Characterization and Homology Modeling of Catalytically Active Recombinant PhaC Ap 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 β-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 ®10G cells harboring A. platensis phaC (rPhaC Ap) was constructed. The overexpressed and purified rPhaC Ap with a predicted molecular mass of 69 kDa exhibited V max , K m , and k cat values of 24.5 ± 2 μmol/min/mg, 31.3 ± 2 µM 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.