Polyhydroxybutyrate Synthase:  Evidence for Covalent Catalysis

Wodzinska, Jola; Snell, Kristi D.; Rhomberg, A.; Sinskey, Anthony J.; Biemann, K.; Stubbe, JoAnne

doi:10.1021/ja961108a

Cited by 112 publications

(209 citation statements)

References 10 publications

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“…The mass of peptide 1 (3669.6 Da) agrees well with that predicted for the peptide EAH-GVDKVNLLGICQGGAFSLMYSALHPDKVR (3426.98 Da) plus a saturated trimer moiety (Table 3). Peptide 1 contains C149, which is the only universally conserved cysteine among PHA synthases and the most likely site of acylation (10). C149 is in a sequence context very similar to C319 previously identified in the R. eutropha enzyme as the most likely site of acylation.…”

Section: Pha Synthase Expression and Purificationmentioning

confidence: 55%

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PHA Synthase from Chromatium vinosum: Cysteine 149 Is Involved in Covalent Catalysis

et al. 1998

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Polyhydroxyalkanoate synthase (PHA) from Chromatium Vinosum catalyzes the conversion of 3-hydroxybutyryl-CoA (HB-CoA) to polyhydroxybutyrate (PHB) and CoA. The synthase is composed of a ∼1:1 mixture of two subunits, PhaC and PhaE. Size-exclusion chromatography indicates that in solution PhaC and PhaE exist as large molecular weight aggregates. The holo-enzyme, PhaEC, has a specific activity of 150 units/mg. Each subunit was cloned, expressed, and purified as a (His) 6 -tagged construct. The PhaC-(His) 6 protein catalyzed polymerization with a specific activity of 0.9 unit/mg; the PhaE-(His) 6 protein was inactive (specific activity <0.001 unit/mg). Addition of PhaE-(His) 6 to PhaC-(His) 6 increased the activity several 100-fold. To investigate the priming step of the polymerization process, the PhaEC was incubated with a trimer of HB-CoA in which the terminal hydroxyl was replaced with tritium ( Sequencing by ion trap mass spectrometry showed that they were identical and that they each contained an altered cysteine (C149). One peptide contained the [ 3 H]-sT while the other two contained, in addition to the [ 3 H]-sT, one and two additional monomeric HBs, respectively. Mutation of C149 to alanine gave inactive synthase. The remaining two cysteines of PhaC, 292 and 130, were also mutated to alanine. The former had wild-type (wt) activity, while the latter had 0.004 wt % activity and was capable of making polymer. A mechanism is proposed in which PhaC contains all the elements essential for catalysis and the polymerization proceeds by covalent catalysis using C149 and potentially C130.Polyhydroxyalkanoates (PHAs 1 ) are polyoxoesters with properties that range from elastomers to thermoplastics (1-4). They are produced by a wide range of bacteria when they are placed in an environment of nutrient limitation (5). Copolymers of polyhydroxybutyrate (PHB) and polyhydroxyvalerate in the correct ratio have properties similar to the petrochemically based polypropylenes, the major component of bulk plastics (6). In 1997, the US market for thermoplastics was on the order of 40 million tons per year (7). PHAs have recently received much attention because they are biodegradable and can be generated from biorenewable sources: bacteria and plants (8). The major focus of many investigators is to make their production economically competitive with the polypropylenes. To achieve this goal, the requirements for the polymerization process need to be established. This paper focuses on the PHA synthase from Chromatium Vinosum which catalyzes the formation of PHB from 3-hydroxybutyryl-CoA (HB-CoA). Evidence is presented that two cysteines and covalent catalysis play an important role in the initiation and elongation of the polymerization process.PHA synthases from 20 organisms have now been identified. They have been divided into three classes based on their substrate specificity and subunit composition (9). The class I synthases, with the Ralstonia eutropha synthase as a prototype, are composed of a single polypeptide (∼65 k...

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Section: Pha Synthase Expression and Purificationmentioning

confidence: 55%

“…Interestingly, the lag phase is greatly diminished by preincubation of [ 3 H]-sT-CoA, a putative primer. This decrease of the lag phase is accompanied by conversion of the monomeric to the dimeric form of the enzyme, and only the latter appears to be covalently labeled with 1 equiv of [ 3 H]-sT per protein dimer (10).…”

Section: Discussionmentioning

confidence: 99%

PHA Synthase from Chromatium vinosum: Cysteine 149 Is Involved in Covalent Catalysis

et al. 1998

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“…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).…”

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confidence: 99%

“…Over the last 25 years, biochemical work on these systems has established that all PhaCs require the same residues for catalysis but have distinct kinetics of PHB formation using CoA release to monitor activity (8,11,14,15,22). For CnPhaC, a variable lag phase in CoA release is observed, which can be overcome by priming the enzyme through acylation with synthetic (HB) n -CoA analogs, where n ϭ 2-4.…”

mentioning

confidence: 99%

“…For CnPhaC, a variable lag phase in CoA release is observed, which can be overcome by priming the enzyme through acylation with synthetic (HB) n -CoA analogs, where n ϭ 2-4. This acylation is accompanied by conversion of the predominant monomeric form of CnPhaC to the dimeric form, which is accompanied by an increase in its catalytic activity, suggesting that the dimer is the active form of the enzyme (14). Despite these significant insights, there is much that remains to be understood, including the origin of odd kinetic behaviors, which are distinct within and between different classes of PhaCs (14,17,22,23); the process of chain termination; the molecular basis of substrate specificity; and the ability of the synthase to control polymer length and polydispersity.…”

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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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Metabolic modeling of polyhydroxybutyrate biosynthesis

Leaf

Srienc

1998

Biotechnol. Bioeng.

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A mathematical model describing intracellular polyhydroxybutyrate (PHB) synthesis in Alcaligenes eutrophus has been constructed. The model allows investigation of issues such as the existence of rate‐limiting enzymatic steps, possible regulatory mechanisms in PHB synthesis, and the effects different types of rate expressions have on model behavior. Simulations with the model indicate that activities of all PHB pathway enzymes influence overall PHB flux and that no single enzymatic step can easily be identified as rate limiting. Simulations also support regulatory roles for both thiolase and reductase, mediated through AcCoA/CoASH and NADPH/NADP+ ratios, respectively. To make the model more realistic, complex rate expressions for enzyme‐catalyzed reactions were used which reflect both the reversibility of the reactions and the reaction mechanisms. Use of the complex kinetic expressions dramatically changed the behavior of the system compared to a simple model containing only Michaelis–Menten kinetic expressions; the more complicated model displayed different responses to changes in enzyme activities as well as inhibition of flux by the reaction products CoASH and NADP+. These effects can be attributed to reversible rate expressions, which allow prediction of reaction rates under conditions both near and far from equilibrium. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 557‐570, 1998.

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Polyhydroxybutyrate Synthase: Evidence for Covalent Catalysis

Cited by 112 publications

References 10 publications

PHA Synthase from Chromatium vinosum: Cysteine 149 Is Involved in Covalent Catalysis

PHA Synthase from Chromatium vinosum: Cysteine 149 Is Involved in Covalent Catalysis

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

Metabolic modeling of polyhydroxybutyrate biosynthesis

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