Acetoacetyl CoA reductase

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 kind of enantiomer [d-(-)- or l-(+)-] synthesized in the formation of the C4 intermediate varies. The acetoacetyl-CoA reductase (EC 1.1.1.36), which is NADPH-dependent, stereoselectively reduces acetoacetyl-CoA to d-(-)-3-hydroxybutyryl-CoA (R. eutropha [15]). The NADH-dependent reductase catalyzes the reduction of acetoacetyl-CoA to L-(+)-3-hydroxybutyryl-CoA. Afterwards two stereospecific crotonyl-CoA hydratases, l-(+)- and D-(-)-speci-fic, convert the L-(+)-3-hydroxybutyryl-CoA into the D-(-)-isomer (Rhodo-spirillum rubrum [16]). 

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]. 

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

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... 

The copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-co-PHV) produced by A eutrophus has generated more interest than poly-(R)-3-hydroxybutyrate (PHB) homopolymer. Since these bacterial polyesters are biodegradable thermoplastics, their mechanical and physical properties have received much attention. PHB is a relatively stiff and brittle material because of its high crystallinity. However, the physiochemi-cal and mechanical properties of [P(HB-HV)] vary widely and depend on the molar percentage of 3-hydroxyvalerate (HV) in the copolymer (4,5) as shown inTable 1. Propionic acid is converted by a synthetase to propionyl-CoA, and the biosynthetic P-ketothiolase catalyzes the condensation of propionyl-CoA with acetyl-CoA to 3-ketovaleryl-CoA by the acetoacetyl-CoA reductase. The hydroxyvaleryl moiety is finally covalently linked to the polyester by the PHA synthase (6). 

Fig.l. Biologic and chemical processes for production of R3HB in natural and recombinant bacteria. PhaA,P-ketothiolase PhaB,acetoacetyl-CoA reductase PhaC, PHA synthase Ptb, phosphotransbutyrylase Buk, butyratekinase TCA, tricarboxylic acid. 

The phbA, phbB, and phbC genes from Alcaligenes eutrophus (Ralstonia eutrophus) encoding the biosynthetic enzymes (3-ketothiolase, acetoacetyl-CoA reductase (NADPH-dependent), and PHB synthase, respectively, have been cloned into E. coli (Scheme 19.42).339-342 The use of in vitro evolution using error-prone polymerase chain reaction has led to enhanced accumulation of PHA in a resultant recombinant strain.343 Additional studies to enhance the biosynthesis of PHB through the use of metabolic engineering have been discussed.344... 

Dodds, P.F., Guzman, M.G.F., Chalberg, S.C., Anderson, G.J., Kumar, S. 1981. Acetoacetyl-CoA reductase activity of lactating bovine mammary fatty acid synthase. J. Biol. Chem. 256, 6282-6290. 

Figure 7.4. Transport of fatty acids (FA) and metabolic reactions in P-oxidation for the formation of acetyl CoA and (R)-3-hydroxyacyl CoA, key intermediates for poly-hydroxyalkanoate biosynthesis. Note The gene products PhbA, PhbB, PhbC, and PhaC correspond to the enzymes 3-ketothiolase, acetoacetyl CoA reductase, PHB synthase, and PFIA synthase, respectively.

FIG. 4.2 Malate metabolism in mitochondria from body wall muscle of adult Ascaris smm. (1) Fumarase (2) malic enzyme (3) pyruvate dehydrogenase complex (4) complex I (5) succinate-coenzyme Q reductase (complex II, fumarate reductase) (6) acyl CoA transferase (7) methylmalonyl CoA mutase (8) methyl-malonyl CoA decarboxylase (9) propionyl CoA condensing enzyme (10) 2-methyl acetoacetyl CoA reductase (11) 2-methyl-3-oxo-acyl CoA hydratase (12) electron-transfer flavoprotein (13) 2-methyl branched-chain enoyl CoA reductase (14) acyl CoA transferase. 

The route for P(3HB) synthesis in C. necator is one of the simplest and extensively studied PHA biosynthetic pathway (Fig. 2.3). Via this route, P-ketothiolase (PhaA) condenses two molecules of acetyl-CoA to form ace-toacetyl-CoA. An NADH2-dependent acetoacetyl-CoA reductase (PhaB) then catalyzes the conversion of acetoacetyl-CoA to (f )-3-hydroxybutyryl-CoA. The PhaC catalyzes the polymerization of (/ )-3-hydroxybutyryl-CoA monomers into P(3HB) polymer (Sudesh et al. 2000 Khanna and Srivastava 2005a Suriyamongkol et al. 2007). 

Fig. 2.3 Biosynthesis pathway of A P(3HB) B P(3HB-co-3HV) C P(3HB-co-3HHx) via fatty acid /S-oxidation and D P(3HB-co-3HHx) via fatty acid de novo synthesis. PhaA, f -ketothiolase PhaB, NADPH dependent acetoacetyl-CoA reductase PhaC, PHA synthase PhaG, 3-hydroxyl-ACP-CoA transferase PhaJ, (J )-enoyl-CoA hydratase FabG, 3-ketoacyl-CoA reductase (Sudesh et al. 2000)...

Figure 15.2 1-Butanol production via the CoA-dependent pathway implemented in S. elongatus PCC7942. Gene symbols are accABCD, acetyl-CoA carboxylase nphT7, acetoacetyl-CoA synthase phaB, acetoacetyl-CoA reductase phaJ, R-specific enoyl-CoA...

O.P. Peoples, A.J. Sinskey, Poly-beta-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16. Characterization of the genes encoding beta-ketothiolase and acetoacetyl-CoA reductase, J. Biol. Chem. 264 (26) (1989) 15293-15297. 

Polyhydroxyalkanoates biosynthesis is regulated, on one hand, by the activity of 3-ketothiolase (EC 2.3.1.16), and on the other hand of acetoacetyl-CoA reductase (EC 1.1.1.36) intracellular PHA breakdown is dependent on the activity of 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30). Besides these three enzymes, the following compounds can be pointed out as major factors responsible of the activities of the key enzymes acetyl-CoA, free CoA, NAD(P) + (or NAD(P)H2, respectively) and, to a lower extent, ATP, pyruvate and oxalacetate. In any case, acetyl-CoA can be considered as the central metabolite both for biomass formation and PHB biosynthesis. This compound stems from the catabolic break down of carbon substrates like sugars (mainly catabolized by the 2-Keto-3-desoxy-6-phosphogluconate pathway) or fatty acids (converted by 6-oxidation). 

Free CoA is inhibiting 3-ketothiolase as a consequence, the carbon flux towards PHAs is decreased. To a lower extent, 3-ketothiolase is also inhibited by NAD+ and ATP. Additionally, high amounts of NAD + slow down the reduction steps from acetoacetyl-CoA to 3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase. 

So far, biosynthesis of PHA can be summarized in eight pathways (Fig. 4, Table 1). The first pathway involves the three key enzymes (3-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase, and PHA synthase encoded by genes phaA, phaB, and phaC, respectively. Ralstonia eutropha is the representative of this pathway. An associated pathway involving PHA degradation catalyzed by PHA depolymerase, dimer hydrolase, 3-hydroxybutyrate dehydrogenase, and acetoacetyl-CoA synthase helps regulate PHA synthesis and degradation. The associated pathway was found in strains of Aeromonas hydrophila. Pseudomonas stutzeri, R. eutropha, and Pseudomonas oleovorans (Sudesh et al. 2000). 

Pathway IV NADH-dependent acetoacetyl-CoA reductase Rhizobium (Cicer) sp. CC 1192 Chohan and Copeland (1998)... 

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