0-Ketothiolase

The final step in the /3-oxidation cycle is the cleavage of the /3-ketoacyI-CoA. This reaction, catalyzed by thiolase (also known as j8-ketothiolase), involves the attack of a cysteine thiolate from the enzyme on the /3-carbonyI carbon, followed by cleavage to give the etiolate of acetyl-CoA and an enzyme-thioester intermediate (Figure 24.17). Subsequent attack by the thiol group of a second CoA and departure of the cysteine thiolate yields a new (shorter) acyl-CoA. If the reaction in Figure 24.17 is read in reverse, it is easy to see that it is a Claisen condensation—an attack of the etiolate anion of acetyl-CoA on a thioester. Despite the formation of a second thioester, this reaction has a very favorable A).q, and it drives the three previous reactions of /3-oxidation. 

From this group of microorganisms, so far only Saccharomyces cerevisiae has been transformed to a poly(3HB) accumulating organism by expressing solely phflCRe in the cytoplasm ([137], Table 4). In contrast to E. coli and plants, which synthesize poly(3HB) only if a /3-ketothiolase and an acetoacetyl-CoA reductase... 

Fig. 1. The metabolic cycle for the synthesis and degradation of poly(3HB). (1) 3-ketothiolase (2) NADPH-dependent acetoacetyl-CoA reductase (3) poly(3HB) synthase (4) NADH-dependent acetoacetyl-CoA reductase (5), (6) enolases (7) depolymerase (8) d-(-)-3-hydroxybutyrate dehydrogenase (9) acetoacetyl-CoA synthetase (10) succinyl-CoA transferase (11) citrate synthase (12) see Sect. 3...

The 3-ketothiolase has been purified and investigated from several poly(3HB)-synthesizing bacteria including Azotobacter beijerinckii [10], Ral-stonia eutropha [11], Zoogloea ramigera [12], Rhodococcus ruber [13], and Methylobacterium rhodesianum [14]. In R. eutropha the 3-ketothiolase occurs in two different forms, called A and B, which have different substrate specificities [11,15]. In the thiolytic reaction, enzyme A is only active with C4 and C5 3-ketoacyl-CoA whereas the substrate spectrum of enzyme B is much broader, since it is active with C4 to C10 substrates [11]. Enzyme A seems to be the main biosynthetic enzyme acting in the poly(3HB) synthesis pathway, while enzyme B should rather have a catabolic function in fatty-acid metabolism. However, in vitro studies with reconstituted purified enzyme systems have demonstrated that enzyme B can also contribute to poly(3HB) synthesis [15]. 

In M. rhodesianum no isoenzymes of the 3-ketothiolase have been found [ 14]. The enzyme s capability to react with long-chain acyl-CoAs has not been tested. The enzyme was found to be very similar to the 3-ketothiolases from R. eutropha, A. beijerinckii, Z. ramigera, and R. ruber with respect to molecular weight, optimum pH, and kinetic properties [14]. 

In Aeromonas caviae, 3-ketothiolase and acetoacetyl-CoA reductase are absent. In this species, the synthesis of poly(3HB) proceeds via an enoyl-CoA hy-dratase from either crotonyl-CoA or hexenoyl-CoA. The enoyl-CoA derivatives stem from the fatty-acid oxidation pathway [18]. 

The following examples, found in R. eutropha, illustrate the formation of copolymers (cf. [37]). With propionic acid as an additional carbon source, the 3-ketothiolase catalyzes the condensation of the propionyl-CoA unit with acetyl-CoA to form 3-ketovaleryl-CoA, which is reduced to 3-hydroxyvalerate moieties and polymerized by the synthase [27]. 

If nitrogen (in the form of ammonia) is growth limiting, the potential applications of acetyl-CoA and NAD(P)H are restricted. Liberated NAD(P)H cannot be consumed for reductive syntheses, for instance of amino acids, it remains available and starts to inhibit citrate synthase [45, 46]. To the extent that the TCA cycle is thereby inhibited, acetyl-CoA should become available for the 3-ketothiolase, and could flow into poly(3HB) (Fig. 1, Table 1). 

Organism 3-ketothiolase Citrate synthase Acetoacetyl-CoA reductase 3 -hydroxyb utyryl-CoA dehydrogenase... 

From this perspective it would be interesting to discover if there is a relationship between the substrate used and the concentration of free CoA under conditions of unlimited growth. If there is, depending on the source of carbon and energy used and the K value of the 3-ketothiolase for CoASH, cell multiplication and poly(3HB) accumulation can occur simultaneously [60,61]. If this enzyme is not involved, poly(3HB) [29] and other polyesters [28,29] can also be synthesized during growth. 

Another example of poly(3HB) formation during unlimited growth was reported by Doi et al. [29]. These authors demonstrated that the synthesis of poly(3HB) from butyrate in R. eutropha occurs under balanced growth conditions in the presence of ammonium because the building blocks of poly(3HB) are available without 3-ketothiolase catalyzed conversion. This holds true basically for substrates which are assimilated according to the principle of prefabricated construction [58],e.g.,pentanol/poly(3-hydroxyvalerate) [124]. 

R. eutropha, formerly known as Alcaligenes eutrophus, has been used for the commercial production of P(3HB-co-3HV) [72]. This bacterium grows well in a relatively inexpensive minimal medium and accumulates a large amount of P(3HB) under the unbalanced growth condition. In R. eutropha, acetyl-CoA is converted to P(3HB) by three enzymes (genes) /J-ketothiolase (phaA), aceto-acetyl-CoA reductase (phaB), and PHA synthase (phaC) [6]. 

Poly(3HB) is synthesized in bacteria from acetyl-CoA by a three-step reaction (Fig. 1). The first enzyme of the pathway, 3-ketothiolase, catalyzes the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA. Aceto-acetyl-CoA reductase subsequently reduces acetoacetyl-CoA to R-3-hydroxy-butyryl-CoA, which is then polymerized by the PHA synthase to produce poly(3HB). Since acetyl-CoA is present in plant cells in the cytosol, plastid, mitochondrion, and peroxisome, the synthesis of poly(3HB) in plants could, in... 

Fig. 1. Modification of plant metabolic pathways for the synthesis of poly(3HB) and poly(3HB-co-3HV). The pathways created or enhanced by the expression of transgenes are highlighted in bold, while endogenous plant pathways are in plain letters. The various transgenes expressed in plants are indicated in italics. The ilvA gene encodes a threonine deaminase from E. coli. The phaARe, phaBRe, and phaCRe genes encode a 3-ketothiolase, an aceto-acetyl-CoA reductase, and a PHA synthase from R. eutropha, respectively. The btkBRe gene encodes a second 3-ketothiolase isolated from R. eutropha which shows high affinity for both propionyl-CoA and acetyl-CoA [40]. PDC refers to the endogenous plant pyruvate dehydrogenase complex...

In order to produce PHAs in plants it is necessary to introduce the biosynthetic enzymes from bacteria. PHB represents the best characterized and simplest form of PHA, and the synthetic pathway (Figure 4.2) has been extensively studied in Ralstonia eutropha. 30,31 Starting from acetyl-CoA, a P-ketothiolase is required in order to form acetoacetyl-CoA. This is then reduced by a NADPH-dependent acetoacetyl-CoA reductase, which gives rise to 3-hydroxybutyryl-CoA. The latter intermediate is the substrate for the polymerization reaction catalyzed by polyhydroxybutyrate synthase.30 In Ralstonia eutropha, the thiolase, reductase, and synthase genes make up an operon.31... 

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