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

Simple Summary The cyanobacterium Arthrospira platensis contains 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 from A. platensis into Escherichia cloni ®10G cells to produce rPhaCAp protein. The Vmax, Km, and kcat values for β-3-hydroxybutyryl coenzyme A (3HB-CoA) of the purified rPhaCAp were investigated. Size-exclusion chromatography revealed that rPhaCAp exists 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. 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 (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.


Introduction
Energy demand worldwide is increasing continuously due to human population and economic growth. Higher consumption of fossil fuels leads to higher greenhouse gas emissions, particularly of CO 2 , which contribute to global warming. Petrochemical-based plastics, which have been used in daily life for decades, cannot be decomposed by biological processes and accumulate in the environment. The global demand for biodegradable plastic as a mandatory substitute for synthetic plastics is augmented by considering their biocompatibility, biodegradability, nontoxicity, and renewability properties [1,2]. Among the biopolymers, poly (3-hydroxybutyrate) (PHB) is the most well-characterized of the polyhydroxyalkanoates (PHAs) that are produced by a large variety of microorganisms such as bacteria, fungi, and cyanobacteria under unbalanced growth [3]. The PHB has properties similar to some traditional synthetic plastics such as polypropylene and polyethylene [4,5]. The PHB biosynthetic pathway consists of three enzymatic reactions catalyzed by three distinct enzymes encoded by the phaABC operon in many cyanobacterial species [6]. The first reaction begins with the condensation of two acetyl-CoA molecules into acetoacetyl-CoA by β-ketothiolase (encoded by phaA). The second reaction is the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase (encoded by phaB). Finally, the 3-hydroxybutyryl-CoA monomers are polymerized into poly (3-hydroxybutyrate) (PHB) by PHA synthase (encoded by phaC) ( Figure S1).
PHA synthases (PhaC) are classified into four classes based on subunit composition, primary sequences, and substrate specificity [7,8]. The structure and properties have been characterized for the Class I PhaC proteins from Cupriavidus necator (PhaC Cn ) [7], Chromobacterium sp. USM2 (PhaC Cs ) [9], Rhodovulum sulfidophilum (PhaC Rs ) [10], and Aquitalea sp. USM4 (PhaC As ) [11]. Class II PhaC is characterized from Pseudomonas spp. (PhaC Ps ) [12], Class III PhaC from Chromatium vinosum (PhaC Cv ), and Class IV PhaC from Bacillus spp. (PhaC Bs ) [13]. PHB can be categorized into two primary groups based on the carbon atom count present in the monomer units. The first group is referred to as short-chain-length (SCL) PHB and consists of repeat units made up of hydroxy fatty acids containing carbon atoms ranging from C3 to C5. This type of PHB is generated through PhaC Class I, III, and IV. The second group is known as medium-chain-length (MCL) PHB. The repeat units in this group are hydroxy fatty acids of C6-C14 carbon atoms, and they are produced by PhaC Class II [14].
Arthrospira platensis is a filamentous cyanobacterium that grows naturally in alkaline salt lakes. A. platensis are the most employed because of their high growth rate, simple cultivation, and relatively easy harvesting [15]. A. platensis contains PHA synthase Class III (PhaC Ap ), which can produce SCL-PHB. Most Arthrospira species produce large amounts of PHB under nitrogen-depleted conditions [16]. It is worth noting that the highest content of polyhydroxybutyrate (PHB) was found in Arthrospira platensis cultures that were grown under nitrogen-deprived conditions for three days, while being supplemented with 0.50% (w/v) acetate. This resulted in the maximum PHB content of 19.2% of cell dry weight [17], which is much higher than the unicellular cyanobacterium Synechocystis sp. PCC 6803 under optimized conditions, which showed a PHB content of 13.1% [18]. Therefore, A. platensis is considered to be one of the most viable candidates for industrial-scale production of PHB. To obtain a more comprehensive understanding of the function of PhaC, we created recombinant Escherichia cloni ® 10G cells that contained A. platensis phaC. The expressed A. platensis PhaC protein was purified and evaluated for its enzymatic activity and oligomeric structure in vitro. We also constructed homology 3D models for A. platensis PhaC and analyzed the active site for its ligand-binding properties.

Bacterial Strains and Culture Conditions
The Arthrospira platensis was cultivated in a 250-mL Erlenmeyer flask with 50 mL of Zarrouk medium (pH 10.0) at 32 • C under a continuous fluorescent lamp emitting 40 µmol photon/m 2 /s on a rotatory shaker at 120 rpm [14]. Escherichia cloni ® 10G (Lucigen) was used for cloning and expression analysis. Luria-Bertani (LB) media supplemented with 100 g/mL kanamycin was used to grow and maintain cells containing recombinant plasmids. Following overnight cell growth at 37 • C, the growth of E. cloni ® 10G was measured in terms of turbidity (OD 600 ). E. cloni ® 10G strains were grown in LB medium Biology 2023, 12, 751 3 of 15 at 37 • C and 120 rpm in an incubator. Expresso ® pSol-Tsf vector (Lucigen) was used as a vector for cloning.

Cloning of phaC Ap in pSol-Tsf Plasmid
The open reading frame of phaC Ap gene (1095 bp) encoding PhaC Ap protein was amplified from A. platensis genomic DNA using primers: Fpha: 5 -AAT CTG TAC TTC CAG GGT ATG TTA CCT-3 and Rpha: 5 -GTG GCG GCC GCT CTA TTA TTA CTC TCG-3 (underlined sequence indicates primers that add flanking sequence identical to those found at the ends of linear pSol-Tsf vector). PCR was performed with a temperature program starting at 94 • C for 10 min, followed by 30 cycles of 94 • C for 45 s, 52 • C for 45 s, 72 • C for 45 s, and a final elongation at 72 • C for 10 min. The phaC Ap gene, which was amplified to a length of 1131-bp, was extracted and purified using the Silica Bead DNA Gel Extraction Kit (Thermo Fisher Scientific), and the fragment was cloned into the Expresso ® pSol-Tsf vector (Lucigen). The recombinant plasmid (pSol-Tsf-phaC) harboring the SelecTEV TM Protease, 6x-His-tagged fusion, and terminator recognition sites was transformed into E. cloni ® 10G, and the presence of inserts with correct sequence was verified by colony PCR and further by sequencing.

Expression and Purification of rPhaC Ap
Fresh 500 mL LB broth containing 100 µg/mL kanamycin was inoculated with transformed cells harboring recombinant (pSol-Tsf-phaC) or empty vectors (pSol-Tsf), and allowed to grow at 37 • C. When the OD 600 of the culture reached~0.5, cells were induced with a final concentration of 0.2% (w/v) L-rhamnose. This culture was incubated at 30 • C for 4 h with shaking at 120 rpm. Overexpressed cells were collected and subjected to three MQ water washes. The cell pellets were suspended in buffer pH 7.5 (50 mM Na 2 HPO 4 , 300 mM NaCl) and lysed by sonication (20 kHz) at 32 • C for 10 min, followed by centrifugation to remove cell debris. The supernatant containing recombinant 6x-His-tagged fusion protein (rPhaC Ap ) was loaded onto a Ni-NTA column (Ni Sepharose TM 6 Fast Flow, GE Healthcare Bio-sciences AB), then washed with five column volumes of binding buffer (50 mM Na 2 HPO 4 pH 7.5, 300 mM NaCl, 10 mM imidazole). The rPhaC Ap was eluted from the column with elution buffer (50 mM Na 2 HPO 4 , pH 7.5, 300 mM NaCl, 90 mM imidazole). The protein purity was determined by using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Protein concentration was determined by using a Bio-Rad protein assay kit and BSA as standard [19].

In Vitro Enzymatic Assay of the Purified rPhaC Ap
The activity of the purified rPhaC Ap was determined by estimating the amount of CoA (reaction product) using β-3-hydroxybutyryl coenzyme A (3HB-CoA). This assay was carried out in the reaction mixture (1 mL) containing 50 µM 3HB-CoA, 50 µM 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB), BSA (0.2 mg/mL) in 100 mM Tris-HCl buffer (pH 8.0) and enzyme, which was previously reported by [20]. Incubation was carried out at 30 • C for 10 min. The liberation of CoA was determined by measuring the absorbance at 412 nm with a spectrophotometer. The enzyme activity was expressed as µmol/min/mg. The measurement was carried out in triplicate. Protein concentration was determined by using a Bio-Rad protein assay kit and BSA as standard [19].

Determination of Steady-State Kinetic Constants of rPhaC Ap
The K m and k cat values were calculated using the initial velocity data obtained by varying the concentration of 3HB-CoA from 10 µM to 400 µM. The initial velocity data were fitted to Michaelis-Menten equation V 0 = V max [S]/(K m + [S]) (where V 0 is the initial velocity, V max is the maximum velocity, [S] is the substrate concentration, and K m is the Michaelis constant) with GraphPad Prism 9.5.1 software. The K m represents the equilibrium constant for dissociation of the enzyme from the substrate. The k cat value was calculated from the ratio of V max and enzyme concentration. Each reaction was initiated by the addition of 100 µg of crude enzyme to 1 mL of reaction mixture.

Production of Polyclonal Antibodies against rPhaC Ap
Two healthy, 12-week-old male New Zealand rabbits were maintained in the experimental animal facility. Purified rPhaC Ap (100 µg) was emulsified with an equal volume of Freund's Complete Adjuvant (FCA) and subcutaneously injected into rabbits for the first immunization. Three booster injections of the same protein mixed with FCA were given to each rabbit on the 14 th , 21 st , and 28 th day. The rabbits were bled via the marginal ear vein prior to the first dose and at 7-day intervals, and the serum antibody titer was measured using ELISA. The rabbit anti-rPhaC Ap polyclonal antibody serum was kept for further experiment. The body weight, temperature, complete blood count, and behavior of the rabbits in the home cage, upon handling, and in an open field did not differ significantly among the immunization groups during the 7-week assessment period.

Western Blot Analysis
The 6x-His-tagged fusion rPhaC Ap protein (69 kDa) was purified and analyzed by resolving in a 12% SDS-PAGE gel. The separated protein was then transferred to a nitrocellulose membrane (Bio-Rad) using a Trans-Blot ® Semi-Dry system (Bio-Rad) with an applied voltage of 10V for 60 min. The nitrocellulose membranes were incubated with 5% (w/v) non-fat powdered milk in PBST buffer (25 mM phosphate-buffer pH 7.4, 150 mM NaCl, 0.1% (v/v) Tween 20) at 4 • C overnight. The membranes were washed three times by PBST buffer. Subsequently, the nitrocellulose membranes were incubated with either a 1:5000 dilution of rabbit anti-His-tagged polyclonal antibody (Thermo Fisher Scientific) or a 1:10,000 dilution of rabbit anti-rPhaC Ap polyclonal antibody in PBST buffer for 3 h at room temperature. Next, the membranes were washed three times with PBST buffer and then incubated with a 1:2000 dilution of HRP-conjugated goat anti-rabbit IgG (Sigma-Aldrich) in PBST buffer containing 2% non-fat powdered milk for 90 min at room temperature. Following the washing step, the nitrocellulose membranes were treated with diaminobenzidine (DAB) using a DAB substrate kit (Thermo Fisher Scientific) at room temperature for 5-10 min. A brown band on a nitrocellulose membrane was observed, indicating the presence of the rPhaC Ap protein.

Size-Exclusion Chromatography
The reaction mixture consisting of purified rPhaC Ap and 100 mM sodium phosphate (pH 7.4) was incubated for 10 min. The reaction mixtures were then loaded onto a Sephadex-G50 column (Merck, Darmstadt, Germany) equilibrated with 100 mM sodium phosphate (pH 7.4). The reaction mixture containing rPhaC Ap was eluted with the same buffer at a flow rate of 1 mL/min. The eluted samples were measured for absorbance at 280 nm, and their retention times were compared to those of molecular standards. The standards used for comparison were alcohol dehydrogenase (150 kDa, 18.5 min), BSA (66 kDa, 24.5 min), and ovalbumin (44 kDa, 27.5 min).

Sequence and Multiple Sequence Alignment Analysis
The amino acid sequence of PhaC Ap was retrieved from UniProtKB (UniProt code D4ZNW6). BLAST (Basic Local Alignment Search Tool) was used to identify the structural templates for modeling PhaC Ap . The Protein Data Bank (PDB, http://www.rcsb.org (accessed on 11 May 2020); Berman et al., 2000), which is available at http://www.rcsb.org (accessed on 13 May 2020) and contains a collection of protein structures, was used as the target database. The BLAST searches revealed 4 PhaC structures with expectation values (e-value) below 1 × 10 −17 : two structures from Cupriavidus necator H16 (Ralstonia eutropha) (PDB codes 5T6O and 5HZ2), and Chromobacterium sp. USM2 (PDB codes:5XAV and 6K3C). Next, we created a structure-based sequence alignment of the PhaC structures from Cupriavidus necator H16 (5HZ2) and from Chromobacterium sp. USM2 (6K3C) by superimposing the structures with VERTAA [21]. Since the sequence identity of these structures to PhaC Ap was only around 20%, we searched for more sequences similar to PhaC Ap and aligned the Synechocystis sp. PCC 6803 PhaC ((PhaC Ss ; WP_041425936.1), Bernardetia litoralis PhaC (PhaC Bl ; WP_014797661.1), and Aromatoleum buckelii PhaC (PhaC Ab ; WP_169197587.1) to the pre-aligned structure-based alignment using MALIGN in Bodil [22]. The secondary structure for the PhaC Ap sequence was predicted using Jpred [23]. The final sequence alignment figure was prepared with ESPript 3.0 [24].

Homology Modelling
The 3D model of PhaC Ap was created using the multiple sequence alignment and the crystal structure of PhaC Cs from Chromobacterium sp. USM2 (PDB code: 6K3C) as a template, with a resolution of 1.8 Å. MODELLER generated ten models for PhaC Ap , and the one with the lowest energy, as determined by the MODELLER objective function, was selected for additional structural analysis. The ProSA-wed (https://prosa.services.came. sbg.ac.at/prosa.php (accessed on 10 October 2021)) [25,26] was used to assess the quality of the 3D model for PhaC Ap .

Cloning, Expression, and Purification of rPhaC Ap
A band of approximately 1131 bp on the agarose gel electrophoregram corresponded to the fragment encoding rPhaC Ap ( Figure 1A). The fragment was purified and ligated into the pSol-Tsf vector and successfully verified by colony PCR and DNA sequencing. E. cloni ® 10G was transformed with the recombinant plasmid pSol-Tsf-phaC Ap for the expression of rPhaC Ap with 6x-His-tagged fusion ( Figure 1B). Conditions for rPhaC Ap induction were established with the expression strain E. cloni ® 10G. SDS-PAGE showed that rPhaC Ap was present in soluble fraction (SF) after induction with 0.002% (w/v) Lrhamnose at 30 • C ( perimposing the structures with VERTAA [21]. Since the sequence identity of these str tures to PhaCAp was only around 20%, we searched for more sequences similar to Pha and aligned the Synechocystis sp. PCC 6803 PhaC ((PhaCSs; WP_041425936.1), Bernard litoralis PhaC (PhaCBl; WP_014797661.1), and Aromatoleum buckelii PhaC (Pha WP_169197587.1) to the pre-aligned structure-based alignment using MALIGN in B [22]. The secondary structure for the PhaCAp sequence was predicted using JPred [23]. final sequence alignment figure was prepared with ESPript 3.0 [24].

Homology Modelling
The 3D model of PhaCAp was created using the multiple sequence alignment and crystal structure of PhaCCs from Chromobacterium sp. USM2 (PDB code: 6K3C) as a t plate, with a resolution of 1.8 Å. MODELLER generated ten models for PhaCAp, and one with the lowest energy, as determined by the MODELLER objective function, selected for additional structural analysis. The ProSA-wed (https://prosa. vices.came.sbg.ac.at/prosa.php (accessed on 10 October 2021)) [25,26] was used to as the quality of the 3D model for PhaCAp.

PHA Synthase Activity and Kinetics
The optimal condition for purified rPhaCAp activity was observed at 30 °C in 100 mM Tris-HCl buffer (pH 8.0) in the presence of 3HB-CoA as substrate. The Michaelis-Menten fitting of the data is shown in Figure 3A. The Vmax, Km, and kcat values for 3HB-CoA of the purified rPhaCAp were 24.5 ± 2 μmol/min/mg, 31.3 ± 2 μM, and 412.7 ± 2 1/s, respectively.

Size-exclusion Chromatography
Size-exclusion chromatography was performed to examine the multimeric formation of PhaCAp. Two peaks were detected at 19.0 and 24.5 min ( Figure 3B). According to the calibration curve generated from the elution times of the molecular standard, the molecular weights corresponding to peaks with elution times of 19.0 and 24.5 min were estimated to be 141 and 71 kDa, respectively, which corresponds to the dimeric rPhaCAp (138 kDa) and monomeric rPhaCAp (69 kDa).

PHA Synthase Activity and Kinetics
The optimal condition for purified rPhaC Ap activity was observed at 30 • C in 100 mM Tris-HCl buffer (pH 8.0) in the presence of 3HB-CoA as substrate. The Michaelis-Menten fitting of the data is shown in Figure 3A. The V max , K m , and k cat values for 3HB-CoA of the purified rPhaC Ap were 24.5 ± 2 µmol/min/mg, 31.3 ± 2 µM, and 412.7 ± 2 1/s, respectively.

PHA Synthase Activity and Kinetics
The optimal condition for purified rPhaCAp activity was observed at 30 °C in 100 mM Tris-HCl buffer (pH 8.0) in the presence of 3HB-CoA as substrate. The Michaelis-Menten fitting of the data is shown in Figure 3A. The Vmax, Km, and kcat values for 3HB-CoA of the purified rPhaCAp were 24.5 ± 2 μmol/min/mg, 31.3 ± 2 μM, and 412.7 ± 2 1/s, respectively.

Size-exclusion Chromatography
Size-exclusion chromatography was performed to examine the multimeric formation of PhaCAp. Two peaks were detected at 19.0 and 24.5 min ( Figure 3B). According to the calibration curve generated from the elution times of the molecular standard, the molecular weights corresponding to peaks with elution times of 19.0 and 24.5 min were estimated to be 141 and 71 kDa, respectively, which corresponds to the dimeric rPhaCAp (138 kDa) and monomeric rPhaCAp (69 kDa).

Size-Exclusion Chromatography
Size-exclusion chromatography was performed to examine the multimeric formation of PhaC Ap . Two peaks were detected at 19.0 and 24.5 min ( Figure 3B). According to the calibration curve generated from the elution times of the molecular standard, the molecular weights corresponding to peaks with elution times of 19.0 and 24.5 min were estimated to be 141 and 71 kDa, respectively, which corresponds to the dimeric rPhaC Ap (138 kDa) and monomeric rPhaC Ap (69 kDa).

Sequence Analysis and Multiple Sequence Alignment for Homology Modeling
The gene for PhaC Ap in A. platensis had a length of 1095 base pairs and encoded a protein consisting of 364 amino acid residues with a molecular weight of 42 kDa. Due to the low sequence identity between PhaC Ap and the structurally known PhaC homologs, multiple sequence alignments with intact structural data were made to provide a more reliable basis for the construction of a 3D model for PhaC Ap . A structure-based sequence alignment, pre-aligned for PhaC Cs and PhaC Cn structures, was used to align PhaC Ap and four other related PhaC sequences. The resulting alignment is shown in Figure 4. According to the structure-based alignment, PhaC Cs and PhaC Cn share 51.5% sequence identity with each other, and 20% sequence identity with PhaC Ap . Table 1

Sequence Analysis and Multiple Sequence Alignment for Homology Modeling
The gene for PhaCAp in A. platensis had a length of 1095 base pairs and encoded a protein consisting of 364 amino acid residues with a molecular weight of 42 kDa. Due to the low sequence identity between PhaCAp and the structurally known PhaC homologs, multiple sequence alignments with intact structural data were made to provide a more reliable basis for the construction of a 3D model for PhaCAp. A structure-based sequence alignment, pre-aligned for PhaCCs and PhaCCn structures, was used to align PhaCAp and four other related PhaC sequences. The resulting alignment is shown in Figure 4. According to the structure-based alignment, PhaCCs and PhaCCn share 51.5% sequence identity with each other, and 20% sequence identity with PhaCAp. Table 1 displays the conserved amino acid residues found in PhaCAp, PhaCCn (Cupriavidus necator PhaC), and PhaCCs (Chromobacterium sp. USM2 PhaC).
Since the experimental analysis revealed that rPhaC Ap forms a catalytically active dimer, we used the asymmetric dimer of the catalytic domain of PhaC Cs (6K3C) as a template for modeling to predict the overall fold and pinpoint the catalytically important residues and structural features of rPhaC Ap . Similar to the template structure of PhaC Cs , the 3D model for the PhaC Ap is an asymmetric dimer in which the CoA-free monomer is in the closed conformation and the CoA-bound monomer in the open conformation ( Figure 5A). The α/β hydrolase fold, consisting of the α/β core subdomains and the CAP subdomains (residues Val175-Pro301), forms the dimer interface in both the closed and open monomers of the PhaC Ap dimer. The α/β core subdomain consists of β-sheets (β1-β8) and α-helices (α1-α5) in both monomers. However, the closed and open conformations represent a distinct conformation, particularly in the CAP domain ( Figure 5B). The closed form of the CAP domain is composed of four α-helices (αA-αD) and three 3 10 -helices (ηA-ηC) in the sequence (ηA-αA-αB-αC-αD-ηB-ηC), and make close contacts with the α/β core subdomain. The αB helix, which is made up of 26 amino acid residues (Asp215-Ile240), is a unique feature of the CAP subdomain. The αB helix, αB-αC loop, and αC helix of the CAP subdomain provide protection to the catalytic triad residues (Cys151-His339-Asp310) to a significant extent. On the other hand, the open form of the CAP subdomain comprises five α-helices (αA-αF) and a short 3 10 -helix (ηA-ηF) in the sequence (ηA-αA-αB-αC-αD-ηB-ηF-ηC-ηD). In the open conformation, the fold of the CAP subdomain (residues Val175-Pro301) differs from that of the closed form, and the subdomain does not provide coverage to the catalytic triad residues. The dimer interface is formed by the CAP subdomain of both chains ( Figure 5A,B). Analysis of the homology model for PhaC Ap with the ProSA-web gave a Z-score of −6.24 ( Figure 6A), and the crystal structure of PhaC Cs gave a Z-score of −8.1 ( Figure 6B). These Z-scores are within the range of the typical scores for experimentally determined structures of a similar size. Furthermore, the predicted secondary structure for PhaC Ap matches well with the secondary structure elements in the PhaC Cs and PhaC Cn crystal structures. Hence, the overall fold of PhaC Ap could be modeled with relatively good reliability.

The Active Site and Catalytic Mechanism of PhaC Ap
The overall architecture of the active site in PhaC Ap is very similar to that of PhaC Cs . In the closed monomer of PhaC Cs , the αB-αC loop (Leu380-Lys382) in the CAP subdomain blocks the catalytic triad. In the closed-form monomer of rPhaC Ap , the catalytic triad is blocked by residues Met241-Ser243 of the αB-αC loop in the CAP subdomain ( Figure 7A,B). Residues Met241-Ser243 of the αC-αD loop in the CAP subdomain have a similar role in the open conformation of PhaC Ap (Figure 7C,D). In PhaC Ap , the open conformation exposes the catalytic Cys-His-Asp triad (Cys151-His339-Asp310). Residues Asp86, Leu87, His311, and His339 near the CoA binding site are conserved in PhaC Ap , PhaC Cs , and PhaC Cn ( Table 1). The PhaC Ap catalytic triad (Cys-Asp-His) cleft is situated between the CAP subdomain and the α/β core subdomains. The β5-α3 loop containing Cys151, the β7-α4 loop containing Asp310, and the β8-α5 loop containing His339 are the components that make up the α/β core subdomains. The extended pantetheine arm of CoA attaches to the active site, which is close to the catalytic triad, and the ADP moiety of CoA is positioned there. Cysteamine containing the terminal thiol (SH) group is close to Cys151 ( Figure 7C). The ribose ring is shielded by the hydrophilic residue Glu37 of the extended N-terminal loop, while the Val85 residue from β3-α1 loop interacts with the adenine moiety of CoA. The interaction with CoA is facilitated by Met241-Ser243, which is located in the αC-αD loop of the CAP subdomain. The hydrophobic nature of the cleft is due to the presence of nonpolar residues from the α/β core subdomains, namely Asp86, Val85, Leu312, and Val313. The side chain of Asp310 forms a hydrogen bond with the pantetheine arm of CoA. In addition, the αC-αD loop of the enzyme locks the pantetheine arm of CoA, as shown in Figure 7C, through the side chain carbonyl groups of Leu312 and Val313.

The Active Site and Catalytic Mechanism of PhaCAp
The overall architecture of the active site in PhaCAp is very similar to that of PhaCCs. In the closed monomer of PhaCCs, the αB-αC loop (Leu380-Lys382) in the CAP subdomain blocks the catalytic triad. In the closed-form monomer of rPhaCAp, the catalytic triad is blocked by residues Met241-Ser243 of the αB-αC loop in the CAP subdomain ( Figure 7A,B). Residues Met241-Ser243 of the αC-αD loop in the CAP subdomain have a similar role in the open conformation of PhaCAp ( Figure 7C,D). In PhaCAp, the open conformation exposes the catalytic Cys-His-Asp triad (Cys151-His339-Asp310). Residues Asp86, Leu87, His311, and His339 near the CoA binding site are conserved in PhaCAp, PhaCCs, and PhaCCn ( Table 1). The PhaCAp catalytic triad (Cys-Asp-His) cleft is situated between the CAP subdomain and the α/β core subdomains. The β5-α3 loop containing Cys151, the β7-α4 loop containing Asp310, and the β8-α5 loop containing His339 are the components that make up the α/β core subdomains. The extended pantetheine arm of CoA attaches to the active site, which is close to the catalytic triad, and the ADP moiety of CoA is positioned there. Cysteamine containing the terminal thiol (SH) group is close to Cys151 ( Figure 7C). The ribose ring is shielded by the hydrophilic residue Glu37 of the extended N-terminal loop, while the Val85 residue from β3-α1 loop interacts with the adenine moiety of CoA. The interaction with CoA is facilitated by Met241-Ser243, which is located in the αC-αD loop of the CAP subdomain. The hydrophobic nature of the cleft is due to the presence of nonpolar residues from the α/β core subdomains, namely Asp86, Val85, Leu312, and Val313. The side chain of Asp310 forms a hydrogen bond with the pantetheine arm of CoA. In addition, the αC-αD loop of the enzyme locks the pantetheine arm of CoA, as shown in Figure 7C, through the side chain carbonyl groups of Leu312 and Val313.

Discussion
In this study, we cloned class III PhaC from A. platensis (rPhaC Ap ) and produced rPhaC Ap with hydrophilic His-tagged fusion using a bacterial protein expression system. We demonstrated that His-tagged rPhaC Ap was highly induced with 0.2% (w/v) L-rhamnose at 30 • C. The purified His-tagged rPhaC Ap , which represented a molecular size of 69 kDa, was used directly as an antigen for immunization without further depleting His-tag [27]. The presence of N-terminal His-tagged rPhaC Ap was verified through Western blotting with polyclonal anti-rPhaC Ap antibodies. The lower faint band was the minor protein contaminants. These minor proteins showed no changes without or with induction by increasing concentration of L-rhamnose ( Figure 2A, lanes 2, 3, 4, and 5). In addition, sizeexclusion chromatography revealed the active dimerization of rPhaC Ap subunit (138 kDa). These results suggested that the PhaC Ap exists as a dimeric assembly in solution. On the basis of previous observations, PhaC synthase likely forms either various dimers or multimers other than monomers [28][29][30]. Furthermore, a dimeric form of PhaC synthase was important in its catalytic function, although, in general, the monomeric and dimeric forms of PhaC exist in equilibrium [31][32][33]. The Michaelis-Menten fitting of the data shown in Figure 3A demonstrated that K m and k cat values for 3HB-CoA of the purified rPhaC Ap were 31.3 ± 2 µM and 412.7 ± 2 1/s, respectively. Numerous studies have reported kinetic parameters of PhaC. One of those demonstrated that the K m value for 3HB-CoA of PhaCE from Synechocystis sp. PCC 6803 (rPhaCE) was 478 ± 31 µM [20], whereas the K m value of PhaC from Aeromonas caviae FA440 (rPhaC Ac ) was 77 ± 5 µM [34]. In addition, K m and k cat values of the purified rPhaC Av (Allochromatium vinosum) were 0.11 mM and 508 1/s, respectively [35], while those of the purified rPhaEC Es (Ectothiorhodospira shaposhnikovii) were 0.065 mM and 320 1/s, respectively [36], and 0.13 mM and 3920 1/min, respectively for rPhaEC Av (Allochromatium vinosum [37]. Probably, high values of K m correspond to the structure of PHA synthase. The rPhaCE has a lower enzyme affinity for substrates than rPhaC. The catalytic activity of PHA synthase is easily influenced by abiotic factors, i.e., pH, ionic strength, or temperature [38]. Although this enzyme has frequently been tested for activity using both Tris-HCl and phosphate-buffered solutions, at the same concentration and pH, Tris-HCl buffer exhibits primarily greater enzyme activity [38]. In the present study, the highest rPhaC Ap activity was observed in 100 mM Tris-HCl buffer (pH 8.0) at 30 • C. Additionally, the PHA synthase activity from Ralstonia eutropha was reduced when the concentration of the buffer solution increased [38]. This implies that ionic strength affects PHA synthase activity. Additionally, the cultivation temperature could significantly influence PHB production. In terms of the class III PHA synthase, E. coli JM109 harboring the phaECAB expression plasmid was found to be capable of producing PHB. The maximum PHB production (0.64 g/L) was found at 30 • C among the four temperatures tested (25 • C, 30 • C, 37 • C, and 42 • C), indicating their differences in catalytic activity [39]. In addition, when BSA (0.2 mg/mL) was added in the assay mixture, the PHA synthase activity from Allochromatium vinosum (PhaEC Av ) was increased [35]. BSA has been shown to bind PHB granules in vitro, and it was proposed that BSA interacts with hydrophobic polymers to prevent the increasing chain from obstructing the active site, which led to the observed increase in activity. In the present study, inclusion of BSA (0.2 mg/mL) in the reaction mixture somehow enhanced the activity of rPhaC Ap . Moreover, it was reported that a R. eutropha PHA synthase mutant with a high catalytic activity can synthesize higher molecular weight P (3HB) compared with wild type or mutants with lower catalytic activities, indicating that the catalytic activity of PHA synthase affects the molecular weight of PHA [40].
The PhaC Ap forms are very similar to those of PhaC Cs . The αB-αC loop undergoes conformational changes in the PhaC Cs crystal structure, causing the C-terminal end region of the α/β core subdomains to become disordered. As a result, the αB and αC helices move away from the active site, allowing the CoA molecule to enter the active site cleft. [9]. The movement of the αB helix and αC helix of the CAP subdomain in both PhaC Cs and PhaC Ap is similar to a retracting "Boom gate" mechanism, which covers the catalytic Cys-His-Asp triad and blocks substrate entry [9]. In the open conformation of PhaCCs, the αC-αD loop in the CAP subdomain contains Leu380-Lys382 residues that form polar interactions with CoA, which is critical for PhaC activity. Additionally, the CoA moiety of acyl-CoA substrates plays an important role in this process. Zhang et al. [41] reported that the Class III synthase (PhaC-PhaE) from Allochromatium vinosum (PhaCE Av ) appears to require the CoA molecule for binding to its substrate. These results are in agreement with the hypothesis that the complex between PhaC and the substrate acyl-CoA is formed by the interaction between PhaC and the CoA moiety of the substrate. The CAP domain may play a significant role in the catalytic activity of PHA synthase, because local conformational changes in the α/β core subdomain are caused by conformational changes of the CAP subdomain. The activity of PHA synthase is reliant on the presence of the catalytic triad C-H-D, which refers to the amino acid residues cysteine, histidine, and aspartate. Interestingly, this same triad is also responsible for the catalysis of lipases, but with the substitution of serine in place of cysteine (S-H-D) [8]. In the crystal structure of PhaC Cs , the Cys291, Asp447, and His477 that form the catalytic triad are crucial for its catalytic activity [9]. The most striking difference between the crystal structures of PhaC Cs and PhaC Cn compared to PhaC Ap is the lack of so-called protruding structure regions (PS-region in Figure 4), which are also lacking in some other PhaC proteins [42].
The catalytic triad in PhaC Cs has been proposed to function in PHA biosynthesis in the following way. First, the 3HB-CoA molecule enters the active site tunnel of PhaC, where His477 facilitates deprotonation of the thiol group of the catalytic triad residue Cys291. This results in the formation of a covalent bond between 3HB and Cys291, and the release of CoA from the tunnel. Asp447 plays a role in the PHA elongation process by participating in the attack on the acyl-CoA substrate, specifically targeting the hydroxyl group within the acyl moiety. After the release of CoA, the newly attacked 3HB-CoA enters the active site and the cycle repeats. In this way, the polymer chain is elongated, resulting in the formation of (3HB) n+1 covalently bound to the Cys residue. Therefore, at the end of each cycle, the 3HB polymer is bound to the enzyme [43]. In addition, in the CoA-bound monomer of the PhaC Cs dimer, the replacement of aspartic acid with asparagine (D447N) forms hydrogen bonds with the carbonyl group of CoA [14,44], and it was reported that a D480N mutation in 6x-His-tagged PhaC Cn reduced the reaction rate to the 0.0008 units/mg from 20 unit/mg observed in wild type. Furthermore, the mutation of Phe318 in the pocket in Ralstonia eutropha PHA synthase (PhaC Re ) led to a decrease in synthase activity [42]. Harada et al. [28] demonstrated that mutation of Phe318 in PhaC Ac resulted in a significant reduction in polymer synthesis. Moreover, Müh et al. [45] showed that PhaC from Chromatium Vinosum was capable of in vitro PHA polymerization in the absence of PhaE, and PhaC alone was much more susceptible to such inhibition of PMSF in comparison with PhaEC.

Conclusions
In this study, the recombinant PhaC Ap enzyme was constructed and produced in an E. cloni ® 10G system. Our homology model shows that Cys151, Asp310, and His339 are the important catalytic triad residues that determine the pocket size of PhaC Ap . Therefore, it is reasonable to hypothesize that a mutation at these positions would have a significant influence on the pocket depth and PhaC synthase activity. To better understand the regulation of PHA synthesis in A. platensis, the kinetics of enzymes with catalytic triad (Cys-Asp-His) mutations would be valuable for further investigation. The proper orientation of the substrate may increase the efficiency of the catalytic reaction. Additionally, the model for an asymmetric PhaC Ap homodimer provided insight into the putative mechanism and function of PHA synthase in A. platensis.