PDBsum entry: 1w85

Oxidoreductase

PDB id: 1w85

Contents

Protein chains

359 a.a. *

324 a.a. *

42 a.a. *

35 a.a. *

Ligands

PEG ×3

TDP ×4

Metals

_MG ×6

__K ×4

Waters ×1733

* Residue conservation analysis

PDB id:

1w85

Name:

Oxidoreductase

Title:

The crystal structure of pyruvate dehydrogenase e1 bound to the peripheral subunit binding domain of e2

Structure:

Pyruvate dehydrogenase e1 component, alpha subunit. Chain: a, c, e, g. Engineered: yes. Pyruvate dehydrogenase e1 component, beta subunit. Chain: b, d, f, h. Engineered: yes. Dihydrolipoyllysine-residue acetyltransferase

Source:

Geobacillus stearothermophilus. Organism_taxid: 1422. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_taxid: 562

Biol. unit:

Pentamer (from PDB file)

Resolution:

2.0Å R-factor: 0.178 R-free: 0.215

Authors:

R.A.W.Frank,J.V.Pratap,X.Y.Pei,R.N.Perham,B.F.Luisi

Key ref:

R.A.Frank et al. (2004). A molecular switch and proton wire synchronize the active sites in thiamine enzymes. Science, 306, 872-876. PubMed id: 15514159 DOI: 10.1126/science.1101030

Date:

16-Sep-04 Release date: 02-Nov-04

Protein chains

P21873  (ODPA_GEOSE) -  Pyruvate dehydrogenase E1 component subunit alpha

Seq: 369 a.a.

Struc: 359 a.a.

Protein chains

P21874  (ODPB_GEOSE) -  Pyruvate dehydrogenase E1 component subunit beta

Seq: 325 a.a.

Struc: 324 a.a.

Protein chain

P11961  (ODP2_GEOSE) -  Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex

Seq: 428 a.a.

Struc: 42 a.a.

Protein chain

P11961  (ODP2_GEOSE) -  Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex

Seq: 428 a.a.

Struc: 35 a.a.

Enzyme reactions

• Enzyme class 1: Chains A, B, C, D, E, F, G, H: E.C.1.2.4.1 - Pyruvate dehydrogenase (acetyl-transferring).
• Pathway: Oxo-acid dehydrogenase complexes
• Reaction: Pyruvate + [dihydrolipoyllysine-residue acetyltransferase] lipoyllysine = [dihydrolipoyllysine-residue acetyltransferase] S-acetyldihydrolipoyllysine + CO₂

Pyruvate (Bound ligand (Het Group name = PEG) matches with 44.00% similarity) + [dihydrolipoyllysine-residue acetyltransferase] lipoyllysine = [dihydrolipoyllysine-residue acetyltransferase] S-acetyldihydrolipoyllysine + CO(2)

• Cofactor: Thiamine diphosphate

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

• Enzyme class 2: Chains I, J: E.C.2.3.1.12 - Dihydrolipoyllysine-residue acetyltransferase.

Pathway:

• Reaction: Acetyl-CoA + enzyme N₆-(dihydrolipoyl)lysine = CoA + enzyme N₆- (S-acetyldihydrolipoyl)lysine

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

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

 Gene Ontology (GO) functional annotation 

• Biological process: metabolic process (3 terms)
• Biochemical function: catalytic activity (6 terms)

reference

DOI no: 10.1126/science.1101030

Science 306:872-876 (2004)

PubMed id: 15514159

A molecular switch and proton wire synchronize the active sites in thiamine enzymes.

R.A.Frank, C.M.Titman, J.V.Pratap, B.F.Luisi, R.N.Perham.

ABSTRACT

Thiamine diphosphate (ThDP) is used as a cofactor in many key metabolic enzymes. We present evidence that the ThDPs in the two active sites of the E1 (EC 1.2.4.1) component of the pyruvate dehydrogenase complex communicate over a distance of 20 angstroms by reversibly shuttling a proton through an acidic tunnel in the protein. This "proton wire" permits the co-factors to serve reciprocally as general acid/base in catalysis and to switch the conformation of crucial active-site peptide loops. This synchronizes the progression of chemical events and can account for the oligomeric organization, conformational asymmetry, and "ping-pong" kinetic properties of E1 and other thiamine-dependent enzymes.

Selected figure(s)

Figure 2.
Fig. 2. B. stearothermophilus E1 active-site asymmetry and proposed proton wire. (A) Ribbon diagram of E1 taken from its complex with the PSBD of E2 colored by temperature (B-) factor. The ThDP cofactors are represented by space-filling atoms and three Mg2+ ions (red spheres), one in each of the two active sites and one midway in the protein tunnel-like cavity that links them. The dyad axis of E1 is oriented vertically. E1 is symmetric except for residues in the active sites, which are non-equivalent. Two peptide loops ( 275 to 293 and 203 to 212) at the entrance to one of the two active sites are disordered. Two arrows indicate the entrance to the active sites, and the N-termini of the two subunits are labeled. Image (B) is a close-up of image (C). A solvent-accessible surface is shown for E1, which is clipped with a bounding plane to expose the interior. The figure was made with VMD, MSMS, and Raster3D (23).

Figure 4.
Fig. 4. Catalytic mechanism of ThDP-dependent enzymes. (A to D) are the steps of ThDP activation in both active sites and (E) is the slinky cycle. (A) ThDP binds fast/tightly to the first site and is activated to generate a ThDP C2-carbanion. The ThDP C2 proton is relayed via the amino N4' group to the N1' atom of the ThDP aminopyrimidine ring and onward through a proton wire to the open, apo-active site. (B) Two loops (represented by a blue shape) preorganize the active site by folding around the zwitterionic thiazolium. After the first site is activated, the second site has no route for abstracting a second proton, so its ThDP binds but remains dormant. (C) Substrate (in this example, pyruvate) reacts with the activated C2. The ThDP of the second active site is a general acid, donating a proton to the first site. (D) This results in decarboxylation at the first site (not shown, see Fig. 4E), which forms the enamine intermediate, and activation of the second site. Both active sites now gain entry into the slinky cycle, shown in panel (E). The entry points into the slinky cycle are nonequivalent for each active site and are highlighted by an asterisk and a green border. At the first and last steps of ping-pong catalysis, both ThDPs separated by a 20 Å proton wire are mutually obligated as general acid-base catalysts in a slinky-like progression of chemical events. The dithiolane ring of the lipoyl domain is the second substrate in this example and requires activation by ßHis128 in the active site (10, 11).

The above figures are reprinted by permission from the AAAs: Science (2004, 306, 872-876) copyright 2004.

Figures were selected by the author.