Structure of polyhydroxyalkanoate (PHA) synthase PhaC from Chromobacterium sp. USM2, producing biodegradable plastics.
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 ?/? hydrolase fold comprising ?/? 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.
Project description:Biodegradable polyester polyhydroxyalkanoate (PHA) is a promising bioplastic material for industrial use as a replacement for petroleum-based plastics. PHA synthase PhaC forms an active dimer to polymerize acyl moieties from the substrate acyl-coenzyme A (CoA) into PHA polymers. Here we present the crystal structure of the catalytic domain of PhaC from Chromobacterium sp. USM2, bound to CoA. The structure reveals an asymmetric dimer, in which one protomer adopts an open conformation bound to CoA, whereas the other adopts a closed conformation in a CoA-free form. The open conformation is stabilized by the asymmetric dimerization and enables PhaC to accommodate CoA and also to create the product egress path. The bound CoA molecule has its ?-mercaptoethanolamine moiety extended into the active site with the terminal SH group close to active center Cys291, enabling formation of the reaction intermediate by acylation of Cys291.
Project description:The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC(Cs)). PhaC(Cs) showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. An in vitro assay of recombinant PhaC(Cs) expressed in Escherichia coli showed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strain C. necator (307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaC(Cs) was 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC from C. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation in Escherichia coli expressing PhaC(Cs) of up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaC(Cs) is a naturally occurring, highly active PHA synthase with superior polymerizing ability.
Project description:Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaC(Cs)) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaC(Cs) for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C(4) and C(5)) or MCL (C(6)) substrates substantiates the fundamental classification of PHA synthases.
Project description:PHA synthases (PhaC) are grouped into four classes based on the kinetics and mechanisms of reaction. The grouping of PhaC enzymes into four classes is dependent on substrate specificity, according to the preference in forming short-chain-length (scl) or medium-chain-length (mcl) polymers: Class I, Class III and Class IV produce scl-PHAs depending on propionate, butyrate, valerate and hexanoate precursors, while Class II PhaC synthesize mcl-PHAs based on the alkane (C6 to C14) precursors. PHA synthases of Class I, in particular PhaCCs from Chromobacterium USM2 and PhaCCn/RePhaC1 from Cupriavidus necator/Ralstonia eutropha, have been analysed and the crystal structures of the C-domains have been determined. PhaCCn/RePhaC1 was also studied by X-ray absorption fine-structure (XAFS) analysis. Models have been proposed for dimerization, catalysis mechanism, substrate recognition and affinity, product formation, and product egress route. The assays based on amino acid substitution by mutagenesis have been useful to validate the hypothesis on the role of amino acids in catalysis and in accommodation of bulky substrates, and for the synthesis of PHB copolymers and medium-chain-length PHA polymers with optimized chemical properties.
Project description:The thermophile Cupriavidus sp. strain S-6 accumulated polyhydroxybutyrate (PHB) from glucose at 50°C. A 9.0-kbp EcoRI fragment cloned from the genomic DNA of Cupriavidus sp. S-6 enabled Escherichia coli XL1-Blue to synthesize PHB at 45°C. Nucleotide sequence analysis showed a pha locus in the clone. The thermophilic polyhydroxyalkanoate (PHA) synthase (PhaC(Csp)) shared 81% identity with mesophilic PhaC of Cupriavidus necator H16. The diversity between these two strains was found dominantly on their N and C termini, while the middle regions were highly homologous (92% identity). We constructed four chimeras of mesophilic and thermophilic phaC genes to explore the mutations related to its thermostability. Among the chimeras, only PhaC(H16?), which was PhaC(H16) bearing 30 point mutations derived from the middle region of PhaC(Csp), accumulated a high content of PHB (65% [dry weight]) at 45°C. The chimera phaC(H16)(?) and two parental PHA synthase genes were overexpressed in E. coli BLR(DE3) cells and purified. At 30°C, the specific activity of the chimera PhaC(H16?) (172 ± 17.8 U/mg) was 3.45-fold higher than that of the parental enzyme PhaC(H16) (50 ± 5.2 U/mg). At 45°C, the half-life of the chimera PhaC(H16?) (11.2 h) was 127-fold longer than that of PhaC(H16) (5.3 min). Furthermore, the chimera PhaC(H16?) accumulated 1.55-fold (59% [dry weight]) more PHA content than the parental enzyme PhaC(H16) (38% [dry weight]) at 37°C. This study reveals a limited number of point mutations which enhance not only thermostability but also PhaC(H16) activity. The highly thermostable and active PHA synthase will provide advantages for its promising applications to in vitro PHA synthesis and recombinant E. coli PHA fermentation.
Project description:Polyhydroxyalkanoic acids (PHAs) are a class of polyesters stored in inclusion bodies and found in many bacteria and in some archaea. The terminal step in the synthesis of PHA is catalyzed by PHA synthase. Genes encoding this enzyme have been cloned, and the primary sequence of the protein, PhaC, is deduced from the nucleotide sequences of more than 30 organisms. PHA synthases are grouped into three classes based on substrate range, molecular mass, and whether or not there is a requirement for phaE in addition to the phaC gene product. Here we report the results of an analysis of a PHA synthase that does not fit any of the described classes. This novel PHA synthase from Bacillus megaterium required PhaC (PhaC(Bm)) and PhaR (PhaR(Bm)) for activity in vivo and in vitro. PhaC(Bm) showed greatest similarity to the PhaCs of class III in both size and sequence. Unlike those in class III, the 40-kDa PhaE was not required, and furthermore, the 22-kDa PhaR(Bm) had no obvious homology to PhaE. Previously we showed that PhaC(Bm), and here we show that PhaR(Bm), is localized to inclusion bodies in living cells. We show that two forms of PHA synthase exist, an active form in PHA-accumulating cells and an inactive form in nonaccumulating cells. PhaC was constitutively produced in both cell types but was more susceptible to protease degradation in the latter type. Our data show that the role of PhaR is posttranscriptional and that it functions directly or indirectly with PhaC(Bm) to produce an active PHA synthase.
Project description:Many bacteria accumulate granules of polyhydroxyalkanoate (PHA) within their cells, which confer resistance to nutritional depletion and other environmental stresses. Here, we report an unexpected involvement of the bacterial endocellular storage polymer, PHA, in an insect-bacterium symbiotic association. The bean bug Riptortus pedestris harbors a beneficial and specific gut symbiont of the ?-proteobacterial genus Burkholderia, which is orally acquired by host nymphs from the environment every generation and easily cultivable and genetically manipulatable. Biochemical and cytological comparisons between symbiotic and cultured Burkholderia detected more PHA granules consisting of poly-3-hydroxybutyrate and associated phasin (PhaP) protein in the symbiotic Burkholderia. Among major PHA synthesis genes, phaB and phaC were disrupted by homologous recombination together with the phaP gene, whereby ?phaB, ?phaC, and ?phaP mutants were generated. Both in culture and in symbiosis, accumulation of PHA granules was strongly suppressed in ?phaB and ?phaC, but only moderately in ?phaP. In symbiosis, the host insects infected with ?phaB and ?phaC exhibited significantly lower symbiont densities and smaller body sizes. These deficient phenotypes associated with ?phaB and ?phaC were restored by complementation of the mutants with plasmids encoding a functional phaB/phaC gene. Retention analysis of the plasmids revealed positive selection acting on the functional phaB/phaC in symbiosis. These results indicate that the PHA synthesis genes of the Burkholderia symbiont are required for normal symbiotic association with the Riptortus host. In vitro culturing analyses confirmed vulnerability of the PHA gene mutants to environmental stresses, suggesting that PHA may play a role in resisting stress under symbiotic conditions.
Project description:The polyhydroxyalkanoic acid synthase gene from Chromobacterium violaceum (phaC(Cv)) was cloned and characterized. A 6.3-kb BamHI fragment was found to contain both phaC(Cv) and the polyhydroxyalkanoic acid (PHA)-specific 3-ketothiolase (phaA(Cv)). Escherichia coli strains harboring this fragment produced significant levels of PHA synthase and 3-ketothiolase, as judged by their activities. While C. violaceum accumulated poly(3-hydroxybutyrate) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) when grown on a fatty acid carbon source, Klebsiella aerogenes and Ralstonia eutropha (formerly Alcaligenes eutrophus), harboring phaC(Cv), accumulated the above-mentioned polymers and, additionally, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) when even-chain-length fatty acids were utilized as the carbon source. This finding suggests that the metabolic environments of these organisms are sufficiently different to alter the product range of the C. violaceum PHA synthase. Neither recombinant E. coli nor recombinant Pseudomonas putida harboring phaC(Cv) accumulated significant levels of PHA. Sequence analysis of the phaC(Cv) product shows homology with several PHA synthases, most notably a 48% identity with that of Alcaligenes latus (GenBank accession no. AAD10274).
Project description:The allosteric transition of glycogen phosphorylase promoted by protein phosphorylation is accompanied by the association of a pair of functional dimers to form a tetramer. The conformational changes within the dimer that lead to the creation of a protein recognition surface have been analyzed from a comparison of the crystal structures of T-state dimeric phosphorylase b and R-state tetrameric phosphorylase a. Regions of the structure that participate in the tetramer interface are situated within structural subdomains. These include the glycogen storage subdomain, the C-terminal subdomain and the tower helix. The subdomains undergo concerted conformational transitions on conversion from the T to the R state (overall r.m.s. shifts between 1 and 1.7 A) and, together with the quaternary conformational change within the functional dimer, create the tetramer interface. The glycogen storage subdomain and the C-terminal subdomain are distinct from those regions that contribute to the dimer interface, but shifts in the subdomains are correlated with the allosteric transitions that are mediated by the dimer interface. The structural properties of the tetramer interface are atypical of an oligomeric protein interface and are more similar to protein recognition surfaces observed in protease inhibitors and antibody-protein antigen complexes. There is a preponderance of polar and charged residues at the tetramer interface and a high number of H-bonds per surface area (one H-bond per 130 A2). In addition, the surface area made inaccessible at the interface is relatively small (1,142 A2 per subunit on dimer to tetramer association compared with 2,217 A2 per subunit on monomer-to-dimer association).
Project description:Polyhydroxyalkanoates (PHAs) are accumulated as intracellular carbon and energy storage polymers by various bacteria and a few haloarchaea. In this study, 28 strains belonging to 15 genera in the family Halobacteriaceae were investigated with respect to their ability to synthesize PHAs and the types of their PHA synthases. Fermentation results showed that 18 strains from 12 genera could synthesize polyhydroxybutyrate (PHB) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). For most of these haloarchaea, selected regions of the phaE and phaC genes encoding PHA synthases (type III) were cloned via PCR with consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and were sequenced. The PHA synthases were also examined by Western blotting using haloarchaeal Haloarcula marismortui PhaC (PhaC(Hm)) antisera. Phylogenetic analysis showed that the type III PHA synthases from species of the Halobacteriaceae and the Bacteria domain clustered separately. Comparison of their amino acid sequences revealed that haloarchaeal PHA synthases differed greatly in both molecular weight and certain conserved motifs. The longer C terminus of haloarchaeal PhaC was found to be indispensable for its enzymatic activity, and two additional amino acid residues (C143 and C190) of PhaC(Hm) were proved to be important for its in vivo function. Thus, we conclude that a novel subtype (IIIA) of type III PHA synthase with unique features that distinguish it from the bacterial subtype (IIIB) is widely distributed in haloarchaea and appears to be involved in PHA biosynthesis.