Poly(3-Hydroxybutyrate) Degradation in Ralstonia eutropha H16 Is Mediated Stereoselectively to (S)-3-Hydroxybutyryl Coenzyme A (CoA) via Crotonyl-CoA

Abstract

bDegradation of poly(3-hydroxybutyrate) (PHB) by the thiolytic activity of the PHB depolymerase PhaZ1 from Ralstonia eutropha H16 was analyzed in the presence of different phasins. An Escherichia coli strain was constructed that harbored the genes for PHB synthesis (phaCAB), the phasin PhaP1, and the PHB depolymerase PhaZ1. PHB was isolated in the native form (nPHB) from this recombinant E. coli strain, and the in vitro degradation of the polyester was examined. Degradation resulted in the formation of the expected 3-hydroxybutyryl coenzyme A (3HB-CoA) and in the formation of a second product, which occurred in significantly higher concentrations than 3HB-CoA. This second product was identified by liquid chromatography mass spectrometry (LC-MS) as crotonyl-CoA. Replacement of PhaP1 by PhaP2 or PhaP4 resulted in a lower degradation rate, whereas the absence of the phasins prevented the degradation of nPHB by the PHB depolymerase PhaZ1 almost completely. In addition, the in vitro degradation of nPHB granules isolated from R. eutropha H16 (wild type) and from the R. eutropha ⌬phaP1 and ⌬phaP1-4 deletion mutants was examined. In contrast to the results obtained with nPHB granules isolated from E. coli, degradation of nPHB granules isolated from the wild type of R. eutropha yielded high concentrations of 3HB-CoA and low concentrations of crotonyl-CoA. The degradation of nPHB granules isolated from the ⌬phaP1 and ⌬phaP1-4 deletion mutants of R. eutropha was significantly reduced in comparison to that of nPHB granules isolated from wild-type R. eutropha. Stereochemical analyses of 3HB-CoA revealed that the (R) stereoisomer was collected after degradation of granules isolated from E. coli, whereas the (S) stereoisomer was collected after degradation of granules isolated from R. eutropha. Based on these results, a newly observed mechanism in the degradation pathway for PHB in R. eutropha is proposed which is connected by crotonyl-CoA to the ␤-oxidation cycle. According to this model, the NADPH-dependent synthesis of PHB with (R)-3HB-CoA as the intermediate and the PHB degradation yielding (S)-3HB-CoA, which is further converted in an NAD-dependent reaction, are separated. P oly(3-hydroxybutyrate) (PHB) is a short-carbon-chainlength polyester belonging to the bacterial polyhydroxyalkanoates (PHAs), which are formed by a wide range of Bacteria and Archaea and comprise a large variety of constituents (1-3). PHAs are stored as granules in the cytoplasm when one nutrient is limited and a carbon source is provided in excess. They serve as carbon and energy storage compounds and are degraded if no carbon source is available and if the limiting element is provided again (4). Due to their thermoplastic properties and biodegradability, PHAs are of great interest for industry (5-7).The model organism for PHB metabolism is the Gram-negative facultative chemolithoautotrophic bacterium Ralstonia eutropha H16, which was isolated in 1961 (8-10). About 50 years of research unraveled many aspects of the synthesis and composition of PHB. T...

Comparative Proteome Analysis Reveals Four Novel Polyhydroxybutyrate (PHB) Granule-Associated Proteins in Ralstonia eutropha H16

“…4). In summary, our data indicate that PHB granules of NB-gluconategrown R. eutropha cells have two other PHB depolymerases (A0671 and B1632) that could contribute to the PHB mobilization ability of R. eutropha, in addition to PhaZa1 (22,37,43,61). However, B1632 and A0671 do not have amino acid similarities to PhaZa1 or to other known intra-or extracellular PHB depolymerases and probably represent-together with Avin03910 -a novel type of intracellular PHB depolymerase with high molecular mass (80 to 90 kDa).…”

Degradation of Polyhydroxyalkanoate (PHA): a Review

“…The intracellular degradation of P(3HB) results in the liberation of 3-hydroxybutyric acid which is then oxidized by a dehydrogenase to acetoacetylCoA, which is finally converted into acetyl-CoA by β-ketothiolase (Eggers and Steinbüchel, 2013;Lemes et al, 2015). The breakdown products of PHA are naturally found in animals.…”

A Patatin-Like Protein Associated with the Polyhydroxyalkanoate (PHA) Granules of Haloferax mediterranei Acts as an Efficient Depolymerase in the Degradation of Native PHA

“…Notably, the PHA degradation process is not simply along the reverse pathway of PHA synthesis. Crotonyl-CoA and (S)-3HB-CoA (one stereoisomer of 3HB-CoA) were recently found to be involved in nPHB degradation, which indicated that PHB degradation is separate from PHB synthesis and is connected to the ␤-oxidation cycle in Ralstonia eutropha (9).…”

“…PhaZa1 degrades nPHB to 3HB-CoA in the presence of CoA (8,9), whereas PhaZb and PhaZc mainly hydrolyze 3HB oligomer to 3HB (12,13). PhaZd degrades artificial PHB (aPHB) and nPHB granules and releases mainly 3HB oligomer with only a small amount of 3HB monomer (6,7).…”

“…The extracellular PHA depolymerase degradation process is well known, but the mechanism underlying the metabolic pathway and regulation of PHA degradation in vivo remains poorly understood (5). Native polyhydroxybutyrate (nPHB) is degraded by iPhaZs in vitro to 3-hydroxybutyrate (3HB) (6, 7) or 3-hydroxybutyryl-coenzyme A (3HB-CoA) in the presence of CoA (8,9). The PHB degradation product, 3HB-CoA, is the precursor of PHB synthesis; hence, the simultaneous synthesis and mobilization of PHB (10,11) (8).…”