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. 2017 Jul 13;7(1):5312.
doi: 10.1038/s41598-017-05509-4.

Structure of polyhydroxyalkanoate (PHA) synthase PhaC from Chromobacterium sp. USM2, producing biodegradable plastics

Min Fey Chek  1 , Sun-Yong Kim  1 , Tomoyuki Mori  1 , Hasni Arsad  2 , Mohammed Razip Samian  3 , Kumar Sudesh  3 , Toshio Hakoshima  4
Affiliations

Structure of polyhydroxyalkanoate (PHA) synthase PhaC from Chromobacterium sp. USM2, producing biodegradable plastics

Min Fey Chek et al. Sci Rep. .

Abstract

Polyhydroxyalkanoate (PHA) is a promising candidate for use as an alternative bioplastic to replace petroleum-based plastics. Our understanding of PHA synthase PhaC is poor due to the paucity of available three-dimensional structural information. Here we present a high-resolution crystal structure of the catalytic domain of PhaC from Chromobacterium sp. USM2, PhaC Cs -CAT. The structure shows that PhaC Cs -CAT forms an α/β hydrolase fold comprising α/β core and CAP subdomains. The active site containing Cys291, Asp447 and His477 is located at the bottom of the cavity, which is filled with water molecules and is covered by the partly disordered CAP subdomain. We designated our structure as the closed form, which is distinct from the recently reported catalytic domain from Cupriavidus necator (PhaC Cn -CAT). Structural comparison showed PhaC Cn -CAT adopting a partially open form maintaining a narrow substrate access channel to the active site, but no product egress. PhaC Cs -CAT forms a face-to-face dimer mediated by the CAP subdomains. This arrangement of the dimer is also distinct from that of the PhaC Cn -CAT dimer. These findings suggest that the CAP subdomain should undergo a conformational change during catalytic activity that involves rearrangement of the dimer to facilitate substrate entry and product formation and egress from the active site.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Structure of PhaCCs-CAT. (a) Polymerization reaction catalyzed by PhaC. Polymerization of the substrate, 3-hydroxybutyryl coenzyme A (3HB-CoA), into poly 3-hydroxylbutyrate (PHB) is catalyzed by PhaC with release of CoA. The chemical structure of 3HB-CoA is shown in the inset. (b) Domain organization of PhaCCs. PhaCCs consists of two domains, the N-terminal domain which is important for stabilizing dimeric PhaC and the C-terminal catalytic (CAT) domain containing conserved active site including triad residues Cys, His and Asp. (c) A side view of PhaCCs-CAT. The catalytic domain of PhaCCs comprises the α/β core subdomain (residues 175–318, 439–562 in cyan) and the CAP subdomain (residues 319–438 in violet). The side chains of the catalytic triad (Cys291, His477 and Asp447) are shown in orange. (d) As in b, but a top view of PhaCCs-CAT. The active site is covered by the CAP subdomain. (e) Schematic presentation of the secondary structure topology of PhaCCs-CAT (mol B). The CAT domain contains the CAP and core subdomains. The core subdomain comprises 13 strands and 10 helices. Nucleophilic Cys291 is located between β6 and α3, conserved His477 is located between β9 and α5, and Asp447 is located between β8 and α4. The CAP subdomain is connected from β7 and back to the core domain through β8. The catalytic triad is covered by the CAP subdomain which blocks the substrate entry pathway. The other strands (orange) and helices (red) shown represent additional secondary structures observed when comparing the canonical α/β hydrolase fold.
Figure 2
Figure 2
The catalytic residues of PhaCCs-CAT. (a) A close-up view of the catalytic site of PhaCCs-CAT. The catalytic triad residues (orange) comprise Cys291, His477 and Asp447, with a hydrogen bond between His477 and Asp447. The catalytic center Cys291 is located at the nucleophilic elbow sandwiched with nonpolar residues Phe290 and Val292 (green). Hydrogen bonds are indicated by broken lines. (b) Overlay of the catalytic triad of PhaCCs-CAT (orange) onto human gastric lipase (1HLG in green). (c) Overlay of the active site with the catalytic triad of PhaCCs-CAT (orange) onto those of PhaCCn-CAT (5T6O in yellow; 5HZ2 in magenta). The imidazole ring of the His residue of PhaCCn-CAT (magenta) is flipped from the other two.
Figure 3
Figure 3
Water molecules at both sides of the nucleophilic elbow of PhaCCs-CAT. (a) A cluster of water molecules (red balls with dotted surfaces) is located at the active site cavity around the nucleophilic elbow. The cavity is covered by the LID region of the CAP subdomain (magenta). Other water molecules are shown as red crosses. Color codes are the same as in Fig. 2. The hydrogen bond between Glu329 and Arg365 is indicated by a broken line. (b) As in a, but without the LID region. Hydrogen bonds involving the catalytic residues are shown as broken lines. Water molecules are divided into two groups, one group at Site A and the other at Site B. The cavity is mostly hydrophobic although Tyr412 and His324 are located at Site A and Asn220 at Site B. Hydrogen bonds are indicated by broken lines.
Figure 4
Figure 4
Conformational changes in the CAP subdomain as revealed by structural comparison of PhaCCs-CAT and PhaCCn-CAT structures. (a) A side-view of the overlay between PhaCCs-CAT (cyan and magenta) and PhaCCn-CAT (gray and yellow) with an overall r.m.s. deviation of 2.7 Å for Cα carbon atoms. The major conformational deviations are observed in part of the CAP subdomain, the LID region (residues Pro327–Pro386 in magenta) of PhaCCs-CAT, which corresponds to the segment Thr355–Pro419 (yellow) of PhaCCn-CAT. Arrows indicate conformational transitions of the LID region from PhaCCs-CAT to PhaCCn-CAT. (b) As in a, but a top-view down to the active site. A narrow path to the active site was found in PhaCCn-CAT, whereas the path is covered by the LID region in PhaCCs-CAT. (c) To clarify the conformational transitions found by the structural comparison of PhaCCs-CAT and PhaCCn-CAT, overlay of the LID regions of the two structures are shown. Unfolding of ηA, αA and ηB helices of PhaCCs-CAT into a long flexible D-loop found in PhaCCn-CAT is indicated by magenta arrows, and refolding of αB’ and ηB’ helices and their linker loop of PhaCCs-CAT into a long α4 helix in PhaCCn-CAT by red arrows. The disulfide bond between Cys328 and Cys438 of PhaCCn-CAT is shown in green.
Figure 5
Figure 5
PhaCCs-CAT forms a dimer in the crystal. (a) A top-view of the PhaCCs-CAT dimer along the pseudo-dyad axis. The color codes of mol B are the same as in Fig. 1, with mol A core (gray) and CAP (yellow) subdomains shown. The distance between the catalytic cysteine residues is 28.1 Å (red double-headed arrow). (b) As in a, but the side-view. Contact areas I (red) and II (orange) are indicated by broken circles. The N-termini of both protomers are located at the same side of the dimer. (c) Contact area I forms a hydrophobic cluster with nonpolar residues from ηB’-αC loop (Leu369, Trp371) and αC helix (Pro386, Phe387, Phe390) from both protomers. The segment (Asn372-Thr383) between ηB’-αC loop (Leu369, Trp371) and αC helix are disordered in our crystal (dashed lines). (d) Contact area II contains salt bridges (Arg365–Glu329) buried inside the interface formed by nonpolar residues of ηA helix (Phe332, Phe333) and β8-α4 loop (His448, Leu451) from both protomers. Hydrogen bonds are indicated by broken lines.
Figure 6
Figure 6
Conformational changes in the CAP subdomain induce dimer organization as revealed by comparison of PhaCCs-CAT and PhaCCn-CAT dimers. (a) Overlay of the PhaCCs-CAT dimer (cyan and green) on the PhaCCn-CAT dimer (gray and purple). One protomer (cyan) of the PhaCCs-CAT dimer is superimposed on one protomer (gray) of the PhaCCn-CAT dimer. The distance between the catalytic cysteine residues in the PhaCCs-CAT dimer is 28.1 Å, while that in the PhaCCn-CAT dimer is 33.3 Å. (b) The PhaCCs-CAT dimer. The distance between the N-termini is 19.2 Å. (c) The PhaCCn-CAT dimer with mol A in the same orientation as mol A in the PhaCCs-CAT dimer as in (b). The dimer interface is reorganized by refolding/unfolding of the LID region with accompanying rotation of one protomer. The distance between the N-termini is 55.1 Å.
Figure 7
Figure 7
A model of PhaC activation in dimeric form. (a) The closed form as observed in our PhaCCs-CAT structure. The N domain is proposed to contribute to stabilization of the dimer. (b) The partially open form as observed in the PhaCCn-CAT structure. The structure provides a possible path to the active site, but no apparent path for product. (c) The single active site provides a full active site architecture for initiation of acylation and chain elongation. (d) The Cys-bound product in one protomer attacks the 3HB-Cys thioester of the other protomer in the dimer for chain elongation.
Figure 8
Figure 8
The channel of the active site of PhaCCs. (a) A close-up view of the β9-α5-β10 segment forming part of the active site of PhaCCs-CAT. Ala479 of PhaCCs is located at α5 helix, where His477 of the catalytic triad is also located. Asp368 of the LID region stabilizes α5 helix by forming hydrogen bonds to the main chain amide groups of Ile478 and Ala479 residues which form the helix. Ala479 corresponds to Ala510 of PhaCCn. (b) A view of the channel at the active site of PhaCCs-CAT. Trp392 of PhaCCs is located in the αC helix of the CAP subdomain and faces Site B of the channel where a water cluster is present. Tyr412 and Ile413 are located in the αD helix of the CAP subdomain. Tyr412 projects into Site A of the channel, while Ile413 forms a hydrophobic core with other aliphatic residues.
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