Structure of the Catalytic Domain of the Class I Polyhydroxybutyrate Synthase from Cupriavidus necator

Abstract

Polyhydroxybutyrate synthase (PhaC) catalyzes the polymerization of 3-(R)-hydroxybutyryl-coenzyme A as a means of carbon storage in many bacteria. The resulting polymers can be used to make biodegradable materials with properties similar to those of thermoplastics and are an environmentally friendly alternative to traditional petroleum-based plastics. A full biochemical and mechanistic understanding of this process has been hindered in part by a lack of structural information on PhaC. Here we present the first structure of the catalytic domain (residues 201-589) of the class I PhaC from Cupriavidus necator (formerly Ralstonia eutropha) to 1.80 Å resolution. We observe a symmetrical dimeric architecture in which the active site of each monomer is separated from the other by ∼33 Å across an extensive dimer interface, suggesting a mechanism in which polyhydroxybutyrate biosynthesis occurs at a single active site. The structure additionally highlights key side chain interactions within the active site that play likely roles in facilitating catalysis, leading to the proposal of a modified mechanistic scheme involving two distinct roles for the active site histidine. We also identify putative substrate entrance and product egress routes within the enzyme, which are discussed in the context of previously reported biochemical observations. Our structure lays a foundation for further biochemical and structural characterization of PhaC, which could assist in engineering efforts for the production of eco-friendly materials.

Keywords: X-ray crystallography; bacterial metabolism; biosynthesis; biotechnology; enzyme mechanism.

FIGURE 1.

Proposed mechanisms for polyhydroxybutyrate formation catalyzed by PhaC. A, mechanism invoking the use of two PhaC active sites, suggesting that catalysis must take place at the dimer interface. B, mechanism invoking the use of a single PhaC active site with a CoA-bound thioester as an intermediate in catalysis. The inset shows the structure of CoA. We note that in both mechanisms, regeneration of the catalytic bases (i.e. histidine and aspartate) is ultimately achieved through proton transfer to the CoASH leaving group. Proton transfer steps have been omitted from the mechanisms as drawn for simplicity.

FIGURE 2.

The catalytic domain of CnPhaC has an α/β-hydrolase fold and is structurally similar to lipases. A, overall structure of the catalytic domain of CnPhaC(C319A). The monomer is shown in ribbon representation in violet with active site residues shown as balls and sticks with black and purple carbons for the green and violet monomers, respectively. The active site cysteine has been modeled for illustrative purposes. The second protomer of the dimer is colored green. Disordered residues within the catalytic domain are indicated as black dashed lines. The N and C termini are shown as spheres. The distance between the active site cysteine residues of the two monomers is ∼33 Å and is indicated as a black solid line. B, topology diagram of the CnPhaC catalytic domain monomer, colored as in A with active site residues indicated as colored circles. Disordered residues are indicated as a dashed line. C, overlay of the CnPhaC catalytic domain colored as in A with the gastric lipase from Canis lupus (Protein Data Bank code 1K8Q) colored in gray (Cα root mean square deviation = 3.8 Å). D, stereo view of the overall Cα trace of the structure colored in rainbow mode with the N terminus in blue and the C terminus in red. Active site residues are shown as spheres and labeled with single-letter amino acid codes.

FIGURE 3.

The active site of PhaC is composed of Cys³¹⁹, Asp⁴⁸⁰, and His⁵⁰⁸. A, view of the CnPhaC(C319A) active site with electron density. 2F_o − F_c density (teal) contoured to 1σ. No F_o − F_c density is visible in this orientation when contoured to ±3σ. B, interactions within the active site with Cys³¹⁹ modeled for illustrative purposes. Catalytic residues are shown in purple; residues on the Asp⁴⁸⁰ turn are shown in violet. Hydrogen bonds are indicated by black dashed lines.

FIGURE 4.

The active site of CnPhaC is accessible via a water-filled channel. A, overall view of the CnPhaC catalytic domain with the substrate channel as visualized using CAVER (blue surface). The PhaC monomer is shown in violet; the second protomer of the dimer is in green. Active site residues are shown as balls and sticks in purple. Arg³⁹⁸ from each monomer are shown as sticks. Dashed lines represent the disordered region of the structure. B, view of the solvent channel from the dimer interface. Protein is shown in surface representation with underlying ribbons. Waters are shown as red spheres. C, view of the channel as in B with visualization from CAVER (blue surface). D, model of HB-CoA (carbon atoms in cyan) bound in the proposed substrate channel. Modeled interactions of Arg³⁹⁸ and His⁴⁸¹ with HB-CoA are shown as gray dashes.

FIGURE 5.

Conserved residues in CnPhaC form structural motifs that appear to stabilize a proposed PHB exit channel. A, stereo view of the CnPhaC catalytic domain, colored as in Fig. 4A, showing the proposed substrate channel (blue surface) and product egress channel (yellow surface). Conserved structural motifs are represented as sticks and colored as in B–E. Nonconserved residues that participate in structural motifs through backbone atoms are shown in violet. Asp⁴²¹ is shown in pink. Cys³¹⁹, Asp⁴⁸⁰, and His⁵⁰⁸ are shown in purple. B, sequence of the C-terminal domain of CnPhaC. Strictly conserved residues are in bold; active site residues are colored in purple. Residues forming structural motifs are colored as follows: Asp²⁵⁴–Ser²⁶⁰ motif in orange, WNXD motif in teal, and GSWW motif in green. Arg³⁹⁸, Asp⁴²¹, and His⁴⁸¹ are in pink. C–E, close-up views of structural motifs as colored in A and B.

FIGURE 6.

Stereo views of HB-CoA modeled into the CnPhaC catalytic domain. A, active site of CnPhaC(C319A) in the absence of substrate. Black dashed arrow indicates deprotonation of Cys³¹⁹ by His⁵⁰⁸. The position of the Cys³¹⁹ sulfur atom is modeled. Blue and yellow dashed arrows represent the directions of the proposed substrate entrance and product egress channels, respectively. B, active site of CnPhaC(C319A) with HB-CoA (carbon atoms in cyan) modeled for initiation. Black dashed arrow represents path of nucleophilic attack of Cys³¹⁹ on the HB-CoA thioester. The position of the sulfur atom of Cys³¹⁹ is modeled. Gray dashed line represents a modeled hydrogen bond between the carbonyl oxygen of HB-CoA and the amide group of Val³²⁰. Cys²⁴⁶ and Ile²⁴⁷ are located on a loop just behind the nucleophile elbow. The amide group of one of these residues, in combination with the Val³²⁰ amide, could form the oxyanion hole to stabilize the tetrahedral intermediate during catalysis, although a conformational change within the active site upon substrate binding would be necessary to bring the loop close enough to form the appropriate interaction with the substrate carbonyl. C, active site of CnPhaC(C319A) with HB monomer (carbon atoms in gray) modeled onto Cys³¹⁹ and HB-CoA (carbon atoms in cyan) modeled for polymer elongation. Black dashed arrow represents path of nucleophilic attack of the HB-CoA hydroxyl group on the protein-bound thioester.

FIGURE 7.

Modified mechanistic scheme for PhaC. His⁵⁰⁸ deprotonates Cys³¹⁹, allowing for nucleophilic attack on the HB-CoA thioester; the histidine base is regenerated by transfer of the proton to the CoASH leaving group. A second HB-CoA substrate binds, and the HB hydroxyl group is deprotonated by His⁵⁰⁸, facilitated through modulation of the histidine basicity by Asp⁴⁸⁰. The newly formed HB alkoxide attacks the Cys-HB thioester, generating a noncovalent, CoA-bound intermediate. The growing PHB chain is then transferred back to Cys³¹⁹.