Characterization of Site-Specific Mutations in a Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate Synthase:
In Vivo
and
In Vitro
Studies of Enzymatic Activity and Substrate Specificity
Abstract: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 s… Show more
“…We speculate that this loss results in weakening of the interactions between the LID region and the core subdomain, and stabilizes the active form of this enzyme by releasing the LID region from the active site. Interestingly, a maximum 4-fold increase in 3HHx incorporation was observed for the A479S mutation, while the A479T mutation showed ~2.8-fold enhancement 40 . Since Ala479 is surrounded by polar residues (Ser475 and Arg490), it was reasonable to postulate that replacement of Ala479 with Ser or Thr would facilitate hydrogen-bonding interactions with the polar residues and stabilization of α5 helix harboring the active residue His477, which may be important for enzyme activity.Figure 8The channel of the active site of PhaC Cs .…”
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, PhaCCs-CAT. The structure shows that PhaCCs-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 (PhaCCn-CAT). Structural comparison showed PhaCCn-CAT adopting a partially open form maintaining a narrow substrate access channel to the active site, but no product egress. PhaCCs-CAT forms a face-to-face dimer mediated by the CAP subdomains. This arrangement of the dimer is also distinct from that of the PhaCCn-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.
“…We speculate that this loss results in weakening of the interactions between the LID region and the core subdomain, and stabilizes the active form of this enzyme by releasing the LID region from the active site. Interestingly, a maximum 4-fold increase in 3HHx incorporation was observed for the A479S mutation, while the A479T mutation showed ~2.8-fold enhancement 40 . Since Ala479 is surrounded by polar residues (Ser475 and Arg490), it was reasonable to postulate that replacement of Ala479 with Ser or Thr would facilitate hydrogen-bonding interactions with the polar residues and stabilization of α5 helix harboring the active residue His477, which may be important for enzyme activity.Figure 8The channel of the active site of PhaC Cs .…”
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, PhaCCs-CAT. The structure shows that PhaCCs-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 (PhaCCn-CAT). Structural comparison showed PhaCCn-CAT adopting a partially open form maintaining a narrow substrate access channel to the active site, but no product egress. PhaCCs-CAT forms a face-to-face dimer mediated by the CAP subdomains. This arrangement of the dimer is also distinct from that of the PhaCCn-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.
“…33 Since not many PHA synthases have the ability to use both the SCL and MCL monomers, the PhaC Cs ’s capacity to produce copolymers with mixed monomeric chain lengths highlights its great potential for various applications. In addition, the mutant A479S- PhaC Cs was reported to display higher preference to MCL monomers than the wt , 20 suggesting that the mutation may facilitate the enzyme to adapt to side chain modifications. Thus, it was also included for our study.…”
Section: Resultsmentioning
confidence: 99%
“…3-( R )-hydroxyhexanoate coenzyme A (HHxCoA, R’ = Pr), could be obtained either chemically at extremely low yields or enzymatically in limited quantities. 20–23 As far as we know, preparation of PHA substrate analogs with modified side chains has not been reported. However, such analogs are needed for developing biotechnological applications of PhaCs and the produced PHAs.…”
Polyhydroxyalkanoates (PHAs) are carbon and energy storage polymers produced by a variety of microbial organisms under nutrient-limited conditions. They have been considered as an environmentally friendly alternative to oil-based plastics due to their renewability, versatility and biodegradability. PHA synthase (PhaC) plays a central role in PHA biosynthesis, in which its activity and substrate specificity are major factors in determining the productivity and properties of the produced polymers. However, the effects of modifying the substrate side chain are not well understood because of the difficulty to accessing the desired analogs. In this report, a series of 3-(R)-hydroxyacyl coenzyme A (HACoA) analogs were synthesized and tested with class I synthases from Chromobacterium sp. USM2 (PhaCCs and A479S-PhaCCs) and Caulobacter crescentus (PhaCCc) as well as class III synthase from Allochromatium vinosum (PhaECAv). It was found that, while different PHA synthases displayed distinct preference with regard to the length of the alkyl side chains, they could withstand moderate side chain modifications such as terminal unsaturated bonds and the azide group. Specifically, the specific activity of PhaCCs toward propynyl analog (HHxyCoA) was only 5-fold less than that toward the classical substrate HBCoA. The catalytic efficiency (kcat/Km) of PhaECAv toward azide analog (HABCoA) was determined to be 2.86 × 105 M−1s−1, which was 6.2% of the value of HBCoA (4.62 × 106 M−1s−1) measured in the presence of bovine serum albumin (BSA). These side chain modifications may be employed to introduce new material functions to PHAs as well as to study PHA biogenesis via click-chemistry, in which the latter remains unknown and is important for metabolic engineering to produce PHAs economically.
“…75 Contrary to amyloid fibrils, silk β-sheets composed of Gly and Ala did not express significant cytotoxicity, most likely because of a lack of electrical charge. Nano-assembly of silk β-sheets produced microfibrils after incubation for 72 h (Figure 5a), and formation of the nano-assembly was faster than that of Aβ (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28), Aβ (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42) and full-length Aβ(1-42) because of the greater hydrophobicity of the silk β-sheet. 76,77 The control of silk β-sheet formation is key the assembly needed to create artificial tough polymeric materials; however, this control has not yet been established because β-sheets assemble via hydrophobic interactions too quickly to regulate in solution.…”
Section: Poly(amino Acid)/polypeptide As Structural Materials: Crystamentioning
confidence: 98%
“…[4][5][6][7][8][9][10][11][12] Poly(hydroxyalkanoate) has several advantages such as the ability to be directly synthesized from various types of biomass, including lignin and carbon dioxide, [13][14][15][16][17] and excellent biodegradability. 18,19 However, because of a lack of toughness, this biopolyester has limited use and application.…”
Section: Poly(amino Acid) As An Eco-friendly Materialsmentioning
Poly(amino acids) and polypeptides have the potential to contribute significantly to a biomass-based and sustainable society, due to their biomass origin, functionality, and unique physical properties. To realize amino acid-based polymers as eco-friendly alternatives for petroleum materials, the synthesis of poly(amino acid)s/polypeptides through an environmentally friendly process is needed. In this focus review, the author summarizes the recent progress of chemo-enzymatic polymerization, which is a green and atom-economical reaction that provides new insight into the design of materials from polypeptides. Additionally, polypeptides can be designed to serve as functional and structural materials. The use of peptides as carriers of nucleic acids for delivery into target cells and organelles is one important application of such functional materials. Studies on polypeptides as structural materials are also reviewed.
POLY(AMINO ACID) AS AN ECO-FRIENDLY MATERIALEco-friendly polymeric materials have been designed and processed primarily from bio-based polyesters such as poly(hydroxyalkanoate) 1 and poly(lactic acid), 2,3 due to their plasticity, excellent workability, and biomass origin. 4-12 Poly(hydroxyalkanoate) has several advantages such as the ability to be directly synthesized from various types of biomass, including lignin and carbon dioxide, 13-17 and excellent biodegradability. 18,19 However, because of a lack of toughness, this biopolyester has limited use and application. Development of an engineered biopolymer such as a biomass-derived polyamide is an emerging interest in the field of sustainable chemistry and materials engineering. Biopolyamides (for example, nylon 4) have been investigated as candidates for biomass-based engineering plastics, but their narrow processing window for thermo-forming currently prevents them from being considered for practical purposes. 20 Natural high-performance materials, including spider silk and mussel-derived adhesive (Figure 1), are mainly composed of poly(amino acid)s and a small amount of short peptides, suggesting that poly(amino acid)s could serve as an alternative to the petroleum-derived polymers in support of a sustainable society. Although spider dragline silk is a benchmark in terms of tough materials, scientific and technological advances still cannot produce an artificial dragline silk. To create and develop biomass-based tough polymers like this silk, the synthesis and design of materials from poly(amino acid)s is being investigated.A poly(amino acid) is a polymer composed of amino acids as monomeric units. Structural and functional proteins, polypeptides, peptides and polymers derived from amino acids, that is, poly(β-alanine) and ε-poly(lysine), are classified as poly(amino acid)s. The use of poly(amino acid)s as functional materials has been widely studied, resulting in the development of polypeptides that are bioactive and that can have biological applications such as in tissue engineering, regenerative medicine and drug/gene delivery systems. Poly(amin...
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