Hakoshima T

References (9)

Title : N-terminal truncation of PhaC(BP-M-CPF4) and its effect on PHA production - Neoh_2024_Microb.Cell.Fact_23_52
Author(s) : Neoh SZ , Tan HT , Trakunjae C , Chek MF , Vaithanomsat P , Hakoshima T , Sudesh K
Ref : Microb Cell Fact , 23 :52 , 2024
Abstract : BACKGROUND: Among the polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is reported to closely resemble polypropylene and low-density polyethylene. Studies have shown that PHA synthase (PhaC) from mangrove soil (PhaC(BP-M-CPF4)) is an efficient PhaC for P(3HB-co-3HHx) production and N-termini of PhaCs influence its substrate specificity, dimerization, granule morphology, and molecular weights of PHA produced. This study aims to further improve PhaC(BP-M-CPF4) through N-terminal truncation. RESULTS: The N-terminal truncated mutants of PhaC(BP-M-CPF4) were constructed based on the information of the predicted secondary and tertiary structures using PSIPRED server and AlphaFold2 program, respectively. The N-terminal truncated PhaC(BP-M-CPF4) mutants were evaluated in C. necator mutant PHB(-)4 based on the cell dry weight, PHA content, 3HHx molar composition, molecular weights, and granule morphology of the PHA granules. The results showed that most transformants harbouring the N-terminal truncated PhaC(BP-M-CPF4) showed a reduction in PHA content and cell dry weight except for PhaC(BP-M-CPF4) G8. PhaC(BP-M-CPF4) G8 and A27 showed an improved weight-average molecular weight (M(w)) of PHA produced due to lower expression of the truncated PhaC(BP-M-CPF4). Transformants harbouring PhaC(BP-M-CPF4) G8, A27, and T74 showed a reduction in the number of granules. PhaC(BP-M-CPF4) G8 produced higher M(w) PHA in mostly single larger PHA granules with comparable production as the full-length PhaC(BP-M-CPF4). CONCLUSION: This research showed that N-terminal truncation had effects on PHA accumulation, substrate specificity, M(w), and granule morphology. This study also showed that N-terminal truncation of the amino acids that did not adopt any secondary structure can be an alternative to improve PhaCs for the production of PHA with higher M(w) in mostly single larger granules.
ESTHER : Neoh_2024_Microb.Cell.Fact_23_52
PubMedSearch : Neoh_2024_Microb.Cell.Fact_23_52
PubMedID: 38360657
Gene_locus related to this paper: 9bact-a0a2z5dkz9

Title : Polyhydroxyalkanoate synthase (PhaC): The key enzyme for biopolyester synthesis - Neoh_2022_Curr.Res.Biotechnol_4_87
Author(s) : Neoh SZ , Chek MF , Tan HT , Linares-Pasten JA , Nandakumar A , Hakoshima T , Sudesh K
Ref : Current Research in Biotechnology , 4 :87 , 2022
Abstract : Polyhydroxyalkanoates (PHAs) are considered good candidates in replacing commercial petrochemical plastics in certain applications like single-use packaging since they are biodegradable, biocompatible and share similar properties with conventional plastics. PHA synthase (PhaC) is the key enzyme in PHA biosynthesis. There are four classes of PhaC, namely, class I, class II, class III and class IV, each with their distinct characteristics. To date, there are two PhaCs with successfully solved catalytic domain structures. They are PhaC from C. necator (PhaCCn-CAT) (Ser20-Ala589) and PhaC from Chromobacterium sp. USM2 (PhaCCs-CAT) (Phe175-Asn567). Generally, the structure of PhaC consists of an N-terminal domain and a C-terminal catalytic domain. The N-terminal domain is flexible and has not been successfully visualized in any existing structures of PhaC. It is suggested to affect the dimerization and stability of the PhaC dimer, enzymatic activity, substrate specificity, molecular weight of PHA produced, expression of PhaC, and its ability to bind to PHA granules and PHA-related proteins. The C-terminal catalytic domain contains the cap subdomain, substrate entrance channel, active site, and product egress tunnel.
ESTHER : Neoh_2022_Curr.Res.Biotechnol_4_87
PubMedSearch : Neoh_2022_Curr.Res.Biotechnol_4_87

Title : Identification of regions affecting enzyme activity, substrate binding, dimer stabilization and polyhydroxyalkanoate (PHA) granule morphology in the PHA synthase of Aquitalea sp. USM4 - Lim_2021_Int.J.Biol.Macromol_186_414
Author(s) : Lim H , Chuah JA , Chek MF , Tan HT , Hakoshima T , Sudesh K
Ref : Int J Biol Macromol , 186 :414 , 2021
Abstract : Polyhydroxyalkanoates (PHAs) are biopolyesters synthesized by microorganisms as intracellular energy reservoirs under stressful environmental conditions. PHA synthase (PhaC) is the key enzyme responsible for PHA biosynthesis, but the importance of its N- and C-terminal ends still remains elusive. Six plasmid constructs expressing truncation variants of Aquitalea sp. USM4 PhaC (PhaC1(As)) were generated and heterologously expressed in Cupriavidus necator PHB(-)4. Removal of the first six residues at the N-terminus enabled the modulation of PHA composition without altering the PHA content in cells. Meanwhile, deletion of 13 amino acids from the C-terminus greatly affected the catalytic activity of PhaC1(As), retaining only 1.1-7.4% of the total activity. Truncation(s) at the N- and/or C-terminus of PhaC1(As) gradually diminished the incorporation of comonomer units, and revealed that the N-terminal region is essential for PhaC1(As) dimerization whereas the C-terminal region is required for stabilization. Notably, transmission electron microscopy analysis showed that PhaC modification affected the morphology of intracellular PHA granules, which until now is only known to be regulated by phasins. This study provided substantial evidence and highlighted the significance of both the N- and C-termini of PhaC1(As) in regulating intracellular granule morphology, activity, substrate specificity, dimerization and stability of the synthase.
ESTHER : Lim_2021_Int.J.Biol.Macromol_186_414
PubMedSearch : Lim_2021_Int.J.Biol.Macromol_186_414
PubMedID: 34246679
Gene_locus related to this paper: 9neis-a0a0n9wsd0

Title : Evaluation of BP-M-CPF4 polyhydroxyalkanoate (PHA) synthase on the production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from plant oil using Cupriavidus necator transformants - Tan_2020_Int.J.Biol.Macromol_159_250
Author(s) : Tan HT , Chek MF , Lakshmanan M , Foong CP , Hakoshima T , Sudesh K
Ref : Int J Biol Macromol , 159 :250 , 2020
Abstract : Among the various types of polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] has a high potential to serve as commercial bioplastic due to its striking resemblance to petroleum-based plastics. In this study, five different genotypes of Cupriavidusnecator transformants harbouring the phaC(BP-M-CPF4) gene (including PHB4/pBBR1-C(BP-M-CPF4)) were developed to evaluate the efficiency of 3HHx monomer incorporation. The fraction of 3-hydroxyhexanoate (3HHx) monomer that was incorporated into the PHA synthesized by these C. necator transformants using palm oil as the sole carbon source, was examined. Overall, co-expression of enoyl-CoA hydratase gene (phaJ1) from Pseudomonas aeruginosa, along with PHA synthase (PhaC), increased the 3HHx composition in the PHA copolymer. The differences in the enzyme activities of beta-ketothiolase (PhaA(Cn)) and NADPH-dependent acetoacetyl-CoA reductase (PhaB(Cn)) of the C. necator mutant hosts used in this study, were observed to alter the 3HHx composition and molecular weight of the PHA copolymer produced. The 3HHx fractions in the P(3HB-co-3HHx) produced by these C. necator transformants ranged between 1 and 18 mol%, while the weight-average molecular weight ranged from 0.7 x 10(6) to 1.8 x 10(6) Da. PhaC(BP-M-CPF4) displayed a typical initial lag-phase and a relatively low synthase activity in the in vitro enzyme assay, which is thought to be the reason for the higher molecular weights of PHA obtained in this study.
ESTHER : Tan_2020_Int.J.Biol.Macromol_159_250
PubMedSearch : Tan_2020_Int.J.Biol.Macromol_159_250
PubMedID: 32417540
Gene_locus related to this paper: 9bact-a0a2z5dkz9

Title : Asymmetric Open-Closed Dimer Mechanism of Polyhydroxyalkanoate Synthase PhaC - Chek_2020_iScience_23_101084
Author(s) : Chek MF , Kim SY , Mori T , Tan HT , Sudesh K , Hakoshima T
Ref : iScience , 23 :101084 , 2020
Abstract : 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 beta-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.
ESTHER : Chek_2020_iScience_23_101084
PubMedSearch : Chek_2020_iScience_23_101084
PubMedID: 32388399
Gene_locus related to this paper: 9neis-e1apk1

Title : Strigolactone perception and deactivation by a hydrolase receptor DWARF14 - Seto_2019_Nat.Commun_10_191
Author(s) : Seto Y , Yasui R , Kameoka H , Tamiru M , Cao M , Terauchi R , Sakurada A , Hirano R , Kisugi T , Hanada A , Umehara M , Seo E , Akiyama K , Burke J , Takeda-Kamiya N , Li W , Hirano Y , Hakoshima T , Mashiguchi K , Noel JP , Kyozuka J , Yamaguchi S
Ref : Nat Commun , 10 :191 , 2019
Abstract : The perception mechanism for the strigolactone (SL) class of plant hormones has been a subject of debate because their receptor, DWARF14 (D14), is an alpha/beta-hydrolase that can cleave SLs. Here we show via time-course analyses of SL binding and hydrolysis by Arabidopsis thaliana D14, that the level of uncleaved SL strongly correlates with the induction of the active signaling state. In addition, we show that an AtD14(D218A) catalytic mutant that lacks enzymatic activity is still able to complement the atd14 mutant phenotype in an SL-dependent manner. We conclude that the intact SL molecules trigger the D14 active signaling state, and we also describe that D14 deactivates bioactive SLs by the hydrolytic degradation after signal transmission. Together, these results reveal that D14 is a dual-functional receptor, responsible for both the perception and deactivation of bioactive SLs.
ESTHER : Seto_2019_Nat.Commun_10_191
PubMedSearch : Seto_2019_Nat.Commun_10_191
PubMedID: 30643123
Gene_locus related to this paper: arath-AtD14

Title : Structure of polyhydroxyalkanoate (PHA) synthase PhaC from Chromobacterium sp. USM2, producing biodegradable plastics - Chek_2017_Sci.Rep_7_5312
Author(s) : Chek MF , Kim SY , Mori T , Arsad H , Samian MR , Sudesh K , Hakoshima T
Ref : Sci Rep , 7 :5312 , 2017
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 alpha/beta hydrolase fold comprising alpha/beta 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.
ESTHER : Chek_2017_Sci.Rep_7_5312
PubMedSearch : Chek_2017_Sci.Rep_7_5312
PubMedID: 28706283
Gene_locus related to this paper: 9neis-e1apk1

Title : Structures of D14 and D14L in the strigolactone and karrikin signaling pathways - Kagiyama_2013_Genes.Cells_18_147
Author(s) : Kagiyama M , Hirano Y , Mori T , Kim SY , Kyozuka J , Seto Y , Yamaguchi S , Hakoshima T
Ref : Genes Cells , 18 :147 , 2013
Abstract : Strigolactones (SLs) are plant hormones that inhibit shoot branching. DWARF14 (D14) inhibits rice tillering and is an SL receptor candidate in the branching inhibition pathway, whereas the close homologue DWARF14-LIKE (D14L) participates in the signaling pathway of karrikins (KARs), which are derived from burnt vegetation as smoke stimulants of seed germination. We provide the first evidence for direct binding of the bioactive SL analogue GR24 to D14. Isothermal titration calorimetry measurements show a D14-GR24 binding affinity in the sub-micromolar range. Similarly, bioactive KAR1 directly binds D14L in the micromolar range. The crystal structure of rice D14 shows a compact alpha-/beta-fold hydrolase domain forming a deep ligand-binding pocket capable of accommodating GR24. Insertion of four alpha-helices between beta6 strand and alphaD helix forms the helical cap of the pocket, although the pocket is open to the solvent. The pocket contains the conserved catalytic triad Ser-His-Asp aligned with the oxyanion hole, suggesting hydrolase activity. Although these structural characteristics are conserved in D14L, the D14L pocket is smaller than that of D14. The KAR-insensitive mutation kai2-1 is located at the prominent long beta6-alphaD1 loop, which is characteristic in D14 and D14L, but not in related alpha-/beta-fold hydrolases.
ESTHER : Kagiyama_2013_Genes.Cells_18_147
PubMedSearch : Kagiyama_2013_Genes.Cells_18_147
PubMedID: 23301669
Gene_locus related to this paper: arath-KAI2.D14L , orysj-Q10QA5

Title : Gibberellin-induced DELLA recognition by the gibberellin receptor GID1 - Murase_2008_Nature_456_459
Author(s) : Murase K , Hirano Y , Sun TP , Hakoshima T
Ref : Nature , 456 :459 , 2008
Abstract : Gibberellins control a range of growth and developmental processes in higher plants and have been widely used in the agricultural industry. By binding to a nuclear receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1), gibberellins regulate gene expression by promoting degradation of the transcriptional regulator DELLA proteins, including GIBBERELLIN INSENSITIVE (GAI). The precise manner in which GID1 discriminates and becomes activated by bioactive gibberellins for specific binding to DELLA proteins remains unclear. Here we present the crystal structure of a ternary complex of Arabidopsis thaliana GID1A, a bioactive gibberellin and the amino-terminal DELLA domain of GAI. In this complex, GID1A occludes gibberellin in a deep binding pocket covered by its N-terminal helical switch region, which in turn interacts with the DELLA domain containing DELLA, VHYNP and LExLE motifs. Our results establish a structural model of a plant hormone receptor that is distinct from the mechanism of the hormone perception and effector recognition of the known auxin receptors.
ESTHER : Murase_2008_Nature_456_459
PubMedSearch : Murase_2008_Nature_456_459
PubMedID: 19037309
Gene_locus related to this paper: arath-GID1B