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371 a.a.
336 a.a.
__K
_MN
 3-methyl-2-oxobutanoate |
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[dihydrolipoyllysine-residue (2- methylpropanoyl)transferase] lipoyllysine |
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[dihydrolipoyllysine-residue (2-methylpropanoyl)transferase] S-(2-methylpropanoyl)dihydrolipoyllysine |
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 CO(2) |
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Bound ligand (Het Group name = TDP) corresponds exactly |
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Key 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-
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
The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 32968-32978) copyright 2004. Figures were selected by the author.
Literature references that cite this PDB file's key reference
PubMed id Reference
18004749 V.I.Bunik, and D.Degtyarev (2008).
Structure-function relationships in the 2-oxo acid dehydrogenase family: substrate-specific signatures and functional predictions for the 2-oxoglutarate dehydrogenase-like proteins.Proteins, 71, 874-890.
16579849 G.E.Homanics, K.Skvorak, C.Ferguson, S.Watkins, and H.S.Paul (2006).
Production and characterization of murine models of classic and intermediate maple syrup urine disease.BMC Med Genet, 7, 33.
16954186 J.Stetefeld, M.Jenny, and P.Burkhard (2006).
Intersubunit signaling in glutamate-1-semialdehyde-aminomutase.Proc Natl Acad Sci U S A, 103, 13688-13693.
PDB codes: 2hoy 2hoz 2hp1 2hp2 The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.
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