Critical residues of class II PHA synthase for expanding the substrate specificity and enhancing the biosynthesis of polyhydroxyalkanoate

2014 ◽  
Vol 56 ◽  
pp. 60-66 ◽  
Author(s):  
Yi-Jr Chen ◽  
Pei-Chien Tsai ◽  
Chun-Hua Hsu ◽  
Chia-Yin Lee
Keyword(s):  
Substrate Specificity ◽  
Class Ii ◽  
Pha Synthase ◽  
Critical Residues

scholarly journals Rational Engineering of Hydratase from Lactobacillus acidophilus Reveals Critical Residues Directing Substrate Specificity and Regioselectivity

ChemBioChem ◽  
2019 ◽  
Vol 21 (4) ◽  
pp. 550-563 ◽  
Author(s):  
Bekir Engin Eser ◽  
Michal Poborsky ◽  
Rongrong Dai ◽  
Shigenobu Kishino ◽  
Anita Ljubic ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Lactobacillus Acidophilus ◽  
Rational Engineering ◽  
Critical Residues

Probing the Molecular Basis of Substrate Specificity, Stereospecificity, and Catalysis in the Class II Pyruvate Aldolase, BphI

Biochemistry ◽  
2011 ◽  
Vol 50 (17) ◽  
pp. 3559-3569 ◽  
Author(s):  
Perrin Baker ◽  
Jason Carere ◽  
Stephen Y. K. Seah
Keyword(s):  
Substrate Specificity ◽  
Molecular Basis ◽  
Class Ii

scholarly journals Characterisation and modelling of a polyhydroxyalkanoate synthase fromAquitaleasp. USM4 reveals a mechanism for polymer elongation

2017 ◽  
Author(s):  
Aik-Hong Teh ◽  
Nyet-Cheng Chiam ◽  
Go Furusawa ◽  
Kumar Sudesh
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Substrate Specificity ◽  
Class Ii ◽  
Class I ◽  
Common Mechanism ◽  
The Third ◽  
Dimeric Interface ◽  
Key Enzyme ◽  
Polyhydroxyalkanoate Synthase ◽  
Key Residues

AbstractPolyhydroxyalkanoate synthase, PhaC, is a key enzyme in the biosynthesis of PHA, a type of bioplastics with huge potential to replace conventional petroleum-based plastics. While two PhaC structures have been determined recently, the exact mechanism remains unclear partly due to the absence of a tunnel for product passage. The PhaC fromAquitaleasp. USM4, PhaCAq, was characterised and showed aKmof 394 µM and akcatof 476.4 s−1on the 3HB-CoA substrate. A model based on the structure of the closely related PhaC fromCupriavidus necator, PhaCCnrevealed a three-branched tunnel at the dimeric interface. Two of the branches open to the solvent and serve as the putative routes for substrate entrance and product exit, while the third branch is elongated in a PhaC1 model fromPseudomonas aeruginosa, indicating a function of accommodating the hydroxyalkanoate (HA) moiety of the HA-CoA substrate. Docking of the two tetrahedral intermediates formed during catalysis suggests a PHA elongation mechanism that requires the HA moiety of the ligand to rotate ~180°. Both classes I and II PhaCs share a common mechanism for polymer elongation, and substrate specificity is determined in part by a bulky Phe/Tyr/Trp residue in the third branch in class I, which is conserved as Ala in class II to create room for longer substrates. The PhaCAqmodel provides fresh insights into a general PhaC mechanism, pinpointing key residues for potential engineering of PhaCs with desirable characteristics.

scholarly journals Altering the Substrate Specificity of Polyhydroxyalkanoate Synthase 1 Derived from Pseudomonas putida GPo1 by Localized Semirandom Mutagenesis

2004 ◽  
Vol 186 (13) ◽  
pp. 4177-4184 ◽  
Author(s):  
Der-Shyan Sheu ◽  
Chia-Yin Lee
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Chain Length ◽  
Ralstonia Eutropha ◽  
Pha Synthase ◽  
Multiple Point ◽  
Evolution System ◽  
Degenerate Primers ◽  
Short Chain Length ◽  
Single Polymer Chain

ABSTRACT The substrate specificity of polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 Pp , class II) from Pseudomonas putida GPo1 (formerly known as Pseudomonas oleovorans GPo1) was successfully altered by localized semirandom mutagenesis. The enzyme evolution system introduces multiple point mutations, designed on the basis of the conserved regions of the PHA synthase family, by using PCR-based gene fragmentation with degenerate primers and a reassembly PCR. According to the opaqueness of the colony, indicating the accumulation of large amounts of PHA granules in the cells, 13 PHA-accumulating candidates were screened from a mutant library, with Pseudomonas putida GPp104 PHA− as the host. The in vivo substrate specificity of five candidates, L1-6, D7-47, PS-A2, PS-C2, and PS-E1, was evaluated by the heterologous expression in Ralstonia eutropha PHB−4 supplemented with octanoate. Notably, the amount of 3-hydroxybutyrate (short-chain-length [SCL] 3-hydroxyalkanoate [3-HA] unit) was drastically increased in recombinants that expressed evolved mutant enzymes L1-6, PS-A2, PS-C2, and PS-E1 (up to 60, 36, 50, and 49 mol%, respectively), relative to the amount in the wild type (12 mol%). Evolved enzyme PS-E1, in which 14 amino acids had been changed and which was heterologously expressed in R. eutropha PHB−4, not only exhibited broad substrate specificity (49 mol% SCL 3-HA and 51 mol% medium-chain-length [MCL] 3-HA) but also conferred the highest PHA production (45% dry weight) among the candidates. The 3-HA and MCL 3-HA units of the PHA produced by R. eutropha PHB−4/pPS-E1 were randomly copolymerized in a single polymer chain, as analytically confirmed by acetone fractionation and the 13C nuclear magnetic resonance spectrum.

Matrix-assisted in vitro refolding of Pseudomonas aeruginosa class II polyhydroxyalkanoate synthase from inclusion bodies produced in recombinant Escherichia coli

2001 ◽  
Vol 358 (1) ◽  
pp. 263-268 ◽  
Author(s):  
Bernd H. A. REHM ◽  
Qingsheng QI ◽  
Br. Bernd BEERMANN ◽  
Hans-Jürgen HINZ ◽  
Alexander STEINBÜCHEL
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Fusion Protein ◽  
Molecular Mass ◽  
Inclusion Bodies ◽  
Specific Activity ◽  
Guanidine Hydrochloride ◽  
Class Ii ◽  
Random Coil ◽  
Pha Synthase

In order to facilitate the large-scale preparation of active class II polyhydroxyalkanoate (PHA) synthase, we constructed a vector pT7-7 derivative that contains a modified phaC1 gene encoding a PHA synthase from Pseudomonas aeruginosa possessing six N-terminally fused histidine residues. Overexpression of this phaC1 gene under control of the strong Ø10 promoter was achieved in Escherichia coli BL21(DE3). The fusion protein was deposited as inactive inclusion bodies in recombinant E. coli, and contributed approx. 30% of total protein. The inclusion bodies were purified by selective solubilization, resulting in approx. 70–80% pure PHA synthase, then dissolved and denatured by 6M guanidine hydrochloride. The denatured PHA synthase was reversibly immobilized on a Ni2+-nitrilotriacetate–agarose matrix. The matrix-bound fusion protein was refolded by gradual removal of the chaotropic reagent. This procedure avoided the aggregation of folding intermediates which often decreases the efficiency of refolding experiments. Finally, the refolded fusion protein was eluted with imidazole. The purified and refolded PHA synthase protein showed a specific enzyme activity of 10.8m-units/mg employing (R/S)-3-hydroxydecanoyl-CoA as substrate, which corresponds to 27% of the maximum specific activity of the native enzyme. The refolding of the enzyme was confirmed by CD spectroscopy. Deconvolution of the spectrum resulted in the following secondary structure prediction: 10% α-helix, 50% β-sheet and 40% random coil. Gel filtration chromatography indicated an apparent molecular mass of 69kDa for the refolded PHA synthase. However, light-scattering analysis of a 10-fold concentrated sample indicated a molecular mass of 128kDa. These data suggest that the class II PHA synthase is present in an equilibrium of monomer and dimer.

scholarly journals Functional metagenomics using Pseudomonas putida expands the known diversity of polyhydroxyalkanoate synthases and enables the production of novel polyhydroxyalkanoate copolymers

2016 ◽  
Author(s):  
Jiujun Cheng ◽  
Trevor Charles
Keyword(s):  
Pseudomonas Putida ◽  
Class Ii ◽  
Class I ◽  
Pha Synthase ◽  
Cosmid Library ◽  
Functional Metagenomics ◽  
Growth Substrates ◽  
Pha Production ◽  
Close Sequence

Bacterially produced biodegradable polyhydroxyalkanoates with versatile properties can be achieved using different PHA synthase enzymes. This work aims to expand the diversity of known PHA synthases via functional metagenomics, and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either Class I, Class II or unclassified PHA synthases, and many did not have close sequence matches to known PHA synthases. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of SCL- and MCL-PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics, and their use for the production of a variety of PHA polymer and copolymer mixtures.

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