Biochemistry 51, 2276–2288 (2012)

Credit: MIMI CHO

Polyhydroxybutyrate (PHB) synthase (PhaC) from Ralstonia eutropha synthesizes PHB polymers to store carbon. Studies with recombinant PhaC have shown that the polymerization reaction proceeds with a slow initiation phase and a fast elongation phase. Though initiation can be increased by the addition of the coenzyme A ester of a hydroxybutyrate trimer, the slow kinetics suggested that another factor primes PhaC for activity in vivo. To search for this factor, Cho et al. created a Strep2-tagged PhaC that could be directly purified from the host organism. Isolation of PhaC after 4 h or 24 h of growth yielded a highly active species, suggesting the authors had succeeded in isolating the primed enzyme. SDS-PAGE analysis identified the known PHB-associated protein PhaP1 as purifying together with PhaC, but this protein slowed initiation in in vitro assays. Size-exclusion chromatography to monitor enzyme oligomerization unexpectedly identified a soluble, high-molecular-weight species that contained PhaP1, PhaC and PHB in addition to the expected monomer and dimer of PhaC. As the soluble complex was highly active and was not formed with mutant PhaC, the authors concluded that PHB itself is the likely priming factor. These results led to a 'modified micelle' model, in which a PhaC dimer is covalently attached to a single PHB chain and several PhaP1s associate with this species in soluble form prior to conversion to insoluble granules.