PDBsum entry 1umc

Oxidoreductase

PDB id: 1umc

Contents

Protein chains

362 a.a. *

323 a.a. *

Ligands

TPP ×2

4MV

Metals

_MG ×2

Waters ×735

* Residue conservation analysis

PDB id:

1umc

Name:

Oxidoreductase

Title:

Branched-chain 2-oxo acid dehydrogenase (e1) from thermus thermophilus hb8 with 4-methylpentanoate

Structure:

2-oxo acid dehydrogenase alpha subunit. Chain: a, c. Synonym: e1-alpha. Engineered: yes. 2-oxo acid dehydrogenase beta subunit. Chain: b, d. Synonym: e1-beta. Engineered: yes

Source:

Thermus thermophilus. Organism_taxid: 274. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Tetramer (from PQS)

Resolution:

2.40Å R-factor: 0.172 R-free: 0.206

Authors:

T.Nakai,N.Nakagawa,N.Maoka,R.Masui,S.Kuramitsu,N.Kamiya,Riken Structural Genomics/proteomics Initiative (Rsgi)

Key ref:

T.Nakai et al. (2004). Ligand-induced conformational changes and a reaction intermediate in branched-chain 2-oxo acid dehydrogenase (E1) from Thermus thermophilus HB8, as revealed by X-ray crystallography. J Mol Biol, 337, 1011-1033. PubMed id: 15033367 DOI: 10.1016/j.jmb.2004.02.011

Date:

25-Sep-03 Release date: 30-Mar-04

Protein chains

Q5SLR4  (ODBA_THET8) -  2-oxoisovalerate dehydrogenase subunit alpha from Thermus thermophilus (strain ATCC 27634 / DSM 579 / HB8)

Seq: 367 a.a.

Struc: 362 a.a.

Protein chains

Q5SLR3  (ODBB_THET8) -  2-oxoisovalerate dehydrogenase subunit beta from Thermus thermophilus (strain ATCC 27634 / DSM 579 / HB8)

Seq: 324 a.a.

Struc: 323 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

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 = 4MV) matches with 60.00% similarity) + H(+) = N(6)-[(R)-S(8)-2-methylpropanoyldihydrolipoyl]-L-lysyl-[protein] + CO2

• Cofactor: Thiamine diphosphate

Thiamine diphosphate (Bound ligand (Het Group name = TPP) corresponds exactly)

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

reference

DOI no: 10.1016/j.jmb.2004.02.011

J Mol Biol 337:1011-1033 (2004)

PubMed id: 15033367

Ligand-induced conformational changes and a reaction intermediate in branched-chain 2-oxo acid dehydrogenase (E1) from Thermus thermophilus HB8, as revealed by X-ray crystallography.

T.Nakai, N.Nakagawa, N.Maoka, R.Masui, S.Kuramitsu, N.Kamiya.

ABSTRACT

The alpha(2)beta(2) tetrameric E1 component of the branched-chain 2-oxo acid (BCOA) dehydrogenase multienzyme complex is a thiamin diphosphate (ThDP)-dependent enzyme. E1 catalyzes the decarboxylation of a BCOA concomitant with the formation of the alpha-carbanion/enamine intermediate, 2-(1-hydroxyalkyl)-ThDP, followed by transfer of the 1-hydroxyalkyl group to the distal sulfur atom on the lipoamide of the E2 component. In order to elucidate the catalytic mechanism of E1, the alpha- and beta-subunits of E1 from Thermus thermophilus HB8 have been co-expressed in Escherichia coli, purified and crystallized as a stable complex, and the following crystal structures have been analyzed: the apoenzyme (E1(apo)), the holoenzyme (E1(holo)), E1(holo) in complex with the substrate analogue 4-methylpentanoate (MPA) as an ES complex model, and E1(holo) in complex with 4-methyl-2-oxopentanoate (MOPA) as the alpha-carbanion/enamine intermediate (E1(ceim)). Binding of cofactors to E1(apo) induces a disorder-order transition in two loops adjacent to the active site. Furthermore, upon binding of MPA to E1(holo), the loop comprised of Gly121beta-Gln131beta moves close to the active site and interacts with MPA. The carboxylate group of MPA is recognized mainly by Tyr86beta and N4' of ThDP. The hydrophobic moiety of MPA is recognized by Phe66alpha, Tyr95alpha, Met128alpha and His131alpha. As an intermediate, MOPA is decarboxylated and covalently linked to ThDP, and the conformation of the protein loop is almost the same as in the substrate-free (holoenzyme) form. These results suggest that E1 undergoes an open-closed conformational change upon formation of the ES complex with a BCOA, and the mobile region participates in the recognition of the carboxylate group of the BCOA. ES complex models of E1(holo).MOPA and of E1(ceim).lipoamide built from the above structures suggest that His273alpha and His129beta' are potential proton donors to the carbonyl group of a BCOA and to the proximal sulfur atom on the lipoamide, respectively.

Selected figure(s)

Figure 1.
Figure 1. Stereoview of the Tth E1 structure. A, Overall structure of the a[2]b[2] tetramer in the a-carbanion/enamine intermediate; B, a-subunit; C, b-subunit. The orientations in B and C are the same as that in A. Ligand molecules are represented by spheres (orange for Mg2+, yellow for ThDP and green for HMB group) and those in B and C are shown as semi-transparent to clarify inner structures. Disordered regions in E1[apo], which include the STSH motif (see the text and Figure 3D), are shown in magenta. The substrate-binding loop (see the text and Figure 6) that undergoes conformational change upon binding of MPA is shown in cyan. The designation of secondary structures of Tth E1 is according to those of Ppu E1, resulting in the absence of helix 1.

Figure 6.
Figure 6. Conformational changes upon binding of the substrate analogue MPA. Stereoview of the structure around the substrate-binding loop for E1[holo] (A), E1[holo]·MPA (B) and E1[ceim] (C). Carbon atoms of each subunit are colored according to Figure 1A, whereas a and b in residue labels are omitted. Water molecules are shown as red spheres. Broken lines represent hydrogen bonds.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2004, 337, 1011-1033) copyright 2004.

Figures were selected by the author.