PHA synthase from Chromatium vinosum: Cysteine 149 is involved in covalentcatalysis

Polyhydroxyalkanoate synthase (PHA) from Chromatium vinosum? catalyzes the conversion of 3-hydroxybutyryl-CoA (HB-CoA) to polyhydroxybutyrate (PHB) an d CoA, The synthase is composed of a similar to 1:1 mixture of two subunits , PhaC and PhaE. Size-exclusion chromatography indicates that in solution P haC and PhaE exist as large molecular weight aggregates, The hole-enzyme, P haEC, has a specific activity of 150 units/mg, Each subunit was cloned, exp ressed, and purified as a (His)(6)-ragged construct. The PhaC-(His)(6) prot ein catalyzed polymerization with a specific activity of 0.9 unit/mg; the P haE-(His)(6) protein was inactive (specific activity <0.001 unit/mg). Addit ion of PhaE-(His)(6) to PhaC(His)(6) increased the activity several 100-fol d, To investigate the priming step of the polymerization process, the PhaEC was incubated with a trimer of HB-CoA in which the terminal hydroxyl was r eplaced with tritium ([H-3]-sT-CoA). After Sephadex G50 chromatography, the synthase contained similar to 0.25 equiv of the labile label per PhaC, Inc ubation of [H-3]-sT-synthase with HB-CoA resulted in production of [H-3]-po lymer. Digestion of [H-3]-sT-synthase with trypsin and HPLC analysis result ed in isolation of three labeled peptides, Sequencing by ion trap mass spec trometry showed that they were identical and that they each contained an al tered cysteine (C149). One peptide contained the [H-3]-sT while the other t wo contained, in addition to the [H-3]-sT, one and two additional monomeric HBs, respectively. Mutation of C149 to alanine gave inactive synthase, The remaining two cysteines of PhaC, 292 and 130, were also mutated to alanine , The former had wild-type (wt) activity, while the latter had 0.004 wt % a ctivity and was capable of making polymer. A mechanism is proposed in which PhaC contains all the elements essential for catalysis and the polymerizat ion proceeds by covalent catalysis using C149 and potentially C130.