The crystal structure and active site location of isocitrate lyase from the fungus Aspergillus nidulans

Britton, K.L.; Langridge, S.J.; Baker, Patrick J.; Weeradechapon, K.; Sedelnikova, S.E.; Lucas, JR De; Dw, Rice; Turner, Geoffrey

doi:10.1016/s0969-2126(00)00117-9

Cited by 69 publications

(63 citation statements)

References 53 publications

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“…This is caused by a linker region between domains I and II in the eukaryotic isocitrate lyases. This linker region might be responsible for sequestration to the peroxisomal microbodies and is therefore not necessary in the bacterial enzyme [18]. However, 2-methylisocitrate lyase from E. coli has an even 35% lower molecular mass than the bacterial isocitrate lyases.…”

Section: Discussionmentioning

confidence: 99%

2‐Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans

Brock

Darley

Textor

et al. 2001

European Journal of Biochemistry

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In Escherichia coli and Aspergillus nidulans, propionate is oxidized to pyruvate via the methylcitrate cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate is catalysed by 2-methylisocitrate lyase. The enzymes from both organisms were assayed with chemically synthesized threo-2-methylisocitrate; the erythrodiastereomer was not active. 2-Methylisocitrate lyase from E. coli corresponds to the PrpB protein of the prp operon involved in propionate oxidation. The purified enzyme has a molecular mass of approximately 32 kDa per subunit, which is lower than those of isocitrate lyases from bacterial sources (< 48 kDa). 2-Methylisocitrate lyase from A. nidulans shows an apparent molecular mass of 66 kDa per subunit, almost equal to that of isocitrate lyase of the same organism. Both 2-methylisocitrate lyases have a native homotetrameric structure as identified by size-exclusion chromatography. The enzymes show no measurable activity with isocitrate. Starting from 250 mm pyruvate, 150 mm succinate and 10 mm PrpB, the enzymatically active stereoisomer could be synthesized in 1% yield. As revealed by chiral HPLC, the product consisted of a single enantiomer. This isomer is cleaved by 2-methylisocitrate lyases from A. nidulans and E. coli. The PrpB protein reacted with stoichiometric amounts of 3-bromopyruvate whereby the activity was lost and one amino-acid residue per subunit became modified, most likely a cysteine as shown for isocitrate lyase of E. coli. PrpB exhibits 34% sequence identity with carboxyphosphoenolpyruvate phosphonomutase from Streptomyces hygroscopicus, in which the essential cysteine residue is conserved.

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Section: Discussionmentioning

confidence: 99%

2‐Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans

Brock

Darley

Textor

et al. 2001

European Journal of Biochemistry

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“…Domain II forms a peripheral head to the subunit. 16 Most sites identified as evolving under positive selection were located between sites 293 and 371 in the protein alignment (Fig. 2), which match with residues belonging to domain II (Fig.…”

Section: Introductionmentioning

confidence: 86%

Identification of positive selection in disease response genes within members of the Poaceae

Rech

Vargas²,

Sukno

et al. 2012

Plant Signaling & Behavior

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“…4C). The bottom of each (␣/␤) 8 barrel is flanked by an additional ␣-helix. Following a barrel 8th helix, the polypeptide chain meanders to form two more ␣-helices and traverses the top of the (␣/␤) 8 barrel of the partner above its active site entrance (indicated as "partner subunit cap" in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Members of this superfamily act on ␣-oxycarboxylate substrates to cleave/form C-C or P-C bonds. All PEPM/ICL superfamily members are oligomeric proteins, mostly tetramers, and each subunit adopts an (␣/␤) 8 barrel fold. The tetramer is composed of a dimer of dimers arranged such that the two molecules within a dimer swap the 8th helix of the ␣/␤ barrel.…”

mentioning

confidence: 99%

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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The crystal structure and active site location of isocitrate lyase from the fungus Aspergillus nidulans

Cited by 69 publications

References 53 publications

2‐Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans

2‐Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans

Identification of positive selection in disease response genes within members of the Poaceae

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

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