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

ABSTRACT: Polyhydroxybutyrate 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 ?33 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.

SUBMITTER: Wittenborn EC

PROVIDER: S-EPMC5122792 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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

Wittenborn Elizabeth C EC Jost Marco M Wei Yifeng Y Stubbe JoAnne J Drennan Catherine L CL

The Journal of biological chemistry 20161014 48

Polyhydroxybutyrate 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 pr ...[more]

PMID: 27742839

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Project description:Background:Pineapple is the third most important tropical fruit produced worldwide, and approximately 24.8 million tons of this fruit are produced annually throughout the world, including in Thailand, which is the fourth largest pineapple producer in the world. Pineapple wastes (peel and core) are generated in a large amount equal to approximately 59.36% based on raw material. In general, the anaerobic digestion of pineapple wastes is associated with a high biochemical oxygen demand and high chemical oxygen demand, and this process generates methane and can cause greenhouse gas emissions if good waste management practices are not enforced. This study aims to fill the research gap by examining the feasibility of pineapple wastes for promoting the high-value-added production of biodegradable polyhydroxybutyrate (PHB) from the available domestic raw materials. The objective of this study was to use agro-industrial residue from the canned pineapple industry for biodegradable PHB production. Results:The results indicated that pretreatment with an alkaline reagent is not necessary. Pineapple core was sized to -?20/+?40 mesh particle and then hydrolyzed with 1.5% (v/v) H2SO4 produced the highest concentration of fermentable sugars, equal to 0.81 g/g dry pineapple core, whereas pineapple core with a +?20 mesh particle size and hydrolyzed with 1.5% (v/v) H3PO4 yielded the highest concentration of PHB substrates (57.2?±?1.0 g/L). The production of PHB from core hydrolysate totaled 35.6?±?0.1% (w/w) PHB content and 5.88?±?0.25 g/L cell dry weight. The use of crude aqueous extract (CAE) of pineapple waste products (peel and core) as a culture medium was investigated. CAE showed very promising results, producing the highest PHB content of 60.00?±?0.5% (w/w), a cell dry weight of 13.6?±?0.2 g/L, a yield ( YP/S ) of 0.45 g PHB/g PHB substrate, and a productivity of 0.160 g/(L h). Conclusions:This study demonstrated the feasibility of utilizing pineapple waste products from the canned pineapple industry as lignocellulosic feedstocks for PHB production. C. necator strain A-04 was able to grow on various sugars and tolerate levulinic acid and 5-hydroxymethyl furfural, and a detoxification step was not required prior to the conversion of cellulose hydrolysate to PHB. In addition to acid hydrolysis, CAE was identified as a potential carbon source and offers a novel method for the low-cost production of PHB from a realistic lignocellulosic biomass feedstock.

Project description:Cupriavidus necator H16 is an ideal strain for polyhydroxybutyrate (PHB) production from CO2. Low-oxygen stress can induce PHB synthesis in C. necator H16 while reducing bacterial growth under chemoautotrophic culture. The optimum growth and PHB synthesis of C. necator H16 cannot be achieved simultaneously, which restricts PHB production. The present study was initiated to address the issue through comparative transcriptome and gene function analysis. First, the comparative transcriptome of C. necator H16 chemoautotrophically cultured under low-oxygen stress and nonstress conditions was studied. Three types of genes were discovered to have differential levels of transcription: those involving PHB enzymatic synthesis, PHB granulation, and regulators. Under low-oxygen stress conditions, acetoacetyl-coenzyme A (CoA) reductase gene phaB2, PHB synthase gene phaC2, phasins genes phaP1 and phaP2, and regulator genes uspA and rpoN were upregulated 3.0-, 2.5-, 1.8-, 2.7-, 3.5-, and 1.6-fold, respectively. Second, the functions of upregulated genes and their applications in PHB synthesis were further studied. It was found that the overexpression of phaP1, phaP2, uspA, and rpoN can induce PHB synthesis under nonstress conditions, while phaB2 and phaC2 have no significant effect. Under the optimum conditions, the PHB percentage content in C. necator H16 was increased by 37.2%, 28.4%, 15.8%, and 41.0%, respectively, with overexpression of phaP1, phaP2, uspA, and rpoN, and the corresponding PHB production increased by 49.8%, 42.9%, 47.0%, and 77.5%, respectively, under nonstress chemoautotrophic conditions. Similar promotion by phaP1, phaP2, uspA, and rpoN was observed in heterotrophically cultured C. necator H16. The PHB percentage content and PHB production were increased by 54.4% and 103.1%, respectively, with the overexpression of rpoN under nonstress heterotrophic conditions. IMPORTANCE Microbial fixation of CO2 is an effective way to reduce greenhouse gases. Some microbes, such as C. necator H16, usually accumulate PHB when they grow under stress. Low-oxygen stress can induce PHB synthesis when C. necator H16 is autotrophically cultured with CO2, H2, and O2, while under stress, growth is restricted, and total PHB yield is reduced. Achieving the optimal bacterial growth and PHB synthesis at the same time is an ideal condition for transforming CO2 into PHB by C. necator H16. The present study was initiated to clarify the molecular basis of low-oxygen stress promoting PHB accumulation and to realize the optimal PHB production by C. necator H16. Genes upregulated under nonstress conditions were identified through comparative transcriptome analysis and overexpression of phasin, and regulator genes were demonstrated to promote PHB synthesis in C. necator H16.

Dataset Information

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

Publications

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

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