Characterization and Homology Modeling of Catalytically Active Recombinant PhaC<sub>Ap</sub> Protein from <i>Arthrospira platensis</i>
Chanchanok Duangsri,
Tiina A. Salminen,
Marion Alix,
Sarawan Kaewmongkol,
Nattaphong Akrimajirachoote,
Wanthanee Khetkorn,
Sathaporn Jittapalapong,
Pirkko Mäenpää,
Aran Incharoensakdi,
Wuttinun Raksajit
Affiliations
Chanchanok Duangsri
Program of Animal Health Technology, Faculty of Veterinary Technology, Kasetsart University, Bangkok 10900, Thailand
Tiina A. Salminen
Structural Bioinformatics Laboratory and InFLAMES Research Flagship Center, Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, 20520 Turku, Finland
Marion Alix
Structural Bioinformatics Laboratory and InFLAMES Research Flagship Center, Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, 20520 Turku, Finland
Sarawan Kaewmongkol
Program of Animal Health Technology, Faculty of Veterinary Technology, Kasetsart University, Bangkok 10900, Thailand
Nattaphong Akrimajirachoote
Department of Physiology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand
Wanthanee Khetkorn
Division of Biology, Faculty of Science and Technology, Rajamangala University of Technology Thanyaburi (RMUTT), Thanyaburi, Pathumthani 12110, Thailand
Sathaporn Jittapalapong
Program of Animal Health Technology, Faculty of Veterinary Technology, Kasetsart University, Bangkok 10900, Thailand
Pirkko Mäenpää
Faculty of Technology, University of Turku, 20014 Turku, Finland
Aran Incharoensakdi
Laboratory of Cyanobacterial Biotechnology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
Wuttinun Raksajit
Program of Animal Health Technology, Faculty of Veterinary Technology, Kasetsart University, Bangkok 10900, Thailand
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 (rPhaCAp) 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 μmol/min/mg, 31.3 ± 2 µM and 412.7 ± 2 1/s, respectively. The catalytically active rPhaCAp was a homodimer. The three-dimensional structural model for the asymmetric PhaCAp homodimer was constructed based on Chromobacterium sp. USM2 PhaC (PhaCCs). The obtained model of PhaCAp 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 PhaCAp involved in the dimerization.
