Molecular characterization of the poly(3-hydroxybutyrate) (PHB) synthase from Ralstonia eutropha: in vitro evolution, site-specific mutagenesis and development of a PHB synthase protein model

Rehm, Bernd H. A.; Antônio, Regina Vasconcellos; Spiekermann, Patricia; Amara, Amro Abd Al Fattah; Steinbüchel, Alexander

doi:10.1016/s0167-4838(01)00299-0

Cited by 83 publications

(81 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2). This architecture is reminiscent of that seen in lipases, as had been suggested based on sequence similarity and threading models (9,10,24,25) (Table 2). Structural comparison of the CnPhaC catalytic domain with these lipases reveals high structural similarity in the ␤-sheet core with variations in the lengths and relative placements of the surrounding ␣-helices (Fig.…”

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

confidence: 68%

“…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: 99%

“…The synthases are divided into four classes depending on their substrate specificity and subunit composition, although all classes are thought to have a common catalytic mechanism and a conserved active site architecture reminiscent of that seen in lipases (7)(8)(9)(10). 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).…”

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).…”

mentioning

confidence: 99%

“…The second, currently favored, mechanism (Fig. 1B) uses a single active site and requires both covalent and noncovalent intermediates during the catalytic cycle (9,10,20,21).…”

mentioning

confidence: 99%

See 4 more Smart Citations

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

Wittenborn¹,

Jost²,

Wei³

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

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.

show abstract

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

confidence: 68%

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

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

“…The second, currently favored, mechanism (Fig. 1B) uses a single active site and requires both covalent and noncovalent intermediates during the catalytic cycle (9,10,20,21).…”

mentioning

confidence: 99%

See 3 more Smart Citations

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

Wittenborn¹,

Jost²,

Wei³

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

Polyhydroxyalkanoate (PHA) Synthases: The Key Enzymes of PHA Synthesis

Rehm

Steinbüchel

2002

Biopolymers Online

View full text Add to dashboard Cite

Introduction Terminology Cloning Strategies for PHA Synthase Genes Organization of PHA Synthase Genes Primary Structures of PHA Synthases Secondary and Quaternary Structures of PHA Synthases In vivo versus in vitro Substrate Specificity of PHA Synthases Development of a Topological Model for PHA Synthases and Analysis of Site‐specific Mutants of the PHA Synthases The Proposed Catalytic Mechanism of PHA Synthases Factors Determining the Molecular Weight and Composition of PHAs PHA Granules In vitro Synthesis of PHA Diversion of Intermediates from Central Pathways to PHA Biosynthesis Conclusions and Outlook Acknowledgments

show abstract

PseudomonasApplications

Rehm¹

2009

Encyclopedia of Industrial Biotechnology

View full text Add to dashboard Cite

The genus Pseudomonas comprises a huge diversity of species which are adapted to very different environments. This capability to thrive in various habitats coincides with an enormous metabolic capacity of this genus which is reflected by the ability to use recalcitrant compounds as carbon source as well as to produce a wide range of secondary metabolites and biopolymers. These properties imply the production of a diversity of enzymes which have been also conceived as biocatalysts for various applications. In this review, an overview will be provided describing the current use as well as the potential use of pseudomonads and their enzymes in various biotechnological production processes. Besides the application of Pseudomonas for the production of biocatalysts and recombinant proteins, the biosynthesis pathways of commercially relevant biopolymers/biomolecules, such as alginates, elastomeric bioplastics, and rhamnolipids, will be described. These biosynthesis pathways have been successfully subjected to metabolic engineering for the production of tailor‐made biomolecules (biomaterials). Finally, environmental applications of various Pseudomonas species in biodegradation of recalcitrant pollutants as well as biocontrol agents in plant growth promotion will be discussed

show abstract

Molecular characterization of the poly(3-hydroxybutyrate) (PHB) synthase from Ralstonia eutropha: in vitro evolution, site-specific mutagenesis and development of a PHB synthase protein model

Cited by 83 publications

References 42 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

Polyhydroxyalkanoate (PHA) Synthases: The Key Enzymes of PHA Synthesis

PseudomonasApplications

Contact Info

Product

Resources

About