Simple SummaryThe cyanobacteriumArthrospira platensiscontains PHA synthase Class III (PhaCAp), which can produce short chain length (SCL) PHB under nitrogen-depleted conditions. In this study, we cloned a gene encoding PhaC fromA. platensisintoEscherichia cloni®10G cells to produce rPhaCApprotein. TheVmax,Km, andkcatvalues for β-3-hydroxybutyryl coenzyme A (3HB-CoA) of the purified rPhaCApwere investigated. Size-exclusion chromatography revealed that rPhaCApexists as an active dimer. The overall fold and catalytic triad residues were predicted using the 3D structural model for rPhaCAp. These results are discussed with respect to the dimerization mechanism of PhaCAp, which has not yet been clarified.AbstractPolyhydroxybutyrate (PHB) is a biocompatible and biodegradable polymer that has the potential to replace fossil-derived polymers. The enzymes involved in the biosynthesis of PHB are β-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC). PhaC inArthrospira platensisis the key enzyme for PHB production. In this study, the recombinantE. cloni®10G cells harboringA. platensis phaC(rPhaCAp) was constructed. The overexpressed and purified rPhaCApwith a predicted molecular mass of 69 kDa exhibitedVmax,Km, andkcatvalues of 24.5 ± 2 μmol/min/mg, 31.3 ± 2 µM and 412.7 ± 2 1/s, respectively. The catalytically active rPhaCApwas a homodimer. The three-dimensional structural model for the asymmetric PhaCAphomodimer was constructed based onChromobacteriumsp. USM2 PhaC (PhaCCs). The obtained model of PhaCAprevealed that the overall fold of one monomer was in the closed, catalytically inactive conformation whereas the other monomer was in the catalytically active, open conformation. In the active conformation, the catalytic triad residues (Cys151-Asp310-His339) were involved in the binding of substrate 3HB-CoA and the CAP domain of PhaCApinvolved in the dimerization.