Phosphoenolpyruvate Mutase Catalysis of Phosphoryl Transfer in Phosphoenolpyruvate:  Kinetics and Mechanism of Phosphorus−Carbon Bond Formation

Kim, Jaebong; Dunaway‐Mariano, Debra

doi:10.1021/bi952944k

Cited by 27 publications

(53 citation statements)

References 34 publications

(59 reference statements)

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“…PEP mutase catalyzes the intramolecular rearrangement of PEP to Ppyr. This reaction is thermodynamically unfavorable (K eq $ 1 Â 10 À3 ) [9]. The ensuing decarboxylation step catalyzed by Ppyr decarboxylase is required to drive the Ppyr-forming reaction forward.…”

Section: Resultsmentioning

confidence: 99%

Enzymatic synthesis of radiolabeled phosphonoacetaldehyde

Allen

Dunaway‐Mariano

2003

Analytical Biochemistry

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Phosphonoacetaldehyde (Pald) is formed in a variety of biosynthetic pathways leading to natural phosphonates and is an intermediate in the degradation pathway of the natural product 2-aminoethylphosphonate. To facilitate the investigation of the enzymes catalyzing these pathways, a method for the synthesis of radiolabeled Pald was developed. The enzyme pyruvate phosphate dikinase was used to prepare phosphoenolpyruvate (PEP) from pyruvate, adenosine triphosphate (ATP), and orthophosphate. Then PEP was converted to phosphonopyruvate (Ppyr) with PEP mutase and then to Pald with Ppyr decarboxylase. By using

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Section: Resultsmentioning

confidence: 99%

Enzymatic synthesis of radiolabeled phosphonoacetaldehyde

Allen

Dunaway‐Mariano

2003

Analytical Biochemistry

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“…side chain of His, Asp, Ser, Thr, Tyr, Cys, or Lys) (16) A structural-based approach was taken to determine whether the active site contains a nucleophilic residue for in-line attack at phosphorus consistent with the double displacement mechanism. The crystal structure of the PEP mutase from the mollusk Mytilus edulis was determined in the presence of Mg(II)-oxalate (10), a tight binding competitive inhibitor (K i ) 25-32 µM for the T. pyriformis PEP mutase (7,8) and K i ) 8.4 µM for the M. edulis PEP mutase (this work)) analogous to pyruvate enolate. Because oxalate is a symmetrical molecule, the location of the PEP's phosphoryl group could be inferred only by considering the active site environment.…”

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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“…In spite of a lack of significant sequence identity between KPHMT and either of the two enzymes (Table 1), structure-based sequence alignment with COMPARER revealed remarkable similarities of both the locations and the properties of important active-site residues. PEPM from Mytilus edulis is a homotetrameric (dimer of dimers) enzyme that catalyzes the conversion of PEP to phosphonopyruvate in the biosynthesis of phosphonates involving cleavage of an OOP bond and formation of a COP bond (10,15). The overall globular architecture of the (␤␣) 8 -barrel is very similar to that in KPHMT.…”

Section: Resultsmentioning

confidence: 99%

Comparative Analysis of the Escherichia coli Ketopantoate Hydroxymethyltransferase Crystal Structure Confirms that It Is a Member of the (βα) ₈ Phosphoenolpyruvate/Pyruvate Superfamily

Schmitzberger

Smith²,

Abell³

et al. 2003

J Bacteriol

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Escherichia coli ketopantoate hydroxymethyltransferase (KPHMT) catalyzes the first step in the biosynthesis pathway of pantothenate (vitamin B 5 ), the transfer of a hydroxymethyl group onto ␣-ketoisovalerate. Here we describe a detailed comparative analysis of the KPHMT crystal structure and the identification of structural homologues, some of which have remarkable similarities in their active sites, modes of binding to substrates, and mechanisms. We show that KPHMT forms a family within the phosphoenolpyruvate/pyruvate superfamily. Based on the analysis, we propose that in this superfamily there should be a subdivision into two groups. This paper completes our structural analysis of the E. coli enzymes in the pantothenate pathway.Pantothenate (vitamin B 5 ) is a vital and central metabolic compound in all organisms. It constitutes the invariable precursor of phosphopantetheine, the moiety of coenzyme A and the acyl carrier protein. However, pantothenate is synthesized only in bacteria, fungi, and plants; animals have lost the corresponding biosynthetic pathway. Consequently, enzymes of the pathway are considered attractive antimicrobial, fungicidal, and herbicidal targets. In Escherichia coli the pantothenate pathway comprises four enzymes ( Fig. 1) (3). Ketopantoate hydroxymethyltransferase (KPHMT; EC 2.1.2.11) catalyzes the first committed reaction, the transfer of the C 11 carbon of 5,10-methylene tetrahydrofolate (THF) onto ␣-ketoisovalerate (␣-KIVA) to form ketopantoate. Subsequently, ketopantoate reductase (EC 1.1.1.169; Protein Data Bank [PDB] code 1ks9) reduces ketopantoate to pantoate, using NADPH as a cofactor. Concomitantly, in a separate branch of the pathway L-aspartate-␣-decarboxylase (EC 4.1.1.11; PDB code 1aw8) converts aspartate to ␤-alanine, which is finally coupled to ATP-activated pantoate by pantothenate synthetase (EC 6.3.2.1; PDB code 1iho).The crystal structure of E. coli KPHMT (PDB code 1m3u) was solved to 1.9-Å resolution in complex with Mg 2ϩ and the product ketopantoate (30). KPHMT adopts a homodecameric quaternary structure. Based on the analysis of both the hydrophobic and polar interactions and the buried surface area between the subunits, it is best described as a pentamer of dimers. The protomeric tertiary structure of the subunits is a classical (␤␣) 8 -or triosephosphate isomerase barrel fold with some minor but important variations (Fig. 2). The helix, usually found between ␤-strands 7 and 8, is here replaced by a loop, and an additional N-terminal ␣-helix preceding the usual first ␤-strand in the sequence is located at the base of the ␤-barrel. The subunits are arranged in an orientation such that the vertical axis of the (␤␣) 8 -barrel is perpendicular to the fivefold axis of the decamer. In the active site of KPHMT, magnesium is coordinated in an octahedral sphere with ketopantoate, occupying an axial and an equatorial coordination site ( Fig. 2a; see also Fig. 5a).Although KPHMT belongs to the common (␤␣) 8 -or triosephosphate isomerase barrel fold, sequence analysis fai...

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Phosphoenolpyruvate Mutase Catalysis of Phosphoryl Transfer in Phosphoenolpyruvate: Kinetics and Mechanism of Phosphorus−Carbon Bond Formation

Cited by 27 publications

References 34 publications

Enzymatic synthesis of radiolabeled phosphonoacetaldehyde

Enzymatic synthesis of radiolabeled phosphonoacetaldehyde

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

Comparative Analysis of the Escherichia coli Ketopantoate Hydroxymethyltransferase Crystal Structure Confirms that It Is a Member of the (βα) ₈ Phosphoenolpyruvate/Pyruvate Superfamily

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Phosphoenolpyruvate Mutase Catalysis of Phosphoryl Transfer in Phosphoenolpyruvate: Kinetics and Mechanism of Phosphorus−Carbon Bond Formation

Cited by 27 publications

References 34 publications

Enzymatic synthesis of radiolabeled phosphonoacetaldehyde

Enzymatic synthesis of radiolabeled phosphonoacetaldehyde

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

Comparative Analysis of the Escherichia coli Ketopantoate Hydroxymethyltransferase Crystal Structure Confirms that It Is a Member of the (βα) 8 Phosphoenolpyruvate/Pyruvate Superfamily

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

Comparative Analysis of the Escherichia coli Ketopantoate Hydroxymethyltransferase Crystal Structure Confirms that It Is a Member of the (βα) ₈ Phosphoenolpyruvate/Pyruvate Superfamily