Polyester synthases

The residues Cys-319, Asp-480 and His-508 of the class I polyester synthase from Cupriavidus necator are conserved in all PHA synthases and were shown to be essential for covalent catalysis [2,4, 5]. Cys-319 is the proposed catalytic nucleophile that is activated by the general base catalyst His-508. 

Rehm, B.H.A. (2003) Polyesters synthases natural catalysts for plastics. Eiochem. J., 376,15-33. 

B.H. Rehm, Genetics and biochemistry of polyhydroxyalkanoate granule self-assembly the key role of polyester synthases, Biotech-nol. Lett. 28 (4) (2006) 207-213. 

The PHAs are deposited as unique water-insoluble inclusions and when a carbon source is accessible in excess, while other nutrients are growth limiting (Figure 3.2). With carbon starvation, the polyester serves as a reserve polymer, which is then mobilized by intracellular PHA depolymerases, found to be attached to the granule surface [7]. At present, over 88 different polyester synthases are identified, cloned, and assigned [15,16]. 

Exhaustive examination of the primary structures of 88 PHA synthases have shown that these enzymes from different bacteria display strong similarity ranging from 8% to 96% identity. Six conserved blocks were identified on the basis of conserved amino acid sequence regions, whereas the N-terminal region (about 100 amino acids relative to class I polyester synthases) is highly variable [37]. 

There are no current structural data of polyester synthases. The secondary structure content was inferred by predictions implementing the multiple alignments of synthases. The use of JPred indicated that polyester synthases are mostly composed of variable loops (49.7%) and a-helical (39.9%) secondary structures, whereas only 10.4% were proposed as P-sheet secondary structures [38]. However, circular dichroism spectroscopy suggested that the class II synthase from P. aeruginosa is comprised of the secondary structures 10% a-helix, 50% P-sheet, and 40% random coil [39]. 

In all of the structural models, the amino acid residues apparently constituting the catalytic triad or involved in covalent catalysis were identified as being adjacent to the core structure with the putative active site nucleophile cysteine located at the elbow of the strand-elbow helix motif. In the class II polyester synthase, the highly conserved histidine residue which functions as a general base catalyst in a/p-hydrolases was functionally replaced by an adjacent histidine residue, which too was close to the core structure. 

Figure 3.4 Model of the catalytic mechanism of polyester synthase. (Ref. [17], reprinted with permission of Horizon Scientific Press.)...

The soluble polyester synthase converts into an amphipathic molecule upon availability of substrate and subsequent hydrophobic polyester chain elongation [15]. This leads to self-assembly of so-called PHA granules with the hydrophobic PHA in the core and the active polyester synthases at the surface, representing the water-polyester interface. Analysis of the granule-associated polyester synthase from R. eutropha showed about 40-fold increased enzyme activity, as compared with the soluble enzyme [46]. This data suggests that interfacial activation occurred and a lid-like structure as found in lipases and also found in the R. eutropha polyester synthase model may also play a role in polyester synthases [37]. 

Gerngross and Martin [46] were the first to demonstrate in vitro synthesis of PHA and self-assembly of spherical granules by only using purified polyester synthase and substrate. Their work fully defined that the polyester synthase possesses... 

With both models of granule formation, the polyester synthase is converted into an amphipathic molecule upon polyester chain synthesis and a self-assembly process occurs either in the membrane or in the cytosol (Figure 3.5). Small water-insoluble and spherical inclusions are formed with an amorphous polyester core and polyester synthase covalently attached to the surface [58, 59], (Figure 3.5). 

These PHA granules grow in size, while the attached polyester synthases constantly converts precursor from the cytosol and into constituents of the growing polyester chain. However, it is unclear whether larger granules occur because of fusion events or whether simple increase in size on the basis of continuous polymerization takes place. Approximately 5-8 PHA granules are formed intracellularly comprising almost the entire cell volume, when maximum PHA accumulation is obtained [22]. 

The non-covalently attached proteins are not vital for PHA granule formation, however, they serve various biological functions, for example, PHA granule structure, PHA biosynthesis gene regulation, and PHA mobilization. Yet, only the covalently attached polyester synthases possess all the inherent properties needed for PHA granule formation. 

Rehm BHA (2003) Polyester synthases natural catalysts for plastics. Biochem J 376(1) 15-33. doi 10.1042/bj20031254... 

---