Characterization and Homology Modeling of Catalytically Active Recombinant PhaCAp Protein from Arthrospira platensis

Polyhydroxybutyrate (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 fi-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC). PhaC in Arthrospira platensis is the key enzyme for PHB production. In this study, the recombinant E. cloni ((R))10G cells harboring A. platensis phaC (rPhaC(Ap)) was constructed. The overexpressed and purified rPhaCAp with a predicted molecular mass of 69 kDa exhibited Vmax, Km, and kcat values of 24.5 +/- 2 mu mol/min/mg, 31.3 +/- 2 mu M and 412.7 +/- 2 1/s, respectively. The catalytically active rPhaC(Ap) was a homodimer. The three-dimensional structural model for the asymmetric PhaC(Ap) homodimer was constructed based on Chromobacterium sp. USM2 PhaC (PhaC(Cs)). The obtained model of PhaC(Ap) revealed 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 PhaC(Ap) involved in the dimerization.