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

Abstract

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.

Keywords: 3HB-CoA; Arthrospira platensis; PHA synthase; PhaC; Polyhydroxybutyrate.

Figure 1

(A) PCR amplification from A. platensis DNA, lane: M marker; lane 1: PCR fragment of A. platensis phaC (1131 bp) (B) Schematic map of pSol-Tsf-phaC_Ap plasmid carrying A. platensis phaC gene).

Figure 2

(A) SDS-PAGE representing rPhaC_Ap protein expression in E. cloni^®10G. The target protein is about 69 kDa. Lane M: protein marker (in kDa); lane 1: crude protein fraction; lane 2: soluble protein fraction (SF) before l-rhamnose induction; lane 3: SF after 0.002% (w/v) l-rhamnose induction; lane 4: SF after 0.02% (w/v) L-rhamnose induction; lane 5: SF after 0.2% (w/v) L-rhamnose induction; lane 6: purified rPhaC_Ap obtained from SF after 0.2% (w/v) l-rhamnose induction. (B) Western blot detection of rPhaC_Ap protein with polyclonal anti-rPhaC_Ap antibody which corresponds to protein band of approximately 69 kDa. Lane M: protein marker (in kDa); lane 1: purified soluble protein fraction containing rPhaC_Ap protein after 0.2% (w/v) L-rhamnose induction.

Figure 3

(A) The Michaelis–Menten plot of purified rPhaC_Ap (B) Size-exclusion chromatography of rPhaC_Ap.

Figure 4

Multiple sequence alignment used for modeling. The PhaC proteins of PhaC_Ap, PhaC_Ss, PhaC_Pl, and PhaC_Ab were aligned to the pre-aligned structure-based alignment of Chromobacterium sp. USM2 PhaC_Cs (PDB ID 6K3C) and Cupriavidus necator PhaC_Cn (PDB ID 5HZ2). The secondary structures of PhaC_Cs and PhaC_Cn are shown above and below the alignment, respectively. The predicted secondary structure of PhaC_Ap is highlighted, with helices colored green and beta-strands colored blue. Residues that are conserved are depicted with a red background. The catalytic triad residues (Cys-His-Asp) are marked with blue boxes. CAP subdomains are highlighted in pink and the Lid area with a pink line. The HTH motif and lacking PS-region are shown with a green line and marked.

Figure 5

(A) Model for the PhaC_Ap homodimer consisting of free form and CoA-bound form. The α-helical CAP subdomains in the dimer are colored in salmon and forest (residues 175-301). The catalytic triad (Cys151-His339-Asp310) is represented by a stick in deep teal color. The CoA is a purple stick. (B) Comparison between CAP subdomains of free form (salmon) and CoA-bound form (forest).

Figure 6

Quality assessment of the PhaC_Ap. The graphs reveal the quality assessment of the model structures using ProSA-web [25,26]. (A) The ProSA-web scores (black dot) for the 3D model of PhaC_Ap are based on the PhaC_Cs. (B) The ProSA-web scores (black dot) for the 3D model of PhaC_Cs.

Figure 7

Side view of the closed and open active site cleft. (A,C) The active site of PhaC_Ap with the catalytic triad Cys151-His339-Asp310 in the closed and open conformation. (B,D) The active site of PhaC_Cs (6K3C) in the closed and open conformation. The catalytic triad residues displayed as deep-teal sticks and the CoA as purple sticks. The residues of the CAP subdomain from the closed and open conformation are presented in salmon and forest color, respectively.