Activity Of acetoacetyl-CoA Reductase Research Articles

Production of Poly(3-Hydroxybutyrate- co -3-Hydroxyhexanoate) from Plant Oil by Engineered Ralstonia eutropha Strains

The polyhydroxyalkanoate (PHA) copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] has been shown to have potential to serve as a commercial bioplastic. Synthesis of P(HB-co-HHx) from plant oil has been demonstrated with recombinant Ralstonia eutropha strains expressing heterologous PHA synthases capable of incorporating HB and HHx into the polymer. With these strains, however, short-chain-length fatty acids had to be included in the medium to generate PHA with high HHx content. Our group has engineered two R. eutropha strains that accumulate high levels of P(HB-co-HHx) with significant HHx content directly from palm oil, one of the world's most abundant plant oils. The strains express a newly characterized PHA synthase gene from the bacterium Rhodococcus aetherivorans I24. Expression of an enoyl coenzyme A (enoyl-CoA) hydratase gene (phaJ) from Pseudomonas aeruginosa was shown to increase PHA accumulation. Furthermore, varying the activity of acetoacetyl-CoA reductase (encoded by phaB) altered the level of HHx in the polymer. The strains with the highest PHA titers utilized plasmids for recombinant gene expression, so an R. eutropha plasmid stability system was developed. In this system, the essential pyrroline-5-carboxylate reductase gene proC was deleted from strain genomes and expressed from a plasmid, making the plasmid necessary for growth in minimal media. This study resulted in two engineered strains for production of P(HB-co-HHx) from palm oil. In palm oil fermentations, one strain accumulated 71% of its cell dry weight as PHA with 17 mol% HHx, while the other strain accumulated 66% of its cell dry weight as PHA with 30 mol% HHx.

Identification of the Polyhydroxyalkanoate (PHA)-Specific Acetoacetyl Coenzyme A Reductase among Multiple FabG Paralogs in Haloarcula hispanica and Reconstruction of the PHA Biosynthetic Pathway in Haloferax volcanii

Genome-wide analysis has revealed abundant FabG (beta-ketoacyl-ACP reductase) paralogs, with uncharacterized biological functions, in several halophilic archaea. In this study, we identified for the first time that the fabG1 gene, but not the other five fabG paralogs, encodes the polyhydroxyalkanoate (PHA)-specific acetoacetyl coenzyme A (acetoacetyl-CoA) reductase in Haloarcula hispanica. Although all of the paralogous fabG genes were actively transcribed, only disruption or knockout of fabG1 abolished PHA synthesis, and complementation of the DeltafabG1 mutant with the fabG1 gene restored both PHA synthesis capability and the NADPH-dependent acetoacetyl-CoA reductase activity. In addition, heterologous coexpression of the PHA synthase genes (phaEC) together with fabG1, but not its five paralogs, reconstructed the PHA biosynthetic pathway in Haloferax volcanii, a PHA-defective haloarchaeon. Taken together, our results indicate that FabG1 in H. hispanica, and possibly its counterpart in Haloarcula marismortui, has evolved the distinct function of supplying precursors for PHA biosynthesis, like PhaB in bacteria. Hence, we suggest the renaming of FabG1 in both genomes as PhaB, the PHA-specific acetoacetyl-CoA reductase of halophilic archaea.

Biochemical and enzymological properties of the polyhydroxybutyrate synthase from the extremely halophilic archaeon strain 56

Some members of the archaebacterial family Halobacteriaceae have been determined to accumulate polyhydroxyalkanoate (PHA) and poly(3-hydroxybutyrate) (PHB). The extremely halophilic archaebacterium strain 56 is capable of accumulating large amounts of PHB. Since measurements of enzyme activities related to archaebacterial PHB biosynthesis have never been achieved, we investigated the enzymology of PHB biosynthesis in strain 56. Crude extracts of strain 56 cultivated under accumulating conditions showed PHB synthase activity, whereas neither β-ketothiolase nor NADH/NADPH-dependent acetoacetyl–CoA reductase activity was detectable. An 80-kDa protein, cross-reacting with the anti-PHB synthase antibodies raised against the PHB synthase from Ralstonia eutropha, was identified in the crude extract and was strongly enriched by purification of PHB granules. The granule-associated PHB synthase was enzymologically characterized. Enzyme kinetics showed a specific activity of about 4.6 U/mg and Hill plot analysis revealed a K 0.5 of 56 μM with ( R)-3-hydroxybutyryl–CoA employed as substrate. A Hill coefficient of 1.75 indicated that the PHB synthase exhibited positive cooperativity. The thioesters 3-hydroxyvaleryl–CoA, 4-hydroxybutyryl–CoA, and 3-hydroxydecanoyl–CoA were not accepted as substrates. Moreover, the PHB synthase was found to be competitively inhibited by CoA, showing an IC 50 of 160 μM . The PHB synthase was stable up to 60 °C and still exhibited about 90% of the maximum enzyme activity, which was obtained at 40 °C. In contrast to the soluble PHB synthase, the granule-bound PHB synthase was almost independent of the salt concentration. The PHB synthase could not be released from the PHB granules, indicating a covalent attachment to the PHB core. This is the first description of an archaebacterial PHA synthase.

Regulatory effects of cellular nicotinamide nucleotides and enzyme activities on poly(3-hydroxybutyrate) synthesis in recombinant Escherichia coli

Regulatory roles of nicotinamide nucleotides and three key enzymes, beta-ketothiolase (KT), NADPH-dependent acetoacetyl-CoA reductase (AAR), and citrate synthase (CS), on poly(3-hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli harboring a plasmid containing the Alcaligenes eutrophus polyhydroxyalkanoate (PHA) biosynthesis genes were examined. Cells were grown in various media and were subsequently compared for PHB concentration, PHB content, the activities of the key enzymes, and the levels of nicotinamide nucleotides. Cells of recombinant E. coli accumulated the largest amount of PHB in LB+glucose medium among those tested. PHB synthesis was not enhanced by limiting inorganic ions. The activity of CS, which competes with KT for acetyl-CoA, was lower when cells were grown in LB+glucose compared with other media. The NADPH level and the NADPH/NADP ratio were high in LB+glucose. Examination of the time profiles of the specific PHB synthesis rate, key enzyme activities, and the levels of nicotinamide nucleotides showed that PHB synthesis is most significantly affected by the NADPH level. Even though the NADH level and the NADH/NAD ratio were also high during the synthesis of PHB, no direct evidence of their positive effect on PHB synthesis was found. Low activity of CS was beneficial for PHB synthesis due to the availability of more acetyl-CoA to PHB biosynthetic pathway. In recombinant E. coli, the level of NADPH and/or the NADPH/NADP ratio seem to be the most critical factor regulating the activity of AAR and, subsequently, PHB synthesis. (c) 1996 John Wiley & Sons, Inc.

Competition between β-ketothiolase and citrate synthase during poly(β-hydroxybutyrate) synthesis inMethylobacterium rhodesianum

The enzymes beta-ketothiolase and citrate synthase from the facultatively methylotrophic Methylobacterium rhodesianum MB 126, which uses the serine pathway, were purified and characterized. The beta-ketothiolase had a relatively high Km for acetyl-CoA (0.5 mM) and was strongly inhibited by CoA (Ki 0.02 mM). The citrate synthase had a much higher affinity for acetyl-CoA (Km 0.07 mM) and was significantly inhibited by NADH (Ki 0.15 mM). The intracellular concentration of CoA metabolites and nucleotides was determined in M. rhodesianum MB 126 during growth on methanol. The level of CoA decreased from about 0.6 nmol (mg dry mass)-1 during growth to the detection limit when poly(beta-hydroxybutyrate) (PHB) accumulated. Nearly unchanged intracellular concentrations of NADH, NADPH, and acetyl-CoA of about 0.5, 0.6-0.7, and 1.0 nmol (mg dry mass)-1, respectively, were determined during growth and PHB synthesis. During growth, the beta-ketothiolase was almost completely inhibited by CoA, and acetyl-CoA was principally consumed by the citrate synthase. During PHB accumulation, the beta-ketothiolase had about 75% of its maximum activity and showed much higher activity than citrate synthase, which at the actual NADH concentration was about 75% inhibited. NADPH concentration was sufficiently high to allow the unlimited activity of acetoacetyl-CoA reductase (Km NADPH 18 microM). PHB synthesis is probably mainly controlled by the CoA concentration in M. rhodesianum MB 126.

The role of β-oxidation of short-chain alkanoates in polyhydroxyalkanoate copolymer synthesis inAzotobacter vinelandiiUWD

Valerate and other short-chain, uneven-length fatty acids promoted the formation of the polyhydroxyalkanoate (PHA) copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Azotobacter vinelandii UWD growing in glucose medium. The uptake of valerate was inducible, being repressed by acetate but not by glucose. A likely route that would direct valerate into PHA synthesis involved the β-oxidation pathway. The short-chain fatty acids butyrate, valerate, trans.-2-pentenoate, crotonate, hexanoate, heptanoate, and octanoate induced the coordinate production of the β-oxidation enzymes enoyl-CoA hydratase (EGH) and L-(+)-3-hydroxybutyryl-CoA dehydrogenase (HAD).trans-3-Pentenoate was the best inducer of these activities, which suggested that the isomerase of the β-oxidation complex also was present. However, 3-hydroxyacyl-CoA epimerase activity of the β-oxidation complex was not detected. 3-Ketoacyl-CoA thiolase activity was constitutive in A. vinelandii and appeared to associate only loosely with the 73 000 Da ECH–HAD complex. Thus, 3-ketoacyl-CoA, the end product of HAD activity, could be directed into PHA synthesis through acetoacetyl-CoA reductase generating the 3-hydroxyvalerate subunit of the polymer. When valerate was the sole carbon source, the incorporation of valerate into the polymer was normal, but most of the valerate was directed into metabolism and very little PHA was formed. When glucose also was present, the β-oxidation of short-chain alkanoates inhibited the specific activity of acetoacetyl-CoA reductase and 3-ketothiolase and the PHA yield. A model for PHA synthesis was developed that suggests that the use of fatty acids to promote PHA copolymer formation in A. vinelandii will inevitably result in decreased PHA yield.Key words: β-oxidation, poly(β-hydroxyalkanoate) synthesis, short chain fatty acids, regulation.

Formation of d-3-hydroxybutyryl-coenzyme A by an acetoacetyl-coenzyme A reductase in Syntrophomonas wolfei subsp. wolfei

Cell-free extracts of Syntrophomonas wolfei subsp. wolfei synthesized d-(-)-3-hydroxybutyryl-coenzyme A (CoA) (the stereoisomer required for the synthesis of poly-β-hydroxyalkanoate) from acetoacetyl-CoA, but not crotonyl-CoA, and NAD(P)H. Ammonium sulfate fractionation and ion exchange chromatography separated an acetoacetyl-CoA reductase activity that formed d-(-)-3-hydroxybutyryl-CoA from the β-oxidation enzyme activity, l-(+)-3-hydroxyacyl-CoA dehydrogenase. The former activity was further purified by hydroxylapatite and affinity chromatography. The most pure acetoacetyl-CoA reductase preparations formed d-(-)-3-hydroxybutyryl-CoA from acetoacetyl-CoA and had high specific activities using either NADH or NADPH as the electron donor. Thus, S. wolfei makes d-(-)-3-hydroxybutyryl-CoA by an acetoacetyl-CoA reductase rather than by a d-isomer specific enoyl-CoA hydratase and the reducing equivalents required for PHA synthesis from acetoacetyl-CoA can be supplied from the NADH made during β-oxidation.

The regulation of poly- -hydroxybutyrate metabolism in Azospirillum brasilense during balanced growth and starvation

Summary: Activities of the enzymes which are involved in the poly-β-hydroxybutyrate (PHB) cycle, in both synthesis and degradation reactions, were assayed in crude extracts of Azospirillum brasilense cells containing different amounts of PHB. The enzymes of the PHB cycle, of both the synthesis and the degradation process, were more active in PHB-rich cells than in PHB-poor cells. During 96 h of starvation of cells suspended in phosphate buffer, enzymes of the PHB cycle were more active in PHB-rich cells. There was a peak of activity of hydroxybutyrate dehydrogenase (BOHB-DH), β-ketothiolase and thiophorase after 24 h of starvation, due to polymer degradation. During the following hours of starvation there was a decrease in the activity of these enzymes. After 24 h of starvation the activity of acetoacetyl-CoA reductase dropped to a minimum level, because the cells could not synthesize PHB under these conditions. The specific activities of BOHB-DH, β-ketothiolase and thiophorase were higher in A. brasilense cells which were grown under low oxygen tension and consequently accumulated high levels of PHB, than in cells grown under high oxygen tension, with a low PHB content. Similarities to the pathway of PHB biosynthesis and degradation and its control in Azotobacter beijerinckii are described.

Rat hepatic microsomal acetoacetyl-CoA reductase. A beta-ketoacyl-CoA reductase distinct from the long chain beta-ketoacyl-CoA reductase component of the microsomal fatty acid chain elongation system.

The present study provides evidence for a new rat liver microsomal enzyme, a short chain beta-ketoacyl (acetoacetyl)-CoA reductase, which is separate from the long chain beta-ketoacyl-CoA reductase component of the microsomal fatty acid chain elongation system. This microsomal reductase converts acetoacetyl-CoA to beta-hydroxybutyryl-CoA at a rate of 70 nmol/min/mg of protein; the enzyme has a specific requirement for NADH and appears to obtain electrons directly from the reduced pyridine nucleotide without the intervention of cytochrome b5 and its flavoprotein reductase. The apparent Km of the enzyme of the acetoacetyl-CoA was 21 microM and for the cofactor, 18 microM. The pH optimum was broad, ranging from 6.5 to 8.0. The product formed is the D-isomer of beta-hydroxybutyryl-CoA. High carbohydrate fat-free diet resulted in a small but significant (35%) increase in microsomal acetoacetyl-CoA reductase activity. The cytosol also contains this enzyme activity, measuring approximately 57% of that found in the microsomes. The mitochondrial activity which is 20-25% higher than the microsomal activity appears to be due to L-beta-hydroxyacyl-CoA dehydrogenase which converts acetoacetyl-CoA to L-beta-hydroxybutyryl-CoA. The microsomal acetoacetyl-CoA reductase activity was extracted from the microsomal membrane by 0.4 M KCl, resulting in an 8- to 10-fold purification; in addition, the long chain fatty acid elongation system was unaffected by this extraction procedure. Employing beta- hydroxyhexanoyl -CoA as a substrate, evidence is also provided for a separate dehydratase which acts on short chain substrates. Lastly, the liver microsomes had no detectable acetoacetyl-CoA synthetase or acetyl-CoA acetyltransferase activities. Hence, the possible involvement of the rat hepatic microsomal short chain beta-ketoacyl-CoA reductase, short chain beta-hydroxyacyl-CoA dehydratase, and the previously reported short chain trans-2-enoyl-CoA reductase in the hepatic utilization of acetoacetyl-CoA and in the synthesis of butyryl-CoA for hepatic lipogenesis is discussed.

Acetoacetyl-CoA reductase activity of lactating bovine mammary fatty acid synthase.

Fatty acid synthase, purified from lactating bovine mammary gland, utilizes coenzyme A esters of acetoacetic, 3-hydroxybutyric, and crotonic acids as substrates for its partial reactions at micromolar concentrations. The NADPH:acetoacetyl-CoA reductase had a Km of 5 microM acetoacetyl-CoA and a Vmax of about 4 mumol of NADPH oxidized min-1 mg-1. In contrast, the Km for the model compound, acetoacetyl pantetheine was 820 microM and that of S-acetoacetyl-N-acetylcysteamine was over 40 mM. The reduction of acetoacetyl-CoA was observed with the enzyme from rat tissues also but not with those from avian tissues or yeast. With the bovine mammary enzyme, the reaction was found to oxidize 2 mol of NADPH for every mol of acetoacetyl-CoA consumed. Butyrate was the major product of reduction. The reductase activity was susceptible to inhibition by several sulfhydryl reagents; it was lost when the synthase was dissociated into one-half molecular weight subunits or when the incubation mixture was depleted of CoA. It was competitively inhibited by acetyl-CoA, butyryl-CoA, methylmalonyl-CoA, and 2-methylcrotonyl-CoA. These results as well as its use as a primer in fatty acid synthesis by the enzyme suggest that the acetoacetyl group from acetoacetyl-CoA is transferred to the enzyme, presumably to its 4'-phosphopantheine prosthetic group. The acyl group is then expected to remain attached to the enzyme while it is reduced, dehydrated, and reduced again to form a butyryl group which can either undergo chain elongation, if malonyl-CoA is present, or be released from the enzyme by hydrolysis or transfer to free CoA.