3-isopropylmalate dehydrogenase

 

Isocitrate dehydrogenase has long been known to be a key enzyme in the Krebs cycle, and isozymes are found in both the mitochondrial matrix and the cytosol of eukaryotes, and in bacteria. In addition to the usual NADP dependent form (EC 1.1.1.42), eukaryotes have an NAD dependent isozyme (EC 1.1.1.41). More recently, two other NAD dependent enzymes - isopropylmalate dehydrogenase (EC 1.1.1.85) and tartrate dehydrogenase (EC 1.1.1.93) have been recognised to be related by sequence and mechanism to isocitrate dehydrogenase - the unusual feature being that all of them decarboxylate the substrate which they oxidise. These are both found in micro-organisms and are involved in leucine biosynthesis and glyoxylate metabolism respectively. All require an Mg2+ ion for catalysis.

 

Reference Protein and Structure

Sequence
Q56268 UniProt (1.1.1.85) IPR004429 (Sequence Homologues) (PDB Homologues)
Biological species
Acidithiobacillus ferrooxidans (Bacteria) Uniprot
PDB
1a05 - CRYSTAL STRUCTURE OF THE COMPLEX OF 3-ISOPROPYLMALATE DEHYDROGENASE FROM THIOBACILLUS FERROOXIDANS WITH 3-ISOPROPYLMALATE (2.0 Å) PDBe PDBsum 1a05
Catalytic CATH Domains
3.40.718.10 CATHdb (see all for 1a05)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:1.1.1.85)

NAD(1-)
CHEBI:57540ChEBI
+
(2R,3S)-3-isopropylmalate(2-)
CHEBI:35121ChEBI
→
hydron
CHEBI:15378ChEBI
+
NADH(2-)
CHEBI:57945ChEBI
+
(2S)-2-isopropyl-3-oxosuccinate(2-)
CHEBI:17214ChEBI
Alternative enzyme names: 3-carboxy-2-hydroxy-4-methylpentanoate:NAD(+) oxidoreductase, Beta-isopropylmalate dehydrogenase, Beta-isopropylmalic enzyme, Threo-D(s)-3-isopropylmalate dehydrogenase, IMDH, IPMDH, Beta-IPM dehydrogenase,

Enzyme Mechanism

Introduction

The oxidation reaction is believed to occur in two steps - dehydrogenation at the alpha-carbon to form a carbonyl and decarboxylation at the beta-carbon. First, Asp 222 B deprotonates the 2-hydroxy oxygen, followed by the formation of a double bond forcing hydride transfer from C2 to NAD(P). In the case of isocitrate dehydrogenase, the intermediate so formed is oxalosuccinate and is stable enough to be isolated. In vivo, however, the beta-carboxylate group is rapidly lost and C3 is stereospecifically protonated to form the product. Lys 190 B is vital for the decarboxylation step in providing the proton, and the Tyr 140 A hydroxyl is very important for the oxidation step in participating in hydride transfer. Magnesium stabilises the negative charge on the hydroxyl oxygen during the dehydrogenation and decarboxylation steps.

Catalytic Residues Roles

UniProt PDB* (1a05)
Lys190 Lys190B Acts as a general acid catalyst in protonation in the decarboxylation step. proton shuttle (general acid/base)
Asp222 Asp222B Acts as a general base catalyst to deprotonate the hydroxyl oxygen of the substrate in the dehydrogenation step proton shuttle (general acid/base), metal ligand
Tyr140 Tyr140A Participates in hydride transfer from the substrate to NAD(P). electron shuttle
Asp246, Asp250 Asp246A, Asp250A Binds the Mg(II) ion. metal ligand
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

References

  1. Hurley JH et al. (1991), Biochemistry, 30, 8671-8678. Catalytic mechanism of NADP+-dependent isocitrate dehydrogenase: implications from the structures of magnesium-isocitrate and NADP+ complexes. DOI:10.1021/bi00099a026. PMID:1888729.
  2. Imada K et al. (2008), Proteins, 70, 63-71. Structure and quantum chemical analysis of NAD+-dependent isocitrate dehydrogenase: Hydride transfer and co-factor specificity. DOI:10.1002/prot.21486. PMID:17634983.
  3. Kalinina OV et al. (2006), Proteins, 64, 1001-1009. Amino acid residues that determine functional specificity of NADP- and NAD-dependent isocitrate and isopropylmalate dehydrogenases. DOI:10.1002/prot.21027. PMID:16767773.
  4. Yasutake Y et al. (2003), J Biol Chem, 278, 36897-36904. Crystal Structure of the Monomeric Isocitrate Dehydrogenase in the Presence of NADP+: INSIGHT INTO THE COFACTOR RECOGNITION, CATALYSIS, AND EVOLUTION. DOI:10.1074/jbc.m304091200. PMID:12855708.
  5. Imada K et al. (1998), Structure, 6, 971-982. Structure of 3-isopropylmalate dehydrogenase in complex with 3-isopropylmalate at 2.0 å resolution: the role of Glu88 in the unique substrate-recognition mechanism. DOI:10.1016/s0969-2126(98)00099-9. PMID:9739088.
  6. Tsuchiya D et al. (1997), J Biochem, 122, 1092-1104. Crystal structure of 3-isopropylmalate dehydrogenase from the moderate facultative thermophile, Bacillus coagulans: two strategies for thermostabilization of protein structures. PMID:9498551.
  7. Wallon G et al. (1997), J Mol Biol, 266, 1016-1031. Crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus. DOI:10.1006/jmbi.1996.0797. PMID:9086278.
  8. Lee ME et al. (1995), Biochemistry, 34, 378-384. Mutational analysis of the catalytic residues lysine 230 and tyrosine 160 in the NADP+-dependent isocitrate dehydrogenase from Escherichia coli. DOI:10.1021/bi00001a046. PMID:7819221.
  9. Kadono S et al. (1995), J Biochem, 118, 745-752. Ligand-induced changes in the conformation of 3-isopropylmalate dehydrogenase from Thermus thermophilus. PMID:8576088.
  10. Dean AM et al. (1995), Protein Sci, 4, 2156-2167. The role of glutamate 87 in the kinetic mechanism ofThermus thermophilusisopropylmalate dehydrogenase. DOI:10.1002/pro.5560041022. PMID:8535253.
  11. Hurley JH et al. (1994), Structure, 2, 1007-1016. Structure of 3-isopropylmalate dehydrogenase in complex with NAD+: ligand-induced loop closing and mechanism for cofactor specificity. PMID:7881901.
  12. Miyazaki K et al. (1993), FEBS Lett, 332, 37-38. Tyr-139 inThermus thermophilus3-isopropylmalate dehydrogenase is involved in catalytic function. DOI:10.1016/0014-5793(93)80478-d. PMID:8405446.
  13. Kawaguchi H et al. (1993), J Biochem, 114, 370-377. 3-Isopropylmalate dehydrogenase from chemolithoautotroph Thiobacillus ferrooxidans: DNA sequence, enzyme purification, and characterization. PMID:8282728.

Step 1.

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Catalytic Residues Roles

Residue Roles
Tyr140A electron shuttle
Asp222B metal ligand, proton shuttle (general acid/base)
Lys190B proton shuttle (general acid/base)
Asp246A metal ligand
Asp250A metal ligand

Chemical Components

Step 1.

Contributors

Gemma L. Holliday, Emma LR Compton, Craig Porter