Helix swapping between two α/β barrels: crystal structure of phosphoenolpyruvate mutase with bound Mg2+–oxalate

Huang, Kangning; Li, Zhong; Jia, Yafei; Dunaway‐Mariano, Debra; Herzberg, Osnat

doi:10.1016/s0969-2126(99)80070-7

Cited by 41 publications

(78 citation statements)

References 43 publications

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“…Access to the active site is further blocked by two loops (colored red in Figure 3). The desolvation of the active site may be required to prevent PEP hydrolysis, while substrate binding and dissociation must be accompanied by conformational changes of the capping structural elements (10).…”

Section: Resultsmentioning

confidence: 99%

“…The structure of the PEP mutase in complex with Mg-(II)-oxalate ( Figure 1) may be considered a model of the reaction intermediate minus the metaphosphate (10). The increased electrostatic interaction that might take place between charged pyruvate enolate and the guanidinium group of Arg159, the Mg(II), and the oxyanion hole provides a significant source of stabilization.…”

Section: Resultsmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

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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“…Even though the reactions catalyzed by these enzymes are different, they are all proposed to proceed via mechanisms that stabilize enol(ate) intermediates (32). Based on the similarities shared by these enzymes, it has been proposed that these proteins represent a new ␣/␤-barrel superfamily (19). The ␣/␤-barrel proteins are known to be a very diverse group of enzymes that may catalyze different reactions by binding substrate differently inside the barrel.…”

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“…The ␣/␤-barrel proteins are known to be a very diverse group of enzymes that may catalyze different reactions by binding substrate differently inside the barrel. The evolutionary link between the mutases and the lyases is strengthened by the fact that the key active-site residues required for PEP mutase catalysis are invariant in the lyases (19). Although the PrpB enzyme catalyzes a reaction similar to that catalyzed by ICL, the PrpB enzyme shares more sequence identity with CPEP and PEP mutases (36 and 30% end-to-end identity, respectively) than with ICL (ca.…”

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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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“…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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Helix swapping between two α/β barrels: crystal structure of phosphoenolpyruvate mutase with bound Mg2+–oxalate

Cited by 41 publications

References 43 publications

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

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

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

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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Helix swapping between two α/β barrels: crystal structure of phosphoenolpyruvate mutase with bound Mg2+–oxalate

Cited by 41 publications

References 43 publications

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

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

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

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^,

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