Isolated Poly(3-Hydroxybutyrate) (PHB) Granules Are Complex Bacterial Organelles Catalyzing Formation of PHB from Acetyl Coenzyme A (CoA) and Degradation of PHB to Acetyl-CoA

Abstract

, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) from PHB. Synthesis of 3HB-CoA was also shown by incubation of artificial (proteinfree) PHB with CoA and PhaZa1, confirming that PhaZa1 is a PHB depolymerase catalyzing the thiolysis reaction. Acetyl-CoA was the major product detectable after incubation of nPHB granules in the presence of NAD ؉ , indicating that downstream mobilizing enzyme activities were also present and active in isolated nPHB granules. We propose that intracellular concentrations of key metabolites (CoA, acetyl-CoA, 3HB-CoA, NAD ؉ / NADH) determine whether a cell accumulates or degrades PHB. Since the degradation product of PHB is 3HB-CoA, the cells do not waste energy by synthesis and degradation of PHB. Thus, our results explain the frequent finding of simultaneous synthesis and breakdown of PHB.Poly(3-hydroxybutyrate) (PHB) is the most prominent member of the bacterial polyhydroxyalkanoates (PHA). PHA are osmotic neutral reservoirs of carbon and energy and are synthesized when more carbon sources are available than can be consumed by the bacteria. PHA are reutilized (mobilized) during times of starvation and greatly enhance the survival of bacteria in the absence of a suitable exogenous carbon source.PHB is synthesized by condensation of two molecules of acetyl coenzyme A (CoA) to acetoacetyl-CoA (with a thiolase encoded by phaA), subsequent reduction to 3-hydroxybutyrylCoA (with a reductase encoded by phaB), and polymerization to PHB (with a synthase encoded by phaC). The polymer can be hydrolyzed to 3-hydroxybutyrate by PHB depolymerases (PhaZs). Biosynthesis and biodegradation of PHA have been investigated by many research groups for about three decades, and a series of books and reviews have been published (11,13,30,34,(39)(40)(41)(42). PHB exists in two different forms. In vivo PHB granules consist of an amorphous polymer and are covered by a dense layer consisting of mainly proteins (phasins, PHB synthase, PHB depolymerases, and other proteins) (14,22,31,38). Such granules are called native PHB (nPHB) granules and can be isolated in the native form by glycerol density gradient centrifugation (12,23,45). Isolated PHB granules that have been treated with solvents or with compounds that remove the surface layer rapidly crystallize and are referred to as denatured PHB granules (23,24). (For more details on the impact of the biophysical state of the polymer granules on their susceptibility to enzymatic hydrolysis see references 5, 8, and 12.) Despite the progress made in understanding the function of individual proteins involved in PHA metabolism, the molecular tools and mechanisms with which a cell decides whether it should synthesize or degrade (mobilize) PHB are not known. Several reports have indicated that PHB synthesis and PHB degradation can happen simultaneously in Ralstonia eutropha, the model organism for PHB...

Production, optimization and characterization of polyhydroxybutyrate, a biodegradable plastic by Bacillus spp.

“…These PHB are reutilized/mobilized during times of starvation and greatly enhance the survival of bacteria in the absence of a suitable exogenous carbon source. 45) The presence of characteristic functional group of the PHA granules is C=O group which has been confirmed by FTIR spectroscopy. Irrespective of the nature and type of bioplastics, carbonyl group (C=O) is a common feature in all structures which confirms the extraction of PHB during the present study.…”

Degradation of Polyhydroxyalkanoate (PHA): a Review

“…It has been proposed that all enzymes necessary for the synthesis of PHA from acetylCoA associate with PHA inclusions in C. necator (Uchino et al, 2007). In C. necator, PHA synthase (PhaC) becomes insoluble by granule binding because the PHA chain remains covalently linked to the enzyme during the synthesis of the polymer (Liebergesell et al, 1991;Gerngross et al, 1993).…”

Exploiting genomic patterns to discover new supramolecular protein assemblies

“…These granules serve as storage sites for PHB polymers, which are surrounded by an amphiphilic layer of structural proteins. 50 The structural proteins include four paralogs of a protein family referred to as phasins, encoded on separate operons. 51 The function of phasins is to control the structure of the PHB granules, but the need for multiple paralogs is unclear.…”