Active Intermediates of Polyhydroxyalkanoate Synthase from Aeromonas caviae in Polymerization Reaction

Numata, Keiji; Motoda, Yoko; Watanabe, Satoru; Tochio, N.; Kigawa, T.; Doi, Yoshiharu

doi:10.1021/bm301276k

Cited by 21 publications

(50 citation statements)

References 30 publications

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“…On the other hand, PhaCs were observed in oligomeric form at the initial stage of PHA polymerization by atomic force microscope imaging (19,30), but the PhaC dimer is thought to be a minimal functional unit (1,15). Based on the observations in this study and previous works (19,(27)(28)(29)(30), we propose a role for PhaPs in the initial stage of PHA polymerization by PhaC Ac , as shown in Fig.…”

Section: Figsupporting

confidence: 76%

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Phasin Proteins Activate Aeromonas caviae Polyhydroxyalkanoate (PHA) Synthase but Not Ralstonia eutropha PHA Synthase

Ushimaru

Motoda

Numata

et al. 2014

Appl Environ Microbiol

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bIn this study, we performed in vitro and in vivo activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from Aeromonas caviae (PhaC Ac ). In in vitro assays, among the three PhaCs tested, PhaC Ac was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaC Ac ), whereas the prepolymerization PhaC Re (derived from Ralstonia eutropha) and PhaC Da (Delftia acidovorans) showed reduced activity with PhaPs. The PhaP-activated PhaC Ac showed a slight shift of substrate preference toward 3-hydroxyhexanoyl-CoA (C 6 ). PhaP Ac also activated PhaC Ac when it was added during polymerization (polymer-elongating PhaC Ac ), while this effect was not observed for PhaC Re . In an in vivo assay using Escherichia coli TOP10 as the host strain, the effect of PhaP Ac expression on PHA synthesis by PhaC Ac or PhaC Re was examined. As PhaP Ac expression increased, PHA production was increased by up to 2.3-fold in the PhaC Ac -expressing strain, whereas it was slightly increased in the PhaC Re -expressing strain. Taken together, this study provides evidence that PhaPs function as activators for PhaC Ac both in vitro and in vivo but do not activate PhaC Re . This activating effect may be attributed to the new role of PhaPs in the polymerization reaction by PhaC Ac .

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Section: Figsupporting

confidence: 76%

“…PhaC Ac with the His tag removed was prepared by using cell-free protein enzymes as reported previously (15). PhaC Ac was eluted with 20 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl.…”

Section: Methodsmentioning

confidence: 99%

Phasin Proteins Activate Aeromonas caviae Polyhydroxyalkanoate (PHA) Synthase but Not Ralstonia eutropha PHA Synthase

Ushimaru

Motoda

Numata

et al. 2014

Appl Environ Microbiol

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“…The results here provide solid evidence of a linear relationship between in vivo and in vitro substrate specificities of synthases, despite conflicting observations in previous studies (38,39). Overall, the affinity of PhaC Cs for the polymerization of (R)-3HV-CoA was found to be superior to those of other class I synthases characterized so far (38,39), and its affinity was comparable to that of PhaC Ac for the polymerization of (R)-3HHx-CoA (40).…”

Section: Discussionsupporting

confidence: 68%

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

Chuah

Tomizawa

Yamada

et al. 2013

Appl Environ Microbiol

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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 wildtype and mutant synthases for either SCL (C 4 and C 5 ) or MCL (C 6 ) substrates substantiates the fundamental classification of PHA synthases.

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“…19 The buffers used for the purification were from our previous study. 19 Briefly, the protein solution was purified with HisTrap [5 mL, nickel-nitrilotriacetic acid (Ni-NTA) column, GE Healthcare]. To remove the tags, TEV (N11 tag) or SUMO protease (N11-SUMO tag) was added to the eluted fraction of the protein at a final concentration of 10 μg/mL.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Co-expression of Two Polyhydroxyalkanoate Synthase Subunits from Synechocystis sp. PCC 6803 by Cell-Free Synthesis and Their Specific Activity for Polymerization of 3-Hydroxybutyryl-Coenzyme A

Numata

Motoda

Watanabe³

et al. 2015

Biochemistry

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Synechocystis sp. PCC 6803 is one of the most studied cyanobacteria for polyhydroxyalkanoate (PHA) synthesis, and its PHA synthase is known to consist of two subunits, namely, PhaC and PhaE. This report is the first to show the specific activity and related biochemical properties of PHA synthase from cyanobacteria. We have cloned and prepared a complex of PhaC and PhaE (PhaCE) from Synechocystis sp. PCC 6803 by the co-expression of PhaC and PhaE using a cell-free synthesis system. The specific activity of PhaCE was comparable to that of the class I PHA synthases, indicating that the low PHA productivity of cyanobacteria is not due to the activity of PHA synthase but may be caused by the other metabolic reactions related to PHA synthesis. The positive Hill coefficient of PhaCE as well as the size exclusion chromatography data indicates that dimeric PhaCE is a major active form that polymerizes 3-hydroxybutyryl-coenzyme A.

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Active Intermediates of Polyhydroxyalkanoate Synthase from Aeromonas caviae in Polymerization Reaction

Cited by 21 publications

References 30 publications

Phasin Proteins Activate Aeromonas caviae Polyhydroxyalkanoate (PHA) Synthase but Not Ralstonia eutropha PHA Synthase

Phasin Proteins Activate Aeromonas caviae Polyhydroxyalkanoate (PHA) Synthase but Not Ralstonia eutropha PHA Synthase

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

Co-expression of Two Polyhydroxyalkanoate Synthase Subunits from Synechocystis sp. PCC 6803 by Cell-Free Synthesis and Their Specific Activity for Polymerization of 3-Hydroxybutyryl-Coenzyme A

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