PDBsum entry: 1cw1

Oxidoreductase

PDB id: 1cw1

Contents

Protein chain

415 a.a. *

Ligands

SO4

ICT

Metals

_MN

Waters ×332

* Residue conservation analysis

PDB id:

1cw1

Name:

Oxidoreductase

Title:

Crystal structure of isocitrate dehydrogenase mutant k230m b isocitrate and mn2+

Structure:

Isocitrate dehydrogenase. Chain: a. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PDB file)

Resolution:

2.10Å R-factor: 0.190 R-free: 0.233

Authors:

R.Stroud,J.Finer-Moore

Key ref:

D.B.Cherbavaz et al. (2000). Active site water molecules revealed in the 2.1 A resolution structure of a site-directed mutant of isocitrate dehydrogenase. J Mol Biol, 295, 377-385. PubMed id: 10623532 DOI: 10.1006/jmbi.1999.3195

Date:

25-Aug-99 Release date: 01-Sep-99

Protein chain

P08200  (IDH_ECOLI) -  Isocitrate dehydrogenase [NADP]

Seq: 416 a.a.

Struc: 415 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.1.1.1.42 - Isocitrate dehydrogenase (NADP(+)).
• Pathway: Citric acid cycle
• Reaction: Isocitrate + NADP⁺ = 2-oxoglutarate + CO₂ + NADPH

Isocitrate (Bound ligand (Het Group name = ICT) corresponds exactly) + NADP(+) = 2-oxoglutarate + CO(2) + NADPH

• Cofactor: Manganese or magnesium

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Biological process: oxidation-reduction process (4 terms)
• Biochemical function: oxidoreductase activity (6 terms)

reference

DOI no: 10.1006/jmbi.1999.3195

J Mol Biol 295:377-385 (2000)

PubMed id: 10623532

Active site water molecules revealed in the 2.1 A resolution structure of a site-directed mutant of isocitrate dehydrogenase.

D.B.Cherbavaz, M.E.Lee, R.M.Stroud, D.E.Koshland.

ABSTRACT

Isocitrate dehydrogenase catalyses the two step, acid base, oxidative decarboxylation of isocitrate to alpha-ketoglutarate. Lysine 230 was suggested to act as proton donor based on geometry and spatial proximity to isocitrate. To clarify further the role of lysine 230, we co-crystallized the lysine-to-methionine mutant (K230M) with isocitrate and with alpha-ketoglutarate. Crystals were flash-frozen and the two structures were determined and refined to 2. 1 A. Several new features were identified relative to the wild-type structure. Seven side-chains previously unplaced in the wild-type structure were identified and included in the model, and the amino acid terminus was extended by an alanine residue. Many additional water molecules were identified.Examination of the K230M active sites (K230M isocitrate and K230M-ketoglutarate) revealed that tyrosine 160 protrudes further into the active site in the presence of either isocitrate or alpha-ketoglutarate in K230 M than it does in the wild-type structure. Also, methionine 230 was not as fully extended, and asparagine 232 rotates approximately 30 degrees toward the ligand permitting polar interactions. Outside the active site cleft a tetragonal volume of density was identified as a sulfate molecule. Its location and interactions suggest it may influence the equilibrium between the tetragonal and the orthorhombic forms of isocitrate dehydrogenase. Differences observed in the active site water structure between the wild-type and K230M structures were due to a single point mutation. A water molecule was located in the position equivalent to that occupied by the wild-type epsilon-amine of lysine 230; a water molecule in that location in K230M suggests it may influence catalysis in the mutant. Comparison of K230M complexed with isocitrate and alpha-ketoglutarate illuminates the influence a ligand has on active site water structure.

Selected figure(s)

Figure 1.
Figure 1. Superposed active site residues from three low temperature structures of isocitrate dehydrogenase illustrates the relative position of designated matched water molecules. Numbers 1 through 13 correspond to water molecules described in Table 2A. Active site residues are labeled with amino acid type and sequence number except for 230, which is lysine in wild-type and methionine in K230M. The red, orange, and yellow residues and water molecules represent wild-type-isocitrate, K230M-isocitrate, and K230M-a-ketoglutarate structures, respectively. (a) Illustration of all active site water molecules outlined in Table 2 and their position relative to active site residues. Water 11 occupies a position in the K230M structures similar to the position the epsilon -anime hydrogen atoms of K230 occupy in the wild-type structure. Water molecules 8, 9, and 11 specifically appear in the K230M mutant structures. (b) Positional differences in the hydroxyl oxygen atom of tyrosine 160 are highlighted in this view. The two views are related by rotation of approximately 50 °.

Figure 2.
Figure 2. Molecular surface of IDH shown with ligands bound. (a) Isocitrate is presented in the cavernous active site pocket. (b) NADP and isocitrate are shown in the extended active site volume. The adenine moiety of NADP+ (top) is bound in a narrow pocket contiguous with the isocitrate active site. The nicotinamide moiety of NADP+ associates with isocitrate and occupies part of the dehydrogenase active site. The sulfate molecule located in the K230M structures is visible, below and exterior to the active site pocket (present in both (a) and (b)). The NADP+ coordinates were supplied by PDB files reported by [Stoddard et al 1993]. The molecular contours corresponding to Van der Waals surfaces were prepared using Grasp [Sridharan et al 1995].

The above figures are reprinted by permission from Elsevier: J Mol Biol (2000, 295, 377-385) copyright 2000.