Isolation and Characterization of the Carbon–Phosphorus Bond-forming Enzyme Phosphoenolpyruvate Mutase from the MolluskMytilus edulis

Kim, Alexander; Kim, Jaebong; Martin, Brian M.; Dunaway‐Mariano, Debra

doi:10.1074/jbc.273.8.4443

Cited by 29 publications

(39 citation statements)

References 30 publications

(26 reference statements)

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“…-690, -913, -922 and --525 mV at pH 7.0 (calculated after Weast et al 1988). Therefore, reduction of phosphate for biosynthesis of organophosphonic acids requires significant energy input in the form of ATP (Kim et al 1998). However, in the past, reduction of phosphate to phosphine was repeatedly assumed to occur also as a respiratory process coupled to biomass oxidation (Barrenscheen and Beckh-Widmannstetter 1923;Rudakow 1929;Devai et al 1988;Gassmann and Glindemann 1993), but these reports could never be substantiated in defined cultures, as had to be expected on the basis of the redox potentials mentioned above.…”

Section: Introductionmentioning

confidence: 87%

Desulfotignum phosphitoxidans sp. nov., a new marine sulfate reducer that oxidizes phosphite to phosphate

Schink

Thiemann

Laue

et al. 2002

Archives of Microbiology

105

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A new sulfate-reducing bacterium was isolated from marine sediment with phosphite as sole electron donor and CO 2 as the only carbon source. Strain FiPS-3 grew slowly, with doubling times of 3-4 days, and oxidized phosphite, hydrogen, formate, acetate, fumarate, pyruvate, glycine, glutamate, and other substrates nearly completely, with concomitant reduction of sulfate to sulfide. Acetate was formed as a side product to a small extent. Glucose, arabinose, and proline were partly oxidized and partly fermented to acetate plus propionate. Growth with phosphite, hydrogen, or formate was autotrophic. Also, in the presence of sulfate, CO dehydrogenase was present, and added acetate did not increase growth rates or growth yields. In the absence of sulfate, phosphite oxidation was coupled to homoacetogenic acetate formation, with growth yields similar to those in the presence of sulfate. Cells were small rods, 0.6-0.8×2-4 µm in size, and gram-negative, with a G+C content of 53.9 mol%. They contained desulforubidin, but no desulfoviridin. Based on sequence analysis of the 16S rRNA gene and the sulfite reductase genes dsrAB, strain FiPS-3 was found to be closely related to Desulfotignum balticum. However, physiological properties differed in many points from those of D. balticum. These findings justify the establishment of a new species, Desulfotignum phosphitoxidans.

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Section: Introductionmentioning

confidence: 87%

Desulfotignum phosphitoxidans sp. nov., a new marine sulfate reducer that oxidizes phosphite to phosphate

Schink

Thiemann

Laue

et al. 2002

Archives of Microbiology

105

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“…Despite intense efforts to determine the mechanism of catalysis by this unique enzyme, the pathway for phosphoryl transfer has remained elusive (4)(5)(6)(7)(8)(9)(10)(11). A great deal is known about enzymic and nonenzymic phosphoryl transfer mechanisms (for reviews see refs 12-21), and it is against this backdrop that we present the unusual case of PEP mutase.…”

Section: )mentioning

confidence: 99%

Dissociative Phosphoryl Transfer in PEP Mutase Catalysis: Structure of the Enzyme/Sulfopyruvate Complex and Kinetic Properties of Mutants^,

Liu

Jia

et al. 2002

Biochemistry

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The crystal structure of PEP mutase from Mytilus edulis in complex with a substrate-analogue inhibitor, sulfopyruvate S-pyr (K i ) 22 µM), has been determined at 2.25 Å resolution. Mg(II)-S-pyr binds in the R/ barrel's central channel, at the C-termini of the -strands. The binding mode of S-pyr's pyruvyl moiety resembles the binding mode of oxalate seen earlier. The location of the sulfo group of S-pyr is postulated to mimic the phosphonyl group of the product phosphonopyruvate (P-pyr). This sulfo group interacts with the guanidinium group of Arg159, but it is not aligned for nucleopilic attack by neighboring basic amino side chains. Kinetic analysis of site directed mutants, probing the key active site residues Asp58, Arg159, Asn122, and His190 correlate well with the structural information. The results presented here rule out a phosphoryl transfer mechanism involving a double displacement, and suggest instead that PEP mutase catalysis proceeds via a dissociative mechanism in which the pyruvyl C(3) adds to the same face of the phosphorus from which the C(2)O departs. We propose that Arg159 and His190 serve to hold the phosphoryl/metaphosphate/phosphonyl group stationary along the reaction pathway, while the pyruvyl C(1)-C(2) bond rotates upon formation of the metaphosphate. In agreement with published data, the phosphoryl group transfer occurs on the Si-face of PEP with retention of configuration at phosphorus.We examine here the mechanism of phosphoryl transfer in the conversion of PEP 1 to P-pyr catalyzed by PEP mutase (1, 2):This P-C bond forming reaction serves as the entry point to all known phosphonate biosynthetic pathways (3). Despite intense efforts to determine the mechanism of catalysis by this unique enzyme, the pathway for phosphoryl transfer has remained elusive (4-11). A great deal is known about enzymic and nonenzymic phosphoryl transfer mechanisms (for reviews see refs 12-21), and it is against this backdrop that we present the unusual case of PEP mutase.A phosphoryl transfer may proceed via one of the following pathways: (i) dissociative, in which a trigonal metaphosphate intermediate is formed, (ii) concerted, associative transfer, involving a trigonal bipyramidal transition state, (iii) stepwise associative transfer (otherwise known as the addition-elimination pathway), involving a trigonal bipyramidal intermediate. While the concerted reaction is thought to proceed with the phosphoryl group donor and acceptor positioned in-line on the trigonal bipyramid apical positions, for the mechanisms involving intermediates, both in-line and adjacent attacks are possible.The preferred pathway is determined by the nature of the phosphorus electrophile, the nucleophile, and the reaction medium (solvent or enzyme active site) (see for examples, refs 22 and 23). Knowledge of the stereochemistry of the phosphoryl transfer at phosphorus can aid in the determination of which pathway is operative for a given reaction (see for examples refs 17, 18, and 24). The stereochemistry of phosphoryl transfer ...

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“…Each 1-ml assay contained 50 mM (K ϩ ) HEPES, pH 8.0, 0.5 mM dithiothreitol, 450 nM T. cruzi recombinant PEP mutase, 1 mM phosphonopyruvate, and varying amounts of MgSO 4 . The production of phosphoenolpyruvate was assayed by the increase in absorbance at 233 nm using a ⌬⑀ ϭ 1.5 mM Ϫ1 cm Ϫ1 (20). Data were fitted by nonlinear regression analysis to either the Michaelis-Menten equation (for phosphonopyruvate) or the Hill equation (for Mg 2ϩ ) using the computer program GraFit.…”

Section: Methodsmentioning

confidence: 99%

Properties of Phosphoenolpyruvate Mutase, the First Enzyme in the Aminoethylphosphonate Biosynthetic Pathway in Trypanosoma cruzi

Sarkar

Hamilton

Fairlamb

2003

Journal of Biological Chemistry

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Isolation and Characterization of the Carbon–Phosphorus Bond-forming Enzyme Phosphoenolpyruvate Mutase from the MolluskMytilus edulis

Cited by 29 publications

References 30 publications

Desulfotignum phosphitoxidans sp. nov., a new marine sulfate reducer that oxidizes phosphite to phosphate

Desulfotignum phosphitoxidans sp. nov., a new marine sulfate reducer that oxidizes phosphite to phosphate

Dissociative Phosphoryl Transfer in PEP Mutase Catalysis: Structure of the Enzyme/Sulfopyruvate Complex and Kinetic Properties of Mutants^,

Properties of Phosphoenolpyruvate Mutase, the First Enzyme in the Aminoethylphosphonate Biosynthetic Pathway in Trypanosoma cruzi

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Isolation and Characterization of the Carbon–Phosphorus Bond-forming Enzyme Phosphoenolpyruvate Mutase from the MolluskMytilus edulis

Cited by 29 publications

References 30 publications

Desulfotignum phosphitoxidans sp. nov., a new marine sulfate reducer that oxidizes phosphite to phosphate

Desulfotignum phosphitoxidans sp. nov., a new marine sulfate reducer that oxidizes phosphite to phosphate

Dissociative Phosphoryl Transfer in PEP Mutase Catalysis: Structure of the Enzyme/Sulfopyruvate Complex and Kinetic Properties of Mutants,

Properties of Phosphoenolpyruvate Mutase, the First Enzyme in the Aminoethylphosphonate Biosynthetic Pathway in Trypanosoma cruzi

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Dissociative Phosphoryl Transfer in PEP Mutase Catalysis: Structure of the Enzyme/Sulfopyruvate Complex and Kinetic Properties of Mutants^,