Crystal Structures of 2-Methylisocitrate Lyase in Complex with Product and with Isocitrate Inhibitor Provide Insight into Lyase Substrate Specificity, Catalysis and Evolution<sup>,</sup>

Liu, Sijiu; Lu, Zuliang; Han, Yuyan; Melamud, Eugene; Dunaway‐Mariano, Debra; Herzberg, Osnat

doi:10.1021/bi0479712

Cited by 32 publications

(108 citation statements)

References 52 publications

(91 reference statements)

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“…The replacement of a Tyr-His pair found in all other lyases with Phe64 and Phe134 in PDP might contribute to its low activity ( Figure 3). The tyrosine hydroxyl group interacts with the substrate, and we previously proposed that the Tyr-His pair may assist proton shuttle (5). It would be interesting to mutate these residues in the future and to determine the effect of the mutations on catalysis.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of the Petal Death Protein from Carnation Flower^,

Teplyakov

Liu

et al. 2005

Biochemistry

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Expression of the PSR132 protein from Dianthus caryophyllus (carnation, clover pink) is induced in response to ethylene production associated with petal senescence, and thus the protein is named petal death protein (PDP). Recent work has established that despite the annotation of PDP in sequence databases as carboxyphosphoenolpyruvate mutase, the enzyme is actually a C-C bond cleaving lyase exhibiting a broad substrate profile. The crystal structure of PDP has been determined at 2.7 Å resolution, revealing a dimer-of-dimers oligomeric association. Consistent with sequence homology, the overall R/ barrel fold of PDP is the same as that of other isocitrate lyase/PEP mutase superfamily members, including a swapped eighth helix within a dimer. Moreover, Mg 2+ binds in the active site of PDP with a coordination pattern similar to that seen in other superfamily members. A compound, covalently bound to the catalytic residue, Cys144, was interpreted as a thiohemiacetal adduct resulting from the reaction of glutaraldehyde used to cross-link the crystals. The Cys144-carrying flexible loop that gates access to the active site is in the closed conformation. Models of bound substrates and comparison with the closed conformation of isocitrate lyase and 2-methylisocitrate lyase revealed the structural basis for the broad substrate profile of PDP.The glyoxylate cycle was discovered in bacteria (1) as a means of converting two-carbon compounds for synthesis of other cell constituents. The same cycle was subsequently found in plants to convert acetyl-CoA from fat degradation into succinate to fuel the tricarboxylic acid cycle (2). The glyoxylate cycle operates in all cells that have the capacity to convert acetate to carbohydrates, including bacteria, plants, fungi, and animals. A key enzyme of the glyoxylate cycle is isocitrate lyase (ICL) 1 which cleaves isocitrate into succinate and glyoxylate. A reaction analogous to that catalyzed by ICL occurs in the metabolism of propionyl-CoA via the 2-methyl isocitrate cycle (3). The corresponding 2-methylisocitrate lyase (MICL) cleaves 2-methylisocitrate into succinate and pyruvate. The amino acid sequences of the two enzymes are homologous and belong to a protein superfamily that also includes phosphoenolpyruvate mutase (PEPM), carboxyphosphoenolpyruvate mutase (carboxyPEPM), phosphonopyruvate hydrolase, oxaloacetate acetylhydrolase, and ketopantoate hydroxymethyl transferase. Even though the reactions catalyzed by these enzymes are different ( Figure 1), they are all proposed to proceed via oxyanion intermediates and/or transition states derived from R-hydroxy-or R-ketocarboxylate substrates (4). The functional diversity within the ICL/PEPM superfamily appears to be even greater than we presently know, because sequence analysis has revealed a number of uncharacterized proteins with potentially different activities (5). The members of the superfamily with known structures, which include PEPM, ICL, MICL, and ketopantoate hydroxymethyltransferase, share a common overall fold...

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Section: Resultsmentioning

confidence: 99%

Crystal Structure of the Petal Death Protein from Carnation Flower^,

Teplyakov

Liu

et al. 2005

Biochemistry

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“…Asn-282 N ␦ anchors the Glu-259 carboxyl group, the group that shares a proton with the DFOA C(4) carboxyl oxygen. A shared proton between two carboxylate groups occurs in the lyase branch of the PEPM/ICL superfamily and is crucial for catalysis (13,16). The DFOA C(4) carboxyl oxygen also interacts with Asn-282 N ␦ .…”

Section: Strainmentioning

confidence: 99%

“…Phylogeny analysis of the PEPM/ICL superfamily highlighted an intriguing clade that contains enzymes with different catalytic activities, in contrast to other phylogeny branches, which include enzymes that perform a single catalytic activity (13). This cluster includes the Petal Death Protein (PDP), shown to be a lyase that cleaves ␣-keto and ␣-hydroxycarboxylic acids with broad substrate specificity (20), carboxy-PEPM (21), 2,3-dimethylmalate lyase (DMML) (14), and OAH (1).…”

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Structure of Oxalacetate Acetylhydrolase, a Virulence Factor of the Chestnut Blight Fungus

Chen

Sun

Narayanan

et al. 2010

Journal of Biological Chemistry

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Oxalacetate acetylhydrolase (OAH), a member of the phosphoenolpyruvate mutase/isocitrate lyase superfamily, catalyzes the hydrolysis of oxalacetate to oxalic acid and acetate. This study shows that knock-out of the oah gene in Cryphonectria parasitica, the chestnut blight fungus, reduces the ability of the fungus to form cankers on chestnut trees, suggesting that OAH plays a key role in virulence. OAH was produced in Escherichia coli and purified, and its catalytic rates were determined. Oxalacetate is the main OAH substrate, but the enzyme also acts as a lyase of (2R,3S)-dimethyl malate with ϳ1000-fold lower efficacy. The crystal structure of OAH was determined alone, in complex with a mechanism-based inhibitor, 3,3-difluorooxalacetate (DFOA), and in complex with the reaction product, oxalate, to a resolution limit of 1.30, 1.55, and 1.65 Å , respectively. OAH assembles into a dimer of dimers with each subunit exhibiting an (␣/␤) 8 barrel fold and each pair swapping the 8th ␣-helix. An active site "gating loop" exhibits conformational disorder in the ligand-free structure. To obtain the structures of the OAH⅐ligand complexes, the ligand-free OAH crystals were soaked briefly with DFOA or oxalacetate. DFOA binding leads to ordering of the gating loop in a conformation that sequesters the ligand from the solvent. DFOA binds in a gem-diol form analogous to the oxalacetate intermediate/transition state. Oxalate binds in a planar conformation, but the gating loop is largely disordered. Comparison between the OAH structure and that of the closely related enzyme, 2,3-dimethylmalate lyase, suggests potential determinants of substrate preference.

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“…PA4872 is a member of the PEP mutase/isocitrate lyase (PEP mutase/isocitrate lyase) superfamily (1). Enzymes of the PEP mutase/isocitrate lyase superfamily catalyze either P-C or C-C bond formation/cleavage.…”

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confidence: 99%

“…An alignment of family sequences identified PA4872 as a representative of a unique clad containing eight members at the time of analysis (now 14 members) and having a novel sequence for the active site gating loop (1). We set forth to discover the reaction catalyzed by PA4872 by employing a functional genomic approach that combines structure determination, activity screening and genome context neighbor analysis.…”

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Structure and Function of PA4872 from Pseudomonas aeruginosa, a Novel Class of Oxaloacetate Decarboxylase from the PEP Mutase/Isocitrate Lyase Superfamily^,

et al. 2007

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Pseudomonas aeruginosa PA4872 was identified by sequence analysis as a structurally and functionally novel member of the PEP mutase/isocitrate lyase superfamily and therefore targeted for investigation. Substrate screens ruled out overlap with known catalytic functions of superfamily members. The crystal structure of PA4872 in complex with oxalate (a stable analog of the shared family α-oxyanion carboxylate intermediate/transition state) and Mg 2+ was determined at 1.9 Å resolution. As with other PEP mutase/isocitrate lyase superfamily members, the protein assembles into a dimer of dimers with each subunit adopting an α/β barrel fold and two subunits swapping their barrel's C-terminal α-helices. Mg 2+ and oxalate bind in the same manner as observed with other superfamily members. The active site gating loop, known to play a catalytic role in the PEP mutase and lyase branches of the superfamily, adopts an open conformation. The N ε of His235, an invariant residue in the PA4872 sequence family, is oriented towards a C(2) oxygen of oxalate analogous to the C(3) of a pyruvyl moiety. Deuterium exchange into α-oxocarboxylate-containing compounds was confirmed by 1 H-NMR spectroscopy. Having ruled out known activities, the involvement of a pyruvate enolate intermediate suggested a decarboxylase activity of an α-oxocarboxylate substrate. Enzymatic assays led to the discovery that PA4872 decarboxylates oxaloacetate (k cat = 7500 s −1 and K m = 2.2 mM) and 3-methyloxaloacetate (k cat = 250 s −1 and K m = 0.63 mM). Genome context of the fourteen sequence family members indicates that the enzyme is used by select group of Gramnegative bacteria to maintain cellular concentrations of bicarbonate and pyruvate; however the decarboxylation activity cannot be attributed to a pathway common to the various bacterial species.

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Crystal Structures of 2-Methylisocitrate Lyase in Complex with Product and with Isocitrate Inhibitor Provide Insight into Lyase Substrate Specificity, Catalysis and Evolution^,

Cited by 32 publications

References 52 publications

Crystal Structure of the Petal Death Protein from Carnation Flower^,

Crystal Structure of the Petal Death Protein from Carnation Flower^,

Structure of Oxalacetate Acetylhydrolase, a Virulence Factor of the Chestnut Blight Fungus

Structure and Function of PA4872 from Pseudomonas aeruginosa, a Novel Class of Oxaloacetate Decarboxylase from the PEP Mutase/Isocitrate Lyase Superfamily^,

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Crystal Structures of 2-Methylisocitrate Lyase in Complex with Product and with Isocitrate Inhibitor Provide Insight into Lyase Substrate Specificity, Catalysis and Evolution,

Cited by 32 publications

References 52 publications

Crystal Structure of the Petal Death Protein from Carnation Flower,

Crystal Structure of the Petal Death Protein from Carnation Flower,

Structure of Oxalacetate Acetylhydrolase, a Virulence Factor of the Chestnut Blight Fungus

Structure and Function of PA4872 from Pseudomonas aeruginosa, a Novel Class of Oxaloacetate Decarboxylase from the PEP Mutase/Isocitrate Lyase Superfamily,

Contact Info

Product

Resources

About

Crystal Structures of 2-Methylisocitrate Lyase in Complex with Product and with Isocitrate Inhibitor Provide Insight into Lyase Substrate Specificity, Catalysis and Evolution^,

Crystal Structure of the Petal Death Protein from Carnation Flower^,

Crystal Structure of the Petal Death Protein from Carnation Flower^,

Structure and Function of PA4872 from Pseudomonas aeruginosa, a Novel Class of Oxaloacetate Decarboxylase from the PEP Mutase/Isocitrate Lyase Superfamily^,