Biochemical and enzymological properties of the polyhydroxybutyrate synthase from the extremely halophilic archaeon strain 56.

Some members of the archaebacterial family Halobacteriaceae have been determined to accumulate polyhydroxyalkanoate (PHA) and poly(3-hydroxybutyrate) (PHB). The extremely halophilic archaebacterium strain 56 is capable of accumulating large amounts of PHB. Since measurements of enzyme activities related to archaebacterial PHB biosynthesis have never been achieved, we investigated the enzymology of PHB biosynthesis in strain 56. Crude extracts of strain 56 cultivated under accumulating conditions showed PHB synthase activity, whereas neither beta-ketothiolase nor NADH/NADPH-dependent acetoacetyl-CoA reductase activity was detectable. An 80-kDa protein, cross-reacting with the anti-PHB synthase antibodies raised against the PHB synthase from Ralstonia eutropha, was identified in the crude extract and was strongly enriched by purification of PHB granules. The granule-associated PHB synthase was enzymologically characterized. Enzyme kinetics showed a specific activity of about 4.6U/mg and Hill plot analysis revealed a K(0.5) of 56 microM with (R)-3-hydroxybutyryl-CoA employed as substrate. A Hill coefficient of 1.75 indicated that the PHB synthase exhibited positive cooperativity. The thioesters 3-hydroxyvaleryl-CoA, 4-hydroxybutyryl-CoA, and 3-hydroxydecanoyl-CoA were not accepted as substrates. Moreover, the PHB synthase was found to be competitively inhibited by CoA, showing an IC(50) of 160 microM. The PHB synthase was stable up to 60 degrees C and still exhibited about 90% of the maximum enzyme activity, which was obtained at 40 degrees C. In contrast to the soluble PHB synthase, the granule-bound PHB synthase was almost independent of the salt concentration. The PHB synthase could not be released from the PHB granules, indicating a covalent attachment to the PHB core. This is the first description of an archaebacterial PHA synthase.