PDBsum entry 1v1r

Oxidoreductase

PDB id: 1v1r

Contents

Protein chains

352 a.a. *

336 a.a. *

Ligands

SO4

GOL ×2

Metals

_CL ×3

__K ×2

_NA ×2

Waters ×531

* Residue conservation analysis

PDB id:

1v1r

Name:

Oxidoreductase

Title:

Crosstalk between cofactor binding and the phosphorylation loop conformation in the bckd machine

Structure:

2-oxoisovalerate dehydrogenase alpha subunit. Chain: a. Synonym: mitochondrial precursor, branched-chain alpha-keto acid dehydrogenase e1, component alpha chain, bckdh e1-alpha, bckde1a. Engineered: yes. 2-oxoisovalerate dehydrogenase beta subunit. Chain: b. Synonym: mitochondrial precursor, branched-chain alpha-keto acid dehydrogenase e1, component beta chain, bckdh e1-beta.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562. Groes. Other_details: expression system used bl-21 cells with overexpressing groel and groes

Biol. unit:

Tetramer (from PDB file)

Resolution:

1.80Å R-factor: 0.158 R-free: 0.183

Authors:

J.Li,R.M.Wynn,M.Machius,J.L.Chuang,S.Karthikeyan,D.R.Tomchick, D.T.Chuang

Key ref:

J.Li et al. (2004). Cross-talk between thiamin diphosphate binding and phosphorylation loop conformation in human branched-chain alpha-keto acid decarboxylase/dehydrogenase. J Biol Chem, 279, 32968-32978. PubMed id: 15166214 DOI: 10.1074/jbc.M403611200

Date:

22-Apr-04 Release date: 03-Jun-04

Protein chain

P12694  (ODBA_HUMAN) -  2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial from Homo sapiens

Seq: 445 a.a.

Struc: 352 a.a.

Protein chain

P21953  (ODBB_HUMAN) -  2-oxoisovalerate dehydrogenase subunit beta, mitochondrial from Homo sapiens

Seq: 392 a.a.

Struc: 336 a.a.

Enzyme reactions

• Enzyme class: Chains A, B: E.C.1.2.4.4 - 3-methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring).
• Pathway: Oxo-acid dehydrogenase complexes
• Reaction: N₆-[(R)-lipoyl]-L-lysyl-[protein] + 3-methyl-2-oxobutanoate + H⁺ = N₆-[(R)-S(8)-2-methylpropanoyldihydrolipoyl]-L-lysyl-[protein] + CO2

N(6)-[(R)-lipoyl]-L-lysyl-[protein] + 3-methyl-2-oxobutanoate (Bound ligand (Het Group name = GOL) matches with 55.56% similarity) + H(+) = N(6)-[(R)-S(8)-2-methylpropanoyldihydrolipoyl]-L-lysyl-[protein] + CO2

• Cofactor: Thiamine diphosphate

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

reference

DOI no: 10.1074/jbc.M403611200

J Biol Chem 279:32968-32978 (2004)

PubMed id: 15166214

Cross-talk between thiamin diphosphate binding and phosphorylation loop conformation in human branched-chain alpha-keto acid decarboxylase/dehydrogenase.

J.Li, R.M.Wynn, M.Machius, J.L.Chuang, S.Karthikeyan, D.R.Tomchick, D.T.Chuang.

ABSTRACT

The decarboxylase/dehydrogenase (E1b) component of the 4-megadalton human branched-chain alpha-keto acid dehydrogenase (BCKD) metabolic machine is a thiamin diphosphate (ThDP)-dependent enzyme with a heterotetrameric cofactor-binding fold. The E1b component catalyzes the decarboxylation of alpha-keto acids and the subsequent reductive acylation of the lipoic acid-bearing domain (LBD) from the 24-meric transacylase (E2b) core. In the present study, we show that the binding of cofactor ThDP to the E1b active site induces a disorder-to-order transition of the conserved phosphorylation loop carrying the two phosphorylation sites Ser(292)-alpha and Ser(302)-alpha, as deduced from the 1.80-1.85 A apoE1b and holoE1b structures. The induced loop conformation is essential for the recognition of lipoylated LBD to initiate E1b-catalyzed reductive acylation. Alterations of invariant Arg(287)-alpha, Asp(295)-alpha, Tyr(300)-alpha, and Arg(301)-alpha that form a hydrogen-bonding network in the phosphorylation loop result in the disordering of the loop conformation as elucidated by limited proteolysis, accompanied by the impaired binding and diminished reductive acylation of lipoylated LBD. In contrast, k(cat) values for E1b-catalyzed decarboxylation of the alpha-keto acid are higher in these E1b mutants than in wild-type E1b, with higher K(m) values for the substrate in the mutants. ThDP binding that orders the loop prevents phosphorylation of E1b by the BCKD kinase and averts the inactivation of wild-type E1b, but not the above mutants, by this covalent modification. Our results establish that the cross-talk between the bound ThDP and the phosphorylation loop conformation serves as a feed-forward switch for multiple reaction steps in the BCKD metabolic machine.

Selected figure(s)

Figure 3.
FIG. 3. Substitutions of residues participating in the hydrogen-bonding network result in markedly decreased reductive acylation (Reaction 3) activity. Invariant residues (Arg287- , Asp295- , Tyr300- , and Arg301- ) that form the hydrogen-bonding network were changed to alanine or phenylalanine in the case of Tyr300- . Reductive acylation of lip-LBD catalyzed by wild-type or mutant E1b was measured with [U-14C]KIV as a substrate as described under "Experimental Procedures." Activity for reductive acylation is expressed as percent relative to the wild type (2.3 min-1). The nonspecific radioactivity incorporated into nonlipoylated LBD and the wild-type or mutant E1b protein served as a blank. Results are averages of two independent experiments.

Figure 7.
FIG. 7. ThDP inhibits the phosphorylation of wild-type but not mutant E1b. A, the reaction mixture contained human apoE1b protein, lipoylated E2b, and maltose-binding protein-tagged rat BCKD kinase in the absence and presence of increasing ThDP concentrations. The phosphorylation reaction was initiated by adding 0.4 mM [ -32P]ATP and was incubated at 25 °C for 1 min. The reaction mixtures were separated by SDS-PAGE. 32P incorporation into the subunit of E1b proteins was quantified by PhosphorImaging. The PhosphorImage counts in wild-type and each mutant E1b in the absence of ThDP was set as 100% with respect to the corresponding E1b protein. B, PhosphorImaging of 32P incorporation into the subunit of the S302A- E1b mutant and E1b double mutants containing the S302A- mutation and a second mutation in the hydrogen-bonding network. The phosphorylation was carried out in the absence of ThDP.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 32968-32978) copyright 2004.

Figures were selected by an automated process.