PDBsum entry 2j9f

Oxidoreductase

PDB id: 2j9f

Contents

Protein chains

383 a.a. *

330 a.a. *

Ligands

THV ×2

GOL ×3

Metals

_MN ×2

__K ×4

Waters ×1069

* Residue conservation analysis

PDB id:

2j9f

Name:

Oxidoreductase

Title:

Human branched-chain alpha-ketoacid dehydrogenase-decarboxylase e1b

Structure:

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

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693. Expressed in: escherichia coli.

Resolution:

1.88Å R-factor: 0.171 R-free: 0.200

Authors:

L.Jun,M.Machius,J.L.Chuang,R.M.Wynn,D.T.Chuang

Key ref:

J.Li et al. (2007). The two active sites in human branched-chain alpha-keto acid dehydrogenase operate independently without an obligatory alternating-site mechanism. J Biol Chem, 282, 11904-11913. PubMed id: 17329260 DOI: 10.1074/jbc.M610843200

Date:

07-Nov-06 Release date: 27-Feb-07

Protein chains

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

Seq: 445 a.a.

Struc: 383 a.a.

Protein chains

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

Seq: 392 a.a.

Struc: 330 a.a.

Enzyme reactions

• Enzyme class: Chains A, B, C, D: 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

Thiamine diphosphate (Bound ligand (Het Group name = THV) matches with 83.87% similarity)

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

reference

DOI no: 10.1074/jbc.M610843200

J Biol Chem 282:11904-11913 (2007)

PubMed id: 17329260

The two active sites in human branched-chain alpha-keto acid dehydrogenase operate independently without an obligatory alternating-site mechanism.

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

ABSTRACT

A long standing controversy is whether an alternating activesite mechanism occurs during catalysis in thiamine diphosphate (ThDP)-dependent enzymes. We address this question by investigating the ThDP-dependent decarboxylase/dehydrogenase (E1b) component of the mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC). Our crystal structure reveals that conformations of the two active sites in the human E1b heterotetramer harboring the reaction intermediate are identical. Acidic residues in the core of the E1b heterotetramer, which align with the proton-wire residues proposed to participate in active-site communication in the related pyruvate dehydrogenase from Bacillus stearothermophilus, are mutated. Enzyme kinetic data show that, except in a few cases because of protein misfolding, these alterations are largely without effect on overall activity of BCKDC, ruling out the requirement of a proton-relay mechanism in E1b. BCKDC overall activity is nullified at 50% phosphorylation of E1b, but it is restored to nearly half of the pre-phosphorylation level after dissociation and reconstitution of BCKDC with the same phosphorylated E1b. The results suggest that the abolition of overall activity likely results from the specific geometry of the half-phosphorylated E1b in the BCKDC assembly and not due to a disruption of the alternating active-site mechanism. Finally, we show that a mutant E1b containing only one functional active site exhibits half of the wild-type BCKDC activity, which directly argues against the obligatory communication between active sites. The above results provide evidence that the two active sites in the E1b heterotetramer operate independently during the ThDP-dependent decarboxylation reaction.

Selected figure(s)

Figure 1.
FIGURE 1. Identical conformation in the two active sites of the human E1b heterotetramer. Ribbon representation of S320P human E1b in the crystal form containing an entire heterotetramer in the asymmetric unit. The N and C termini are indicated. All figures of molecular structures were created with the program PyMol (DeLano Scientific, San Carlos, CA).

Figure 4.
FIGURE 4. SDS-PAGE and Western blotting analysis of affinity-column fractions containing hybrid (S292Q /WT) E1b. The hybrid E1b contains the S292Q mutation in the His[6]-tagged subunit, and the wild-type sequence in the Strep-tagged subunit was prepared, as described under "Experimental Procedures." A, SDS-PAGE of peak fractions from a HisTrap column; B, SDS-PAGE of peak fractions from a consecutive Strep-Tactin Superflow column; C, Western blotting with probes for the His[6] or Strep tag. Hyb, hybrid E1b.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 11904-11913) copyright 2007.

Figures were selected by an automated process.