Overexpression and Purification of the Soluble Polyhydroxyalkanoate Synthase from Alcaligenes eutrophus: Evidence for a Required Posttranslational Modification for Catalytic Activity

Gerngross, Tillman U.; Snell, Kristi D.; Peoples, Oliver P.; Sinskey, Anthony J.; Csuhai, Eva; Masamune, Satoru; Stubbe, JoAnne

doi:10.1021/bi00197a035

Cited by 227 publications

(312 citation statements)

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“…The interface is composed of 66 residues and buries a surface area of ϳ2600 Å 2 from solvent on each monomer. As noted above, CnPhaC is known to exist in an equilibrium between monomer and dimer in solution, with the dimer representing the more catalytically active form (13,14,22 (Fig. 1) (8,10,13,17).…”

Section: The Catalytic Domain Of Cnphac Has An ␣/␤-Hydrolasementioning

confidence: 92%

“…As noted above, CnPhaC is known to exist in an equilibrium between monomer and dimer in solution, with the dimer representing the more catalytically active form (13,14,22 (Fig. 1) (8,10,13,17). Asp 480 has been suggested to deprotonate the hydroxyl group of the second and subsequent HB-CoAs, allowing for ester formation and elongation of the PHB chain (8,9,18).…”

Section: The Catalytic Domain Of Cnphac Has An ␣/␤-Hydrolasementioning

confidence: 98%

“…We propose that these arginine residues bind the 5Ј-pyrophosphate of the CoA nucleotide. Binding of HB-CoA by residues across the dimer interface in PhaC could explain, at least in part, the requirement of dimerization for activity (13,14,22). Furthermore, incubation of PhaC with oligomers of (HB) n -CoA, where n ϭ 2-4, or with (HB) 3 -CoA in which the terminal hydroxyl group is replaced with a hydrogen (saturated trimer, sTCoA), has been shown to induce formation of the dimer (14).…”

Section: Structure Of the Cnphac Catalytic Domainmentioning

confidence: 99%

“…The class I and class III synthases are the best studied and share a common substrate specificity for 3-(R)-hydroxybutyryl-CoA (HB-CoA) to form polyhydroxybutyrate (PHB) of high molecular mass (ϳ1-2 MDa) and low polydispersity (1,2,8,11,12). The class I synthases, as typified by the PhaC from Cupriavidus necator (formerly Ralstonia eutropha), are composed of a single polypeptide chain (65 kDa) and contain an N-terminal domain of unknown function (residues 1-200) and a C-terminal catalytic domain (residues 201-589) (13)(14)(15). The class III synthases are made up of a catalytic PhaC subunit (ϳ40 kDa) and a poorly understood, although functionally necessary, second subunit, PhaE (also ϳ40 kDa), as characterized from Allochromatium vinosum (16,17).…”

mentioning

confidence: 99%

“…1) (1, 2). The active site cysteine has been shown to become acylated with HB units after deprotonation of its thiol, thought to be performed by the histidine residue (8,10,13,17). Esterification is then promoted by deprotonation of the HB hydroxyl group, with the aspartate residue serving as the suggested general base catalyst (18).…”

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confidence: 99%

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Structure of the Catalytic Domain of the Class I Polyhydroxybutyrate Synthase from Cupriavidus necator

Wittenborn¹,

Jost²,

Wei³

et al. 2016

Journal of Biological Chemistry

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Edited by F. Peter GuengerichPolyhydroxybutyrate synthase (PhaC) catalyzes the polymerization of 3-(R)-hydroxybutyryl-coenzyme A as a means of carbon storage in many bacteria. The resulting polymers can be used to make biodegradable materials with properties similar to those of thermoplastics and are an environmentally friendly alternative to traditional petroleum-based plastics. A full biochemical and mechanistic understanding of this process has been hindered in part by a lack of structural information on PhaC. Here we present the first structure of the catalytic domain (residues 201-589) of the class I PhaC from Cupriavidus necator (formerly Ralstonia eutropha) to 1.80 Å resolution. We observe a symmetrical dimeric architecture in which the active site of each monomer is separated from the other by ϳ33 Å across an extensive dimer interface, suggesting a mechanism in which polyhydroxybutyrate biosynthesis occurs at a single active site. The structure additionally highlights key side chain interactions within the active site that play likely roles in facilitating catalysis, leading to the proposal of a modified mechanistic scheme involving two distinct roles for the active site histidine. We also identify putative substrate entrance and product egress routes within the enzyme, which are discussed in the context of previously reported biochemical observations. Our structure lays a foundation for further biochemical and structural characterization of PhaC, which could assist in engineering efforts for the production of eco-friendly materials.

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Section: The Catalytic Domain Of Cnphac Has An ␣/␤-Hydrolasementioning

confidence: 92%

Section: The Catalytic Domain Of Cnphac Has An ␣/␤-Hydrolasementioning

confidence: 98%

Section: Structure Of the Cnphac Catalytic Domainmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Structure of the Catalytic Domain of the Class I Polyhydroxybutyrate Synthase from Cupriavidus necator

Wittenborn¹,

Jost²,

Wei³

et al. 2016

Journal of Biological Chemistry

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Bacterial polyhydroxyalkanoates

Lee

2000

Biotechnol. Bioeng.

762

236

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Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates (HAs) synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. More than 80 HAs have been detected as constituents of PHAs, which allows these thermoplastic materials to have various mechanical properties resembling hard crystalline polymer or elastic rubber depending on the incorporated monomer units. Even though PHAs have been recognized as good candidates for biodegradable plastics, their high price compared with conventional plastics has limited their use in a wide range of applications. A number of bacteria including Alcaligenes eutrophus, Alcaligenes latus, Azotobacter vinelandii, methylotrophs, pseudomonads, and recombinant Escherichia coli have been employed for the production of PHAs, and the productivity of greater than 2 g PHA/L/h has been achieved. Recent advances in understanding metabolism, molecular biology, and genetics of the PHA‐synthesizing bacteria and cloning of more than 20 different PHA biosynthesis genes allowed construction of various recombinant strains that were able to synthesize polyesters having different monomer units and/or to accumulate much more polymers. Also, genetically engineered plants harboring the bacterial PHA biosynthesis genes are being developed for the economical production of PHAs. Improvements in fermentation/separation technology and the development of bacterial strains or plants that more efficiently synthesize PHAs will bring the costs down to make PHAs competitive with the conventional plastics. © 1996 John Wiley & Sons, Inc.

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Production of poly(3-hydroxybutyrate) by high cell density fed-batch culture of Alcaligenes eutrophus with phospate limitation

Ryu

Hahn

Chang

et al. 1997

Biotechnol. Bioeng.

177

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High cell density fed‐batch fermentation of Alcaligenes eutrophus was carried out for the production of poly(3‐hydroxybutyrate) (PHB) in a 60‐L fermentor. During the fermentation, pH was controlled with NH4OH solution and PHB accumulation was induced by phosphate limitation instead of nitrogen limitation. The glucose feeding was controlled by monitoring dissolved oxygen (DO) concentration and glucose concentration in the culture broth. The glucose concentration fluctuated within the range of 0–20 g/L. We have investigated the effect of initial phosphate concentration on the PHB production when the initial volume was fixed. Using an initial phosphate concentration of 5.5 g/L, the fed‐batch fermentation resulted in a final cell concentration of 281 g/L, a PHB concentration of 232 g/L, and a PHB productivity of 3.14 g/L · h, which are the highest values ever reported to date. In this case, PHB content, cell yield from glucose, and PHB yield from glucose were 80, 0.46, and 0.38% (w/w), respectively. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 28–32, 1997.

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Overexpression and Purification of the Soluble Polyhydroxyalkanoate Synthase from Alcaligenes eutrophus: Evidence for a Required Posttranslational Modification for Catalytic Activity

Cited by 227 publications

References 49 publications

Structure of the Catalytic Domain of the Class I Polyhydroxybutyrate Synthase from Cupriavidus necator

Structure of the Catalytic Domain of the Class I Polyhydroxybutyrate Synthase from Cupriavidus necator

Bacterial polyhydroxyalkanoates

Production of poly(3-hydroxybutyrate) by high cell density fed-batch culture of Alcaligenes eutrophus with phospate limitation

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