Isocitrate lyase from<i>Aspergillus nidulans</i>: crystallization and X-ray analysis of a glyoxylate cycle enzyme

Langridge, S.J.; Baker, Patrick J.; Lucas, J. Ramón De; Sedelnikova, S.E.; Turner, Geoffrey; Rice, David W.

doi:10.1107/s0907444997002680

Cited by 2 publications

(1 citation statement)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A. nidulans ICL was purified and crystallized using the hanging-drop method of vapour diffusion with PEG 2000 as the precipitant as described previously [38]. The crystals, which have the morphology of tetragonal bipyramids with maximum dimensions of 0.8 × 0.6 × 0.6 mm, belong to space group P4 2 2 1 2 with cell parameters a = b = 91.9 Å, and c = 152.7 Å with one monomer in the asymmetric unit.…”

Section: Crystallisation Data Collection and Processingmentioning

confidence: 99%

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

et al. 2000

View full text Add to dashboard Cite

Difference Fourier analysis together with the pattern of sequence conservation has led to the identification of both the glyoxylate and metal binding sites and implicates the C-terminal end of the TIM barrel as the active site, which is consistent with studies of other enzymes with this fold. Two disordered regions of the polypeptide chain lie close to the active site, one of which includes a critical cysteine residue suggesting that conformational rearrangements are essential for catalysis. Structural similarities between isocitrate lyase and both PEP mutase and enzymes belonging to the enolase superfamily suggest possible relationships in aspects of the mechanism.

show abstract

Section: Crystallisation Data Collection and Processingmentioning

confidence: 99%

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

et al. 2000

View full text Add to dashboard Cite

show abstract

Insight into the Mechanism of Phosphoenolpyruvate Mutase Catalysis Derived from Site-Directed Mutagenesis Studies of Active Site Residues

Jia

Huang

et al. 1999

Biochemistry

View full text Add to dashboard Cite

PEP mutase catalyzes the conversion of phosphoenolpyruvate (PEP) to phosphonopyruvate in biosynthetic pathways leading to phosphonate secondary metabolites. A recent X-ray structure [Huang, K., Li, Z., Jia, Y., Dunaway-Mariano, D., and Herzberg, O. (1999) Structure (in press)] of the Mytilus edulis enzyme complexed with the Mg(II) cofactor and oxalate inhibitor reveals an alpha/beta-barrel backbone-fold housing an active site in which Mg(II) is bound by the two carboxylate groups of the oxalate ligand and the side chain of D85 and, via bridging water molecules, by the side chains of D58, D85, D87, and E114. The oxalate ligand, in turn, interacts with the side chains of R159, W44, and S46 and the backbone amide NHs of G47 and L48. Modeling studies identified two feasible PEP binding modes: model A in which PEP replaces oxalate with its carboxylate group interacting with R159 and its phosphoryl group positioned close to D58 and Mg(II) shifting slightly from its original position in the crystal structure, and model B in which PEP replaces oxalate with its phosphoryl group interacting with R159 and Mg(II) retaining its original position. Site-directed mutagenesis studies of the key mutase active site residues (R159, D58, D85, D87, and E114) were carried out in order to evaluate the catalytic roles predicted by the two models. The observed retention of low catalytic activity in the mutants R159A, D85A, D87A, and E114A, coupled with the absence of detectable catalytic activity in D58A, was interpreted as evidence for model A in which D58 functions in nucleophilic catalysis (phosphoryl transfer), R159 functions in PEP carboxylate group binding, and the carboxylates of D85, D87 and E114 function in Mg(II) binding. These results also provide evidence against model B in which R159 serves to mediate the phosphoryl transfer. A catalytic motif, which could serve both the phosphoryl transfer and the C-C cleavage enzymes of the PEP mutase superfamily, is proposed.

show abstract

Isocitrate lyase fromAspergillus nidulans: crystallization and X-ray analysis of a glyoxylate cycle enzyme

Cited by 2 publications

References 13 publications

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

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

Insight into the Mechanism of Phosphoenolpyruvate Mutase Catalysis Derived from Site-Directed Mutagenesis Studies of Active Site Residues

Contact Info

Product

Resources

About