The role of NADH- and NADPH-linked acetoacetyl-CoA reductases in the poly-3-hydroxybutyrate synthesizing organism Alcaligenes eutrophus

Haywood, Geoffrey W.

doi:10.1016/0378-1097(88)90372-2

Cited by 72 publications

(107 citation statements)

References 12 publications

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“…The K m for acetoacetyl-CoA determined for the 3-hydroxybutyryl-CoA dehydrogenase of D. biacutus was 18 M, similar to those found for the same enzyme in other PHB-forming bacteria (8,18,29,31). This enzyme, together with the detected 3-enoyl-CoA hydratase (crotonase), is presumably involved in PHB synthesis.…”

supporting

confidence: 77%

Catabolic and anabolic enzyme activities and energetics of acetone metabolism of the sulfate-reducing bacterium Desulfococcus biacutus

Janssen

Schnik

1995

J Bacteriol

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Acetone degradation by cell suspensions of Desulfococcus biacutus was CO 2 dependent, indicating initiation by a carboxylation reaction, while degradation of 3-hydroxybutyrate was not CO 2 dependent. Growth on 3-hydroxybutyrate resulted in acetate accumulation in the medium at a ratio of 1 mol of acetate per mol of substrate degraded. In acetone-grown cultures no coenzyme A (CoA) transferase or CoA ligase appeared to be involved in acetone metabolism, and no acetate accumulated in the medium, suggesting that the carboxylation of acetone and activation to acetoacetyl-CoA may occur without the formation of a free intermediate. Catabolism of 3-hydroxybutyrate occurred after activation by CoA transfer from acetyl-CoA, followed by oxidation to acetoacetyl-CoA. In both acetone-grown cells and 3-hydroxybutyrate-grown cells, acetoacetyl-CoA was thiolytically cleaved to two acetyl-CoA residues and further metabolized through the carbon monoxide dehydrogenase pathway. Comparison of the growth yields on acetone and 3-hydroxybutyrate suggested an additional energy requirement in the catabolism of acetone. This is postulated to be the carboxylation reaction (⌬G؇ for the carboxylation of acetone to acetoacetate, ؉17.1 kJ ⅐ mol ؊1). At the intracellular acyl-CoA concentrations measured, the net free energy change of acetone carboxylation and catabolism to two acetyl-CoA residues would be close to 0 kJ ⅐ mol of acetone ؊1, if one mol of ATP was invested. In the absence of an energy-utilizing step in this catabolic pathway, the predicted intracellular acetoacetyl-CoA concentration would be 10 13 times lower than that measured. Thus, acetone catabolism to two acetyl-CoA residues must be accompanied by the utilization of the energetic equivalent of (at least) one ATP molecule. Measurement of enzyme activities suggested that assimilation of acetyl-CoA occurred through a modified citric acid cycle in which isocitrate was cleaved to succinate and glyoxylate. Malate synthase, condensing glyoxylate and acetyl-CoA, acted as an anaplerotic enzyme. Carboxylation of pyruvate or phosphoenolpyruvate could not be detected.The anaerobic metabolism of acetone appears to involve an initial carboxylation of acetone to acetoacetate (or acetoacetyl coenzyme A [acetoacetyl-CoA]), followed by thiolytic cleavage to two acetyl-CoA residues (3, 32-35). The carboxylation reaction has not been measured in vitro, and evidence has come from 14 C labelling studies (32,33,35). Carboxylation reactions appear to play a role in a number of anaerobic transformations (16, 44), but these reactions have not been well studied. The carboxylation of acetone to acetoacetate, CH 3 COCH 3 ϩ HCO 3), is an endergonic reaction (42). Thus, the energetic equivalent of about 1/3 mol of ATP (⌬GЊЈ of ATP hydrolysis, Ϫ50 kJ ⅐ mol Ϫ1 [42]) is required, but the mechanism of coupling is not known. In addition, attempts to measure the carboxylase in sulfate-reducing bacteria have remained unsuccessful (21).Many genera of sulfate-reducing bacteria are able to oxidize completely (to CO ...

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supporting

confidence: 77%

Catabolic and anabolic enzyme activities and energetics of acetone metabolism of the sulfate-reducing bacterium Desulfococcus biacutus

Janssen

Schnik

1995

J Bacteriol

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“…Lysates were stored on ice while the experiments were conducted. Reductase activity obtained with the use of either NADPH or NADH as the cofactor was measured at pH 6 and 25°C using the published protocol (10). The assay reaction mixtures contained 50 mM potassium phosphate buffer, 0.1 mM NADPH or NADH, and 32 M acetoacetyl-CoA.…”

Section: Methodsmentioning

confidence: 99%

“…Fifteen other potential isologs were also found to encode amino acid sequences that could potentially indicate acetoacetyl-CoA reductase activity (29). The roles of the newly discovered genes in PHB biosynthesis were unclear, especially given the results of an earlier biochemical study that suggested there was a single NADPH-dependent acetoacetyl-CoA reductase in R. eutropha (10). In order to determine the roles of the reductase genes in R. eutropha, we deleted phaB1, phaB2, and phaB3 from the genome both individually and in combination.…”

mentioning

confidence: 99%

Roles of Multiple Acetoacetyl Coenzyme A Reductases in Polyhydroxybutyrate Biosynthesis in Ralstonia eutropha H16

Budde¹,

Mahan²,

Lu³

et al. 2010

J Bacteriol

116

104

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The bacterium Ralstonia eutropha H16 synthesizes polyhydroxybutyrate (PHB) from acetyl coenzyme A (acetyl-CoA) through reactions catalyzed by a ␤-ketothiolase (PhaA), an acetoacetyl-CoA reductase (PhaB), and a polyhydroxyalkanoate synthase (PhaC). An operon of three genes encoding these enzymatic steps was discovered in R. eutropha and has been well studied. Sequencing and analysis of the R. eutropha genome revealed putative isologs for each of the PHB biosynthetic genes, many of which had never been characterized. In addition to the previously identified phaB1 gene, the genome contains the isologs phaB2 and phaB3 as well as 15 other potential acetoacetyl-CoA reductases. We have investigated the roles of the three phaB isologs by deleting them from the genome individually and in combination. It was discovered that the gene products of both phaB1 and phaB3 contribute to PHB biosynthesis in fructose minimal medium but that in plant oil minimal medium and rich medium, phaB3 seems to be unexpressed. This raises interesting questions concerning the regulation of phaB3 expression. Deletion of the gene phaB2 did not result in an observable phenotype under the conditions tested, although this gene does encode an active reductase. Addition of the individual reductase genes to the genome of the ⌬phaB1 ⌬phaB2 ⌬phaB3 strain restored PHB production, and in the course of our complementation experiments, we serendipitously created a PHB-hyperproducing mutant. Measurement of the PhaB and PhaA activities of the mutant strains indicated that the thiolase reaction is the limiting step in PHB biosynthesis in R. eutropha H16 during nitrogen-limited growth on fructose.

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“…The phenotype of this mutant class was referred to as PHB-leaky. PHB-leaky mutants exhibited activities of all three PHBbiosynthetic enzymes; there was also no evidence that isoenzymes of the biosynthetic P-ketothiolase (25) or of the acetoacetyl coenzyme A reductase (26) were affected in these mutants. Measurements with reconstituted enzyme 5844 PRIES ET AL.…”

mentioning

confidence: 91%

Identification and characterization of two Alcaligenes eutrophus gene loci relevant to the poly(beta-hydroxybutyric acid)-leaky phenotype which exhibit homology to ptsH and ptsI of Escherichia coli

et al. 1991

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From genomic libraries of Alcaligenes eutrophus H16 in XL47 and in pVK100, we cloned DNA fragments which restored the wild-type phenotype to poly(j3-hydroxybutyric acid) (PHB)-leaky mutants derived from strains H16 and JMP222. The nucleotide sequence analysis of a 4.5-kb region of one of these fragments revealed two adjacent open reading frames (ORF) which are relevant for the expression of the PHB-leaky phenotype.The 1,799-bp ORF1 represented a gene which was referred to as phbl. The amino acid sequence of the putative protein I (Mr,65,167), which was deduced from phbl, exhibited 38.9% identity with the primary structure of enzyme I of the Escherichia coli phosphoenolpyruvate:carbohydrate phosphotransferase system (PEP-PTS).The upstream 579-bp ORF2 was separated by 50 bp from ORF1. It included the 270-bp phbH gene which encoded protein H (Mrs 9,469). This protein exhibited 34.9% identity to the HPr protein of the E. coli PEP-PTS. Insertions of Tn5 in different PHB-leaky mutants were mapped at eight different positions in phbl and at one position in phbH. Mutants defective in phbH or phbI exhibited no pleiotropic effects and were not altered with respect to the utilization of fructose. However, PHB was degraded at a higher rate in the stationary growth phase. The functions of these HPr-and enzyme I-like proteins in the metabolism of PHB are still unknown. Evidence for the involvement of these proteins in regulation of the metabolism of intracellular PHB was obtained, and a hypothetical model is proposed.The ability to accumulate poly(P-hydroxybutyric acid) (PHB), functioning as a carbon and/or energy source or as a sink for reducing equivalents, is widespread among prokaryotic organisms. The hydrogen-oxidizing bacterium Alcaligenes eutrophus accumulates PHB to more than 80% (wt/wt) of the cellular dry weight (48). PHB and related polyesters are already produced industrially on a small scale by A. eutrophus and are distributed under the trade name Biopol (7,30). Recently, two classes of transposon-induced mutants of A. eutrophus, which are affected in the accumulation of PHB, were isolated (55). PHB-negative mutants were completely impaired in the synthesis of PHB; in this class of mutants TnS::mob (56) mapped within the structural gene of PHB synthase. This fragment encodes the complete A. eutrophus PHB-synthetic pathway and harbors the genes for the biosynthetic 3-ketothiolase (phbA), NADPH-dependent acetoacetyl coenzyme A reductase (phbB), and PHB-synthase (phbC) (39,40,55,58). The genes are organized in one operon (phbCAB) which is transcribed from a &r70-dependent promoter (54, 60). DNA fragments harboring this operon conferred the ability to accumulate PHB to Escherichia coli and to many pseudomonads belonging to rRNA homology group 1 (55, 58, 61, 63, 64 DNA sequence analysis and analysis of sequence data. DNA sequencing was performed by the dideoxy-chain termination method of Sanger et al. (46) with alkali-denatured doublestranded plasmid DNA, 7-deazaguanosine 5'-triphosphate instead of dGTP, and [a-35S]...

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The role of NADH- and NADPH-linked acetoacetyl-CoA reductases in the poly-3-hydroxybutyrate synthesizing organism Alcaligenes eutrophus

Cited by 72 publications

References 12 publications

Catabolic and anabolic enzyme activities and energetics of acetone metabolism of the sulfate-reducing bacterium Desulfococcus biacutus

Catabolic and anabolic enzyme activities and energetics of acetone metabolism of the sulfate-reducing bacterium Desulfococcus biacutus

Roles of Multiple Acetoacetyl Coenzyme A Reductases in Polyhydroxybutyrate Biosynthesis in Ralstonia eutropha H16

Identification and characterization of two Alcaligenes eutrophus gene loci relevant to the poly(beta-hydroxybutyric acid)-leaky phenotype which exhibit homology to ptsH and ptsI of Escherichia coli

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