PDBsum entry 1v11

Oxidoreductase

PDB id: 1v11

Contents

Protein chains

370 a.a. *

336 a.a. *

Ligands

TPP

BEN

GOL ×3

Metals

__K ×2

_MN

_CL ×5

Waters ×529

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Cross-Talk between thiamin diphosphate binding and phosphorylation loop conformation in human branched-Chain alpha-Keto acid decarboxylase/dehydrogenase.

Authors

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

Ref.

J Biol Chem, 2004, 279, 32968-32978. [DOI no: 10.1074/jbc.M403611200]

PubMed id

15166214

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.

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.

Secondary reference #1

Title

Roles of his291-Alpha and his146-Beta' In the reductive acylation reaction catalyzed by human branched-Chain alpha-Ketoacid dehydrogenase: refined phosphorylation loop structure in the active site.

Authors

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

Ref.

J Biol Chem, 2003, 278, 43402-43410. [DOI no: 10.1074/jbc.M306204200]

PubMed id

12902323

Figure 4.
FIG. 4. ITC measurements for lip-LBD binding to wild-type, H146A- and H291A- human E1b. ITC experiments were carried out in a MicroCal VP-ITC microcalorimeter by consecutively injecting aliquots of 1.5 mM lip-LBD or unlipoylated LBD into the reaction cell containing 25 µM wild-type or mutant human E1b. Binding isotherms for wild-type ( ), H146A- ' ( o ), and H291A- ( ) were obtained by plotting heat changes against the molar ratio of lip-LBD, as derived from the integrated raw data. The data were fit using the ORIGIN software supplied by the manufacturer. Wild-type E1b and the His146- ' variant show similar affinity for lip-LBD with dissociation constants (K[d]) of 2.52 x 10^-5 M and 1.56 x 10^-5 M, respectively. The binding of the H291A- mutant to lip-LBD cannot be detected by ITC as indicated by the absence of heat changes. Binding of unlipoylated LBD ( ) to wild-type E1b also cannot be detected.

Figure 5.
FIG. 5. Refined structure of the human E1b active site at the interface between - and '-subunits. 2F[o] - F[c] electron densities (in green) are contoured at 1 . Only two histidine residues are within 5-Å distance from the C2 atom of the bound ThDP. His146- is hydrogenbonded to the O4 water molecular, whereas His291- forms hydrogen bonds to the O1 and O2 water molecules (in red spheres); the former in turn coordinates to the terminal phosphate oxygen of ThDP. The channel leading to the activated C2 atom of ThDP lies at the interface between the - and '-subunits, such that these two histidine residues flank opposite sides of the channel. A Mn2+ ion is bound at the metal ion binding site in place of the common Mg2+ ion. Good electron density is present for Ser292- (phosphorylation site 1), which is positioned at the opening of the channel. Carbon atoms are in gold, ThDP in green, oxygen atoms in red, nitrogen atoms in blue, phosphorous atoms in magenta, and sulfur atoms in yellow. Graphics were generated with the programs BobScript (24) and PovRay (Persistence of Vision, v3.02, POV-Team, www.povray.org).

The above figures are reproduced from the cited reference with permission from the ASBMB

Author's comment:  The crystal structure for the E1 protein shows a dimer (one alpha plus one beta subuint); the functional unit, however, is a tetramer (a2b2).
David Chuang

Secondary reference #2

Title

Roles of active site and novel k+ ion-Binding site residues in human mitochondrial branched-Chain alpha-Ketoacid decarboxylase/dehydrogenase.

Authors

R.M.Wynn, R.Ho, J.L.Chuang, D.T.Chuang.

Ref.

J Biol Chem, 2001, 276, 4168-4174. [DOI no: 10.1074/jbc.M008038200]

PubMed id

11069910

Figure 1.
Fig. 1. Residues in the cofactor TDP binding fold of human BCKD. The inverted V-shaped conformation of cofactor TDP is stabilized by stacking of the aminopyrimidine ring against the side chain of Tyr-102- ' from the ' subunit (in greenish yellow) and the side chain of Leu-164- from the subunit (in magenta). The invariant Glu-76- ' important for cofactor activation coordinates to the N-1' atom of the aminopyrimidine ring (3.4 Å apart). A ketoacid substrate analog (in gray) labeled isocaproate is covalently modeled into the side chain of His-146- ', based on the crystal structure of BCKD from Pseudomonas putida (6). The carboxylate group of the inhibitor interacts with the N-4' amino group of TDP (separated by a distance of 4.3 Å). The side chain of Ser-162- also coordinates to the N-4' amino group (3.0 Å apart) to position the cofactor in the correct conformation. Residue Ser-292- is phosphorylation site 1 of human BCKD. The diphosphate moiety of TDP is stabilized, in part, by an octahedral coordination of the Mg2+ ion. Two of the amino acid ligands Glu-193- and Asn-222- in this coordination are shown. Side chains of Arg-114- , Arg-220- , and His-291- , are, in turn, in direct contact with the distal phosphate oxygens, whereas the side chains of Gln-112- and Tyr-113- (not shown) interact with the proximal phosphate oxygens of the diphosphate moiety of TDP.

Figure 3.
Fig. 3. The K+ ion-binding site on the subunit of human BCKD. The metal ion is bound by two main-chain carbonyl groups and by the side chains of Ser-161- , Thr-166- , and Gln-167- . The side chain of Leu-164- and the main-chain carbonyl group of Ser-162- make direct contacts with cofactor TDP. The octahedral coordination of the metal ion stabilizes the loop structure on the subunit (residues 161-167) that is essential for the efficient binding of the cofactor.

The above figures are reproduced from the cited reference with permission from the ASBMB

Secondary reference #3

Title

Crystal structure of human branched-Chain alpha-Ketoacid dehydrogenase and the molecular basis of multienzyme complex deficiency in maple syrup urine disease.

Authors

A.Aevarsson, J.L.Chuang, R.M.Wynn, S.Turley, D.T.Chuang, W.G.Hol.

Ref.

Structure, 2000, 8, 277-291. [DOI no: 10.1016/S0969-2126(00)00105-2]

PubMed id

10745006

Figure 3.
Figure 3. Cofactor and K+-binding sites in human E1b. (a) Schematic representation of the cofactor-binding site. Gln112-a and protein ligands of the magnesium ion have been omitted for clarity. (b) Potassium site 1 (a subunit). The metal stabilizes a loop involved in cofactor binding. The metal ion is bound by two mainchain carbonyl groups and by the sidechains of Ser161-a, Thr166-a and Gln167-a. The sidechain of Leu164-a and the mainchain carbonyl group of Ser162-a make direct contact with the ThDP cofactor. (c) Potassium site 2 (b subunit). The metal binding at this site stabilizes regions in the b subunit at the interface with the small C-terminal domain in the a subunit. The metal is octahedrally coordinated mainly by mainchain carbonyl groups and interacts favorably with the C-terminal end of a helix dipole as indicated. Several sidechains indicated by an asterisk have been omitted for clarity. This figure was made with LIGPLOT [50], MOLSCRIPT [48] and the Raster3D suite [49].

The above figure is reproduced from the cited reference with permission from Cell Press