PHA synthases

Keywords. Polyhydroxyalkanoic acids, Microbial polyesters, PHA, PHA synthase, Metabolic engineering, PHA granules, Ralstonia eutropha, Pseudomonas aeruginosa... 

This review will provide an update of an overview which was published recently [53] and comprised the knowledge of PHA synthase structural genes and... 

Table 1. Alphabetic list of bacteria strains from which PHA synthase genes were cloned and characterized (updated from [53]) ...

PHA synthases are the key enzymes of PHA biosynthesis. These enzymes catalyze the covalent linkage between the hydroxyl group of one and the carboxyl group of another hydroxyalkanoic acid. The substrates of PHA synthases are the coenzyme A thioesters of hydroxyalkanoic acids there is no evidence that PHA synthases can utilize either free hydroxyalkanoic acids or other derivatives of hydroxyalkanoic acids. With respect to size, structure, and substrate specificity, three different types of PHA synthases (I, II, and III) can be distinguished (see below). 

With respect to size and structure, type I- and type II-PHA synthases are distinguished from type III-PHA synthases. Whereas type I- and type II-PHA synthases consist of only one type of subunit, type III-PHA synthases consist of two different types of subunits. The differences between type I- and type II-PHA synthases regarding the substrate specificity will be described in the next section. 

Type III-PHA synthase is represented by the enzyme of Chromatium vino-sum and is encoded by phaECCv. It consists of the two different subunits PhaCCv and PhaECv exhibiting molecular weights of 39,730 and 40,525 Da, respectively [23]. The native PHA synthase, as isolated from recombinant strains of E. coli, exhibited a molecular weight of approximately 390 and 400 + 20 kDa as revealed in our laboratory [54] or 360 + 50 kDa but also 520 + 50 kDa as revealed in another laboratory [55]. The lower molecular weights for the holoenzyme are... 

Aside from C. vinosum, type III-PHA synthases have so far been detected exclusively in the phototrophic purple sulfur bacteria such as Thiocystis viol-acea [51] and Thiocapsa pfennigii [26, 57] and in cyanobacteria such as Synechocystis sp. PCC6803 [49] or Synechococcus sp. MAI 9 [58]. In contrast, the photosynthetic nonsulfur purple bacteria possess type I-PHA synthases. 

PHA synthases exhibit a remarkably low substrate specificity. In general, the specificity of PHA synthases regarding the length of the hydroxyalkyl moiety of the coenzyme A thioester as well as of the position of the hydroxyl group, the presence of other substituents, and the position of double bonds, is low. In contrast, the stereospecificity of PHA synthases is strict the hydroxyl carbon atom must have the R-configuration. 

There are only very few PHA synthases which can incorporate 3HASCL as well as 3HAmcl into the accumulated PHAs. Examples are the PHA synthases of T. pfennigii and Aeromonas caviae, which synthesize, for example, copolyesters of 3HB, 3HHx plus 3HO [26], or of 3HB plus 3HHx [59], from fatty acids, respectively. Recently it was found that the type I-PHASCL synthase of R. eutropha also conferred the incorporation of 3HHx [60] or of 3HO plus 3HD [61] into PHAs in addition to 3HB to certain recombinant strains of enterobacteria. 

The amino acid sequence similarities of all 36 PHA synthases were pairwise revealed, and the results of this comparison are compiled in Table 2. The data correspond well with phylogenetic tree shown in Fig. 1, and the similarities va-... 

Table 2. Percent identities of primary structures from 36 PHA synthases based on pairwise alignments... 

Fig. 1. Phylogenetic tree of 36 PHA synthases. The branching order and distance score were calculated by the program TREE as described by Feng and Doolittle [63]...

Consistent with this catalytic cycle is the existence of one highly conserved cysteine residue, which o ccurs in any PHA synthase and which is Cys -319 in the PHA synthase of R. eutropha [71] and Cys-149 in the PHA synthase of C. vino-... 

Fig. 2a-d. Multiple alignment of primary structures from 36 PHA synthases. A comparison of amino acid sequences derived from PH A synthase genes is shown. Amino acids are specified by the standard one-letter abbreviations. The consensus sequence represents amino acid residues (shaded) which are present in at least 50% of the PHA synthases. Highly conserved amino acids, which are present in at least 70% of the PHA synthases, are additionally underlined in the consensus sequence and the eight amino acid residues, which are present in all PHA synthases, are indicated as bold letters. See Table 1 for references... 

Intermediates of the metabolism have so far not been identified as inhibitors or activators of PHA synthases. The only exception is coenzyme A which inhibits the PHA synthases of R. eutropha, C. vinosum, and R aeruginosa at relatively low concentrations [73,74]. It is not known whether this inhibition is physiologically relevant. The inhibition by coenzyme A has, however, to be taken into account during the design of in vitro PHA biosynthesis processes, if PHA is being prepared on a preparative scale recycling of coenzyme is then recommended not only to reduce the costs but also to improve the kinetics of PHA formation. 

On the other hand various nonphysiological chemicals were identified as inhibitors of PHA synthases such as, for example, phenazine methosulfonate. None of these chemicals specifically inhibit PHA synthases and they are therefore not suitable for application in strategies to enrich mutants with altered PHA metabolism or altered PHA synthase activity. These inhibitors are only interesting in that they reveal some biochemical features of the enzymes. 

At least 53 PHA synthases have meanwhile been cloned from 42 different bacterial strains, and the nucleotide sequences of 38 different PHA synthases from 31 different bacteria have been obtained (Table 1, Fig. 2). That the number of sequenced PHA synthases exceeds the number of bacteria from which they have been cloned is due to the occurrence of two or even more PHA synthase genes in some bacteria. The organization of PHA synthase genes and other genes of... 

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