Characterization of two 3-ketothiolases possessing differing substrate specificities in the polyhydroxyalkanoate synthesizing organism Alcaligenes eutrophus

Haywood, Geoffrey W.

doi:10.1016/0378-1097(88)90306-0

Cited by 47 publications

(67 citation statements)

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“…Haywood et al reported that 3 to 10% of the monomeric units of PHA accumulated from hexanoic acid are one or more C 2 units longer than the substrate used (10,13). It was suggested that the presence of these longer monomers is due to a condensation reaction of acyl-CoA molecules with acetyl-CoA catalyzed by the ␤-oxidation enzyme 3-ketothiolase (10). An additional copy of fadD in P. putida CA-3 could result in the production of higher concentrations of both fatty acyl-CoA esters and acetyl-CoA due to an increased flux through ␤ oxidation.…”

Section: Discussionmentioning

confidence: 99%

FadD from Pseudomonas putida CA-3 Is a True Long-Chain Fatty Acyl Coenzyme A Synthetase That Activates Phenylalkanoic and Alkanoic Acids

2009

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A fatty acyl coenzyme A synthetase (FadD) from Pseudomonas putida CA-3 is capable of activating a wide range of phenylalkanoic and alkanoic acids. It exhibits the highest rates of reaction and catalytic efficiency with long-chain aromatic and aliphatic substrates. FadD exhibits higher k cat and K m values for aromatic substrates than for the aliphatic equivalents (e.g., 15-phenylpentadecanoic acid versus pentadecanoic acid). FadD is inhibited noncompetitively by both acrylic acid and 2-bromooctanoic acid. The deletion of the fadD gene from P. putida CA-3 resulted in no detectable growth or polyhydroxyalkanoate (PHA) accumulation with 10-phenyldecanoic acid, decanoic acid, and longer-chain substrates. The results suggest that FadD is solely responsible for the activation of long-chain phenylalkanoic and alkanoic acids. While the CA-3⌬fadD mutant could grow on medium-chain substrates, a decrease in growth yield and PHA accumulation was observed. The PHA accumulated by CA-3⌬fadD contained a greater proportion of short-chain monomers than did wild-type PHA. Growth of CA-3⌬fadD was unaffected, but PHA accumulation decreased modestly with shorter-chain substrates. The complemented mutant regained 70% to 90% of the growth and PHA-accumulating ability of the wild-type strain depending on the substrate. The expression of an extra copy of fadD in P. putida CA-3 resulted in increased levels of PHA accumulation (up to 1.6-fold) and an increase in the incorporation of longermonomer units into the PHA polymer.Fatty acyl coenzyme A (CoA) synthetases (FACS; fatty acid: CoA ligases; EC 6.2.1.3) are ATP-, CoA-, and Mg 2ϩ -dependent enzymes that activate alkanoic acids to CoA esters for ␤ oxidation ( Fig. 1) (2, 17). FACS are widely distributed in both prokaryotic and eukaryotic organisms and exhibit a broad substrate specificity (34). FadD is a cytoplasmic membrane-associated FACS (7), with sizes ranging from 47 kDa to 62 kDa (2, 14). There is a lack of biochemical information on FadD with a preference for long-chain aromatic and aliphatic substrates. In the current study we purify and characterize for the first time a true long-chain FadD with activity toward both phenylalkanoic and alkanoic acids.It is known that bacteria such as Pseudomonas putida can accumulate the biological polyester polyhydroxyalkanoate (PHA) from aromatic as well as aliphatic alkanoic acids (5, 6, 42, 45). The presence of aromatic monomers in the PHA polymer suggests that a FadD with activity toward aromatic substrates is present in these PHA-accumulating strains. Garcia et al. knocked out an acyl-CoA synthetase in P. putida U with a high homology to long-chain fadD products from Escherichia coli and Pseudomonas fragi (6). Garcia et al. also showed that the mutant was not capable of growth or PHA accumulation with aromatic and aliphatic substrates having between 5 and 10 carbons in their acyl chain, indicating that it is a general and not a long-chain acyl-CoA ligase (6). In a follow-up study, Olivera et al. showed that the fadD mutants reverted to wild-t...

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Section: Discussionmentioning

confidence: 99%

FadD from Pseudomonas putida CA-3 Is a True Long-Chain Fatty Acyl Coenzyme A Synthetase That Activates Phenylalkanoic and Alkanoic Acids

2009

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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.…”

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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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“…In both Zoogloea ramigera (8,9,15,16,25,27,28) and Alcaligenes eutrophus (19,20), the condensation of two acetyl coenzyme A (CoA) units to form acetoacetyl-CoA by the enzyme 3-ketothiolase is followed by a stereospecific reduction step catalyzed by an NADP+-specific acetoacetyl-CoA reductase to form D-3-hydroxybutyryl-CoA, the substrate for PHB synthase. Both the 13-ketothiolase and acetoacetyl-CoA reductase are soluble cytosolic enzymes, whereas the PHB synthase is found attached to the PHB granules.…”

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confidence: 99%

Immunocytochemical analysis of poly-beta-hydroxybutyrate (PHB) synthase in Alcaligenes eutrophus H16: localization of the synthase enzyme at the surface of PHB granules

et al. 1993

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Antibodies raised against the Alcaligenes eutrophus poly-1-hydroxybutyrate (PHB) synthase polypeptide were used for immunocytochemical localization of the synthase enzyme in whole cells and purified PHB granules. The data presented demonstrate for the first time that the synthase enzyme is located on the surface of the PHB granule rather than being incorporated inside the granule during its formation. From these basic observations and data from the recent literature, a model of granule assembly is proposed.Poly-p-hydroxybutyrate (PHB) accumulates as granular inclusions in the cells of a large number of bacterial species grown under conditions of nutrient limitation (2). In both Zoogloea ramigera (8,9,15,16,25,27,28) and Alcaligenes eutrophus (19,20), the condensation of two acetyl coenzyme A (CoA) units to form acetoacetyl-CoA by the enzyme 3-ketothiolase is followed by a stereospecific reduction step catalyzed by an NADP+-specific acetoacetyl-CoA reductase to form D-3-hydroxybutyryl-CoA, the substrate for PHB synthase. Both the 13-ketothiolase and acetoacetyl-CoA reductase are soluble cytosolic enzymes, whereas the PHB synthase is found attached to the PHB granules.Purified granules contain around 98% PHB by weight, with the remainder being made up of proteins and lipids (17,22). It was previously concluded that the physical state of the polymer inside the granules in vivo was that of a crystalline solid, similar to that of the extracted polymer (12,14,21). More recently, nuclear magnetic resonance studies of whole cells have provided the first evidence that the "granule" is in fact in a mobile, amorphous state in vivo (3,4). Both the presence of plasticizers including water (4, 18) or lipid (22) and the kinetics of the crystallization process (6, 10) have been proposed to describe how the granules are maintained in the amorphous state inside the cell. Additional studies have concluded that the PHB granules inside the cell are surrounded by a membrane containing the synthase enzyme and that the D-3-hydroxybutyryl-CoA monomers are transported across the membrane in conjunction with polymerization (5, 13, 24, 32).Our current research activities are focused on understanding the role of the PHB synthase in the biogenesis of the PHB granules in A. eutrophus. In this regard there are two main issues: (i) the mechanism by which the PHB synthase initiates and carries out the condensation of literally thousands of D-3-hydroxybutyryl-CoA units to form the polymer chains and (ii) the role of this mechanism and the PHB synthase protein in the assembly of the PHB granules. We report here the immunocytochemical localization of the PHB synthase on the surface of the granules and describe a working hypothesis for granule formation. * Corresponding author.Preparation and analysis of anti-PHB synthase antibodies. In order to obtain purified A. eutrophus PHB synthase protein for the purpose of preparing antibodies, the C-terminal region of the phbC gene from plasmid pAeT42 encoding residues 86 to 589 of the PHB synthase (26) was ...

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Characterization of two 3-ketothiolases possessing differing substrate specificities in the polyhydroxyalkanoate synthesizing organism Alcaligenes eutrophus

Cited by 47 publications

References 5 publications

FadD from Pseudomonas putida CA-3 Is a True Long-Chain Fatty Acyl Coenzyme A Synthetase That Activates Phenylalkanoic and Alkanoic Acids

FadD from Pseudomonas putida CA-3 Is a True Long-Chain Fatty Acyl Coenzyme A Synthetase That Activates Phenylalkanoic and Alkanoic Acids

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

Immunocytochemical analysis of poly-beta-hydroxybutyrate (PHB) synthase in Alcaligenes eutrophus H16: localization of the synthase enzyme at the surface of PHB granules

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