PDBsum entry 2ii4

Transferase

PDB id: 2ii4

Contents

Protein chains

(+ 2 more) 234 a.a. *

Ligands

COA ×8

Metals

_CL ×12

Waters ×292

* Residue conservation analysis

PDB id:

2ii4

Name:

Transferase

Title:

Crystal structure of a cubic core of the dihydrolipoamide acyltransferase (e2b) component in the branched-chain alpha-ketoacid dehydrogenase complex (bckdc), coenzyme a-bound form

Structure:

Lipoamide acyltransferase component of branched-chain alpha-keto acid dehydrogenase complex. Chain: a, b, c, d, e, f, g, h. Fragment: core (catalytic) domain. Synonym: dihydrolipoyllysine-residue (2-methylpropanoyl)transferase, e2, dihydrolipoamide branched chain transacylase, bckad e2 subunit. Engineered: yes

Source:

Bos taurus. Cattle. Organism_taxid: 9913. Gene: dbt. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.59Å R-factor: 0.173 R-free: 0.229

Authors:

M.Kato,R.M.Wynn,J.L.Chuang,C.A.Brautigam,M.Custorio,D.T.Chuang

Key ref:

M.Kato et al. (2006). A synchronized substrate-gating mechanism revealed by cubic-core structure of the bovine branched-chain alpha-ketoacid dehydrogenase complex. EMBO J, 25, 5983-5994. PubMed id: 17124494 DOI: 10.1038/sj.emboj.7601444

Date:

27-Sep-06 Release date: 26-Dec-06

Protein chains

P11181  (ODB2_BOVIN) -  Lipoamide acyltransferase component of branched-chain alpha-keto acid dehydrogenase complex, mitochondrial from Bos taurus

Seq: 482 a.a.

Struc: 234 a.a.

Enzyme reactions

• Enzyme class: E.C.2.3.1.168 - dihydrolipoyllysine-residue (2-methylpropanoyl)transferase.
• Pathway: Oxo-acid dehydrogenase complexes
• Reaction: N₆-[(R)-dihydrolipoyl]-L-lysyl-[protein] + 2-methylpropanoyl-CoA = N₆-[(R)-S(8)-2-methylpropanoyldihydrolipoyl]-L-lysyl-[protein] + CoA

N(6)-[(R)-dihydrolipoyl]-L-lysyl-[protein] + 2-methylpropanoyl-CoA = N(6)-[(R)-S(8)-2-methylpropanoyldihydrolipoyl]-L-lysyl-[protein] (Bound ligand (Het Group name = COA) corresponds exactly) + CoA

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

Added reference

DOI no: 10.1038/sj.emboj.7601444

EMBO J 25:5983-5994 (2006)

PubMed id: 17124494

A synchronized substrate-gating mechanism revealed by cubic-core structure of the bovine branched-chain alpha-ketoacid dehydrogenase complex.

M.Kato, R.M.Wynn, J.L.Chuang, C.A.Brautigam, M.Custorio, D.T.Chuang.

ABSTRACT

The dihydrolipoamide acyltransferase (E2b) component of the branched-chain alpha-ketoacid dehydrogenase complex forms a cubic scaffold that catalyzes acyltransfer from S-acyldihydrolipoamide to CoA to produce acyl-CoA. We have determined the first crystal structures of a mammalian (bovine) E2b core domain with and without a bound CoA or acyl-CoA. These structures reveal both hydrophobic and the previously unreported ionic interactions between two-fold-related trimers that build up the cubic core. The entrance of the dihydrolipoamide-binding site in a 30-A long active-site channel is closed in the apo and acyl-CoA-bound structures. CoA binding to one entrance of the channel promotes a conformational change in the channel, resulting in the opening of the opposite dihydrolipoamide gate. Binding experiments show that the affinity of the E2b core for dihydrolipoamide is markedly increased in the presence of CoA. The result buttresses the model that CoA binding is responsible for the opening of the dihydrolipoamide gate. We suggest that this gating mechanism synchronizes the binding of the two substrates to the active-site channel, which serves as a feed-forward switch to coordinate the E2b-catalyzed acyltransfer reaction.

Selected figure(s)

Figure 4.
Figure 4 A CoA-mediated gating mechanism for synchronized dihydrolipoamide binding in the active-site channel of bovine E2bCD. (A) Stereo view of the CoA-binding site of E2bCD-CoA (short soak). The bound CoA in the 'in' conformation and its surrounding residues are shown in stick models. Residues from the three-fold-related subunit are shown in cyan. The residues in magenta are the gatekeepers at the dihydrolipoamide entrance of the active-site channel. Water molecules are shown as small spheres. The 2F[o]-F[c] density of the bound CoA is superimposed at a 1- contour level. (B) The bound IB-CoA in the 'out' conformation in E2bCD-IB-CoA. The 2F[o]-F[c] densities are at the 1- level. (C) The oxidized CoA in the 'in' conformation in E2bCD-CoA (long soak). Three of the eight E2bCD monomers in the asymmetric unit of this crystal show this structure. The omit density of the oxidized CoA was at the 1- level. (D) The disordered pantetheine tail of the CoA (the 'out' conformation) bound to the remaining five monomers in E2bCD-CoA (long soak). The 2F[o]-F[c] density is contoured at the 1- level. (E) Superimposed gating turns and bound cofactors; white: apo, green: E2bCD-CoA (short soak), orange: E2bCD-IB-CoA. The bound CoA (carbon atoms in green) in E2bCD-CoA (short soak) is in the 'in' conformation, whereas the pantetheine tail of the bound IB-CoA (carbons in orange) in E2bCD-IB-CoA is disordered (the 'out' conformation). The gray arrow indicates the direction of the dihydrolipoamide-binding site. The gatekeeper residues are in magenta. (F) Surface model of the closed dihydrolipoamide gate in the apo E2bCD structure. Three gatekeeper residues (Leu293, Val295 and Leu353) are colored in magenta and the gating turn in red. (G) The open gate for the dihydrolipoamide-binding site in E2bCD-CoA (short soak). The thiol group of the bound CoA in the 'in' conformation is shown in yellow. (H) The closed dihydrolipoamide gate in E2bCD-IB-CoA.

Figure 5.
Figure 5 The proposed scheme for the bovine E2bCD-catalyzed acyltransfer reaction cycle. The active-site channel of bovine E2bCD is depicted with the CoA-binding site on the left side and the dihydrolipoamide-binding site on the right. The catalytic resides in the channel (His391' and Ser338) and two important residues on the gating turn (Leu293 and Asp288) are shown. Gray dotted lines: H-bonds, a green oval with 'Ac': acyl group, yellow dots: thiol groups and red dots: water molecules. See the text for details of each reaction step.

The above figures are reprinted by permission from Macmillan Publishers Ltd: EMBO J (2006, 25, 5983-5994) copyright 2006.

Figures were selected by an automated process.