comparison of the relative rate constants of thiolysis by CoA and elongation by PhaCCc, and the size of the PHB polymer generated in vivo, suggests a mechanism for chain termination and reinitiation.

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

“…The interface is composed of 66 residues and buries a surface area of ϳ2600 Å 2 from solvent on each monomer. As noted above, CnPhaC is known to exist in an equilibrium between monomer and dimer in solution, with the dimer representing the more catalytically active form (13,14,22 (Fig. 1) (8,10,13,17).…”

“…We propose that these arginine residues bind the 5Ј-pyrophosphate of the CoA nucleotide. Binding of HB-CoA by residues across the dimer interface in PhaC could explain, at least in part, the requirement of dimerization for activity (13,14,22). Furthermore, incubation of PhaC with oligomers of (HB) n -CoA, where n ϭ 2-4, or with (HB) 3 -CoA in which the terminal hydroxyl group is replaced with a hydrogen (saturated trimer, sTCoA), has been shown to induce formation of the dimer (14).…”

“…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).…”

“…Over the last 25 years, biochemical work on these systems has established that all PhaCs require the same residues for catalysis but have distinct kinetics of PHB formation using CoA release to monitor activity (8,11,14,15,22). For CnPhaC, a variable lag phase in CoA release is observed, which can be overcome by priming the enzyme through acylation with synthetic (HB) n -CoA analogs, where n ϭ 2-4.…”

“…This acylation is accompanied by conversion of the predominant monomeric form of CnPhaC to the dimeric form, which is accompanied by an increase in its catalytic activity, suggesting that the dimer is the active form of the enzyme (14). Despite these significant insights, there is much that remains to be understood, including the origin of odd kinetic behaviors, which are distinct within and between different classes of PhaCs (14,17,22,23); the process of chain termination; the molecular basis of substrate specificity; and the ability of the synthase to control polymer length and polydispersity. A full understanding of these aspects has been hindered in part by a lack of direct structural data on any PhaC.…”