PHB synthase

PHOTO-BIOLOGICAL HYDROGEN PRODUCTION BY THE UPTAKEHYDROGENASE AND PHB SYNTHASE DEFICIENT MUTANT OF RHODOBACTER SPHAEROIDES... 

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... 

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.

Haywood, G.W., Anderson, A.)., and Dawes, E.A. (1989) The importance of PHB-synthase substrate specificuity in polyhydroxyalkanoate synthesis by Alcaligenes eutrophus. FEMS Microbiol. Lett., 57,1-6. 

Sinskey, A.)., and Stubbe,). (2000) Lipases provide a new mechanistic model for polyhydroxybutyrate (PHB) synthases characterization of the functional residues in Chromatium vinosum PHB synthase. Biochemistry, 39, 3927-3936. 

Rehm, B.H.A., Antonio, R.V., Spiekermann, P., Amara, A.A., and Steinbiichel, A. (2002) Molecular characterization of the poly (3-hydroxybutyrate) (PHB) synthase from Ralstonia eutropha in vitro evolution, site-specific mutagenesis and development of a PHB synthase protein model. Biochim. Biophys. Acta, 1594,178-190. 

Enzymatic, surface-initiated polymerizations of aliphatic polyesters was reported for wider clinical use of aliphatic polyesters 84). The hydroxyl terminated SAM acted as an initiation site for lipase B catalyzed ROP of aliphatic polyesters, such as poly(e-caprolactone) and poly (p-dioxanone) (Scheme 3). Another example of enzymatic SIP is the polymerization of poly (3-hydroxybutyrate) (PHB), where PHB synthase, fused with a His-tag at the N-terminus, was immobilized onto solid substrates through transition-metal complexes, Ni (II)-NTA, and the immobilized PHB synthase catalyzed the polymerization of 3-R-hydroxybutyry 1-coenzyme A (3HB-CoA) to PHB 85). 

Cai G., Driscoll B.T. and Charles T.C., 2000, Requirement for the enzymes acetoacetyl Coenzyme A synthase and Poly-3-hydroxybutyrate (PHB) synthase for growth of Sinorhizobium meliloti and PHB cycle interm iates. J. Bacteriol. 182 2113-2118. 

Synthesis of PHB in the cytoplasm of rape leaf cells gave results very similar to Arabidopsis (Poirier 2002). Interestingly, overexpression of the bacterial R)-3-ketothiolase in plants expressing the reductase and PHB synthase did not lead to a significant increase in PHB accumulation, indicating that (/ )-3-ketothiolase activity was probably not limiting PHB synthesis in the cytoplasm, but rather that other factors, such as the low flux of acetyl-CoA, may be important. 

P-Ketothiolase (encoded by phaA) condenses two molecirles of acetyl-CoA to acetoacetyl-CoA, which is then reduced to (S)-3-hydroxybutyryl-CoA by the NADPH-dependent acetoacetyl-CoA reductase (encoded by phaB). The PHB synthase (encoded by phaC in R. eutropha R.) then converts the thioester monomers into the polyoxoester PHB. The polymer aggregates to form a spherical inclusion or granule usually 50-500nm in diameter with the amorphous hydrophobic PHA polyester at the core and attached or embedded proteins at the surface, including the PHA synthase, PHA depolymerases, structural, and regulatory proteins [13,14]. 

The first PHB synthase from an extremely halophilic archaebacterium was identified and characterized by Hezayen etal. [4], representing presumably a new class... 

Gerngross, T.U., Reilly, P., Stubbe, J., Sinskey, A.J., and Peoples, O.P (1993) Immunocytochemical analysis of poly-beta-hydroxybutyrate (PHB) synthase in Alcaligenes eutrophus H16, localization of the synthase enzyme at the surface of PHB granules. /. Bacteriol, 175, 5289 - 5293. 

Note CDW Cell dry weight vgb Gene encoding Vitreoscilla hemoglobin PHB synthesis genes encoding p-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase Ac Aeromonas caviae 1,4-BD 1,4-butanediol phaC c- PHA synthase gene phaC from Aeromonas caviae. 

Several microorganisms are known to accumulate PHB within the cells as an intracellular storage material for carbon and energy source. In microbes PHB synthesis takes place by a sequence of reactions catalyzed by three enzymes— 3-ketothiolase, acetoacetyl-coA reductase and PHB synthase. The first step is catalyzed by the enzyme 3-ketothiolase (E.C. 2.3.1.16) which condenses acetyl-CoA to acetoacetyl-CoA. This intermediate is reduced to D (-)-P-hydroxybutyryl-CoA by an Nicotinamide Adenine Dinucleotide Phosphate (NADPH)-dependent acetoacetyl-CoA reductase (E.C.1.1.1.36) and the enzyme PHB synthase catalyzes the head to tail polymerization of the monomer to PHB (Figure 17.3). 

Their biosynthetic pathway involves uptake of carbon sources followed by conversion into precursor molecules, which are afterwards polymerized by the PHB synthase [321,322]. Other biosynthetic pathways are involved in fatty acid metabolism. The... 

The PHB synthesis is nothing but the conversion of acetyl-Co A to PHB as a mechanism for storing carbon. The P(3HB) biosynthesis, is the three step biosynthesis pathway, mainly consists of three enzymatic reactions catalyzed by three distinct enzymes. The enzymes are P-ketothiolase, acetoacetyl-CoA reductase and PHB synthase. 

Expression of PHB biosynthesis genes in animal cells has been achieved in the cells of insect Spodoptera frugiperda and Trichopulsiani. In Spodoptera, an alternative pathway for the biosynthesis of PHB is created. The dehydratase domain mutant rat fatty acid synthase cDNA and phaC were expressed simultaneously which resulted into PHB synthesis (Wilhams et al., 1996). AhSiCv o vm, Autographa califomica nuclear polyhedrosis vims system has been also used to express PhaCj in Trichopulsiani cells. These cells accmnttlated PHB synthase in 50% of the total cell protein. 

E. A. (1989). The importanee of PHB-synthase substrate speeifieity in polyhydroxyalcanoate synthesis by Alealigenes eutrophus. FEMS Microbiology Letters il, 1. ... 

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