Carboxyphosphonoenolpyruvate phosphonomutase, a novel enzyme catalyzing C-P bond formation

Hidaka, Tomomi; Imai, Satoshi; Hara, Osamu; Anzai, Hiroyuki; Murakami, Takeshi; Nagaoka, Kozo; Seto, Haruo

doi:10.1128/jb.172.6.3066-3072.1990

Cited by 49 publications

(32 citation statements)

References 16 publications

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“…(i) PrpB. The amino acid sequence of PrpB showed homology to the carboxyphosphonoenolpyruvate phosphonomutase (CPEP mutase) of Streptomyces hygroscopicus (14,24). The CPEP mutase and PrpB proteins were the same length and showed 35% end-to-end identity (Fig.…”

Section: Resultsmentioning

confidence: 99%

Propionate catabolism in Salmonella typhimurium LT2: two divergently transcribed units comprise the prp locus at 8.5 centisomes, prpR encodes a member of the sigma-54 family of activators, and the prpBCDE genes constitute an operon

1997

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We present the initial genetic and biochemical characterization of the propionate (prp) locus at 8.5 centisomes of the Salmonella typhimurium LT2 chromosome (T. A. Hammelman et al., FEMS Microbiol. Lett. 137: 233-239, 1996). In this paper, we report the nucleotide sequences of two divergently transcribed transcriptional units. One unit is comprised of the prpR gene (1,626 bp) encoding a member of the sigma-54 family of transcriptional activators; the second unit contains an operon of four genes designated prpB (888 bp), prpC (1,170 bp), prpD (1,452 bp), and prpE (1,923 bp). The heme biosynthetic gene hemB was shown by DNA sequencing to be located immediately downstream of the prpBCDE operon; hemB is divergently transcribed from prpBCDE and is separated from prpE by a 66-bp gap. In addition, we demonstrate the involvement of PrpB, PrpC, and PrpD in propionate catabolism by complementation analysis of mutants using plasmids carrying a single prp gene under the control of the arabinose-responsive P BAD promoter. Expression of prpB to high levels was deleterious to the growth of a prp ؉ strain on minimal medium supplemented with propionate as a carbon and energy source. We also report the cloning and overexpression of prpB, prpC, prpD, and prpE in the T7 system. PrpB, PrpC, PrpD, and PrpE had molecular masses of ca. 32, ca. 44, ca. 53, and ca. 70 kDa, respectively. PrpB showed homology to carboxyphosphonoenolpyruvate phosphonomutase of Streptomyces hygroscopicus and to its homolog in the carnation Dianthus caryophyllus; PrpC was homologous to both archaeal and bacterial citrate synthases; PrpD showed homology to yeast and Bacillus subtilis proteins of unknown function; PrpE showed homology to acetyl coenzyme A synthetases. We identified a sigma-54 (RpoN)-dependent promoter with a consensus RpoN binding site upstream of the initiating methionine codon of prpB, the promoter-proximal gene of the prp operon. Consistent with this finding, an rpoN prp ؉ mutant failed to use propionate as carbon and energy source. Finally, we report the location of MudI1734 elements inserted in prpC or prpD and of a Tn10⌬16⌬17 element in prpB and provide genetic evidence supporting the conclusion that the prpBCDE genes constitute an operon.

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Section: Resultsmentioning

confidence: 99%

Propionate catabolism in Salmonella typhimurium LT2: two divergently transcribed units comprise the prp locus at 8.5 centisomes, prpR encodes a member of the sigma-54 family of activators, and the prpBCDE genes constitute an operon

1997

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“…This is particularly true with regard to the biochemical characterization of enzymes involved in reduced phosphorus metabolism. A few of the enzymes involved in the biosynthesis of the reduced phosphorus antibiotic bialaphos (3,11,12) as well as the enzyme phosphoenolpyruvate phosphonomutase from Tetrahymena (13) have been purified and characterized. However, these carbonphosphorus bond-synthesizing enzymes catalyze phosphorus reduction indirectly via intramolecular rearrangements; they do not catalyze direct redox reactions of phosphorus moieties.…”

Section: ؊1mentioning

confidence: 99%

Purification and Characterization of a Novel Phosphorus-oxidizing Enzyme from Pseudomonas stutzeri WM88

Costas¹,

White²,

Metcalf

2001

Journal of Biological Chemistry

153

155

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The ptxD gene from Pseudomonas stutzeri WM88 encoding the novel phosphorus oxidizing enzyme NAD:phosphite oxidoreductase (trivial name phosphite dehydrogenase, PtxD) was cloned into an expression vector and overproduced in Escherichia coli. The heterologously produced enzyme is indistinguishable from the native enzyme based on mass spectrometry, amino-terminal sequencing, and specific activity analyses. Recombinant PtxD was purified to homogeneity via a two-step affinity protocol and characterized. The enzyme stoichiometrically produces NADH and phosphate from NAD and phosphite. The reverse reaction was not observed. Gel filtration analysis of the purified protein is consistent with PtxD acting as a homodimer. PtxD has a high affinity for its substrates with K m values of 53.1 ؎ 6.7 M and 54.6 ؎ 6.7 M, for phosphite and NAD, respectively. V max and k cat were determined to be 12.2 ؎ 0.3 mol min ؊1 mg ؊1 and 440 min ؊1. NADP can substitute poorly for NAD; however, none of the numerous compounds examined were able to substitute for phosphite. Initial rate studies in the absence or presence of products and in the presence of the dead end inhibitor sulfite are most consistent with a sequential ordered mechanism for the PtxD reaction, with NAD binding first and NADH being released last. Amino acid sequence comparisons place PtxD as a new member of the D-2-hydroxyacid NAD-dependent dehydrogenases, the only one to have an inorganic substrate. To our knowledge, this is the first detailed biochemical study on an enzyme capable of direct oxidation of a reduced phosphorus compound.Phosphorus is widely reported to be a redox conservative element in biological systems, with the sum total of phosphorus biochemistry consisting of the formation and hydrolysis of phosphate-ester bonds. These reports imply that reduced phosphorus compounds are not important in living systems and that enzymatically catalyzed redox reactions of phosphorus compounds do not occur; however, an increasing body of evidence indicates that this is not the case. Although it is true that inorganic phosphate (P valence ϩ5) is the principal form of phosphorus in living systems and that phosphate-esters play a critical role in phosphate biochemistry, it is now clear that reduced phosphorus compounds of both natural and xenobiotic origin play important roles in numerous biological systems. Accordingly, many organisms have been shown to possess metabolic pathways for reduction of phosphate to a variety of reduced phosphorus compounds (1-3); others have been shown to possess metabolic pathways for oxidation of reduced phosphorus compounds (4 -9). Among the most striking of these is a recently isolated sulfate-reducing bacterium that obtains all of the energy it requires for growth from the oxidation of phosphite (ϩ3 valence) to phosphate (10).Unfortunately, detailed studies examining the mechanisms of biological phosphorus oxidation and reduction are scarce. This is particularly true with regard to the biochemical characterization of enzymes involved in reduced p...

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“…The mutases are anabolic enzymes used for the production of secondary metabolites and the only enzymes known to create a carbon-phosphorus bond (13,27), while the lyases are catabolic enzymes (18,24,43). Even though the reactions catalyzed by these enzymes are different, they are all proposed to proceed via mechanisms that stabilize enol(ate) intermediates (32).…”

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confidence: 99%

Residues C123 and D58 of the 2-Methylisocitrate Lyase (PrpB) Enzyme ofSalmonella entericaAre Essential for Catalysis

Grimek¹,

Holden

Rayment

et al. 2003

J Bacteriol

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The prpB gene of Salmonella enterica serovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits. Optimal 2-MIC lyase activity was measured at pH 7.5 and 50°C, and the reaction required Mg 2؉ ions; equimolar concentrations of Mn 2؉ ions were a poor substitute for Mg 2؉ (28% specific activity). Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH. The K m of PrpB for 2-MIC was measured at 19 M, with a k cat of 105 s ؊1 . Mutations in the prpB gene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes. Residues D58, K121, C123, and H125 of PrpB were changed to alanine, and residue R122 was changed to lysine. Nondenaturing polyacrylamide gel electrophoresis indicated that all mutant PrpB proteins retained the same oligomeric state of the wild-type enzyme, which is known to form tetramers. The PrpB K121A , PrpB H125A , and PrpB R122K mutant proteins formed enzymes that had 1,050-, 750-, and 2-fold decreases in k cat for 2-MIC lyase activity, respectively. The PrpB D58A and PrpB C123A proteins formed tetramers that displayed no detectable 2-MIC lyase activity indicating that both of these residues are essential for catalysis. Based on the proposed mechanism of the closely related isocitrate lyases, PrpB residue C123 is proposed to serve as the active site base, and residue D58 is critical for the coordination of a required Mg 2؉ ion.Propionate catabolism in Salmonella enterica serovar Typhimurium LT2 (herein referred to as serovar Typhimurium) occurs via the 2-methylcitric acid (2-MC) cycle (Fig. 1) (18). The 2-MC cycle of propionate metabolism was first identified in the yeast Yarrowia lipolytica and several years later was found to exist in prokaryotes (8,18,36,37). The prokaryotic enzymes of this pathway have been characterized. PrpE is the propionyl coenzyme A synthetase (14, 17), PrpC is the 2-MC synthase (18, 39), PrpD is the 2-MC dehydratase (8, 15), and PrpB is the 2-methylisocitrate (2-MIC) lyase (7,18). This work focuses on the serovar Typhimurium PrpB enzyme, which catalyzes the cleavage of 2-MIC into pyruvate and succinate (15).Comparisons of the amino acid sequence of the PrpB protein to other protein sequences identified it as a homolog of carboxyphosphonoenolpyruvate (CPEP) mutase, phosphoenolpyruvate (PEP) mutase, and isocitrate lyases (ICL) ...

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Carboxyphosphonoenolpyruvate phosphonomutase, a novel enzyme catalyzing C-P bond formation

Cited by 49 publications

References 16 publications

Propionate catabolism in Salmonella typhimurium LT2: two divergently transcribed units comprise the prp locus at 8.5 centisomes, prpR encodes a member of the sigma-54 family of activators, and the prpBCDE genes constitute an operon

Propionate catabolism in Salmonella typhimurium LT2: two divergently transcribed units comprise the prp locus at 8.5 centisomes, prpR encodes a member of the sigma-54 family of activators, and the prpBCDE genes constitute an operon

Purification and Characterization of a Novel Phosphorus-oxidizing Enzyme from Pseudomonas stutzeri WM88

Residues C123 and D58 of the 2-Methylisocitrate Lyase (PrpB) Enzyme ofSalmonella entericaAre Essential for Catalysis

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