PDBsum entry 1olx

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Oxidoreductase PDB id
Protein chains
391 a.a. *
335 a.a. *
GOL ×2
__K ×2
Waters ×371
* Residue conservation analysis
PDB id:
Name: Oxidoreductase
Title: Roles of his291-alpha and his146-beta' in the reductive acylation reaction catalyzed by human branched-chain alpha-ketoacid dehydrogenase
Structure: 2-oxoisovalerate dehydrogenase alpha subunit. Chain: a. Synonym: branched-chain alpha-ketoacid dehydrogenase e1 component alpha chain, bckdh e1-alpha. Engineered: yes. 2-oxoisovalerate dehydrogenase beta subunit. Chain: b. Synonym: branched-chain alpha-keto acid dehydrogenase e1 component beta chain, bckdh e1-beta.
Source: Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Tetramer (from PDB file)
2.25Å     R-factor:   0.161     R-free:   0.214
Authors: R.M.Wynn,M.Machius,J.L.Chuang,J.Li,D.R.Tomchick,D.T.Chuang
Key ref:
R.M.Wynn et al. (2003). 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. J Biol Chem, 278, 43402-43410. PubMed id: 12902323 DOI: 10.1074/jbc.M306204200
18-Aug-03     Release date:   28-Aug-03    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P12694  (ODBA_HUMAN) -  2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial
445 a.a.
391 a.a.
Protein chain
Pfam   ArchSchema ?
P21953  (ODBB_HUMAN) -  2-oxoisovalerate dehydrogenase subunit beta, mitochondrial
392 a.a.
335 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     mitochondrion   3 terms 
  Biological process     metabolic process   5 terms 
  Biochemical function     catalytic activity     8 terms  


DOI no: 10.1074/jbc.M306204200 J Biol Chem 278:43402-43410 (2003)
PubMed id: 12902323  
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.
R.M.Wynn, M.Machius, J.L.Chuang, J.Li, D.R.Tomchick, D.T.Chuang.
We report here that alterations of either His291-alpha or His146-beta' in the active site of human branched-chain alpha-ketoacid dehydrogenase (E1b) impede both the decarboxylation and the reductive acylation reactions catalyzed by E1b as well as the binding of cofactor thiamin diphosphate (ThDP). In a refined human E1b active-site structure, His291-alpha, which aligns with His407 in Escherichia coli pyruvate dehydrogenase and His263 in yeast transketolase, is on a largely ordered phosphorylation loop. The imidazole ring of His291-alpha in E1b coordinates to the terminal phosphate oxygen atoms of bound ThDP. The N3 atom of wild-type His146-beta', which can be protonated, binds a water molecule and points toward the aminopyrimidine ring of ThDP. Remarkably, the H291A-alpha mutation results in a complete order-to-disorder transition of the loop region, which precludes the binding of the substrate lipoyl-bearing domain to E1b. The H146A-beta' mutation, on the other hand, does not alter the loop structure, but nullifies the reductive acylation activity of E1b. Our results suggest that: 1) His291-alpha plays a structural rather than a catalytic role in the binding of cofactor ThDP and the lipoyl-bearing domain to E1b, and 2) His146-beta' is an essential catalytic residue, probably functioning as a proton donor in the reductive acylation of lipoamide on the lipoyl-bearing domain.
  Selected figure(s)  
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,
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2003, 278, 43402-43410) copyright 2003.  
  Figures were selected by the author.  
    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

Literature references that cite this PDB file's key reference

  PubMed id Reference
19081061 M.Kato, R.M.Wynn, J.L.Chuang, S.C.Tso, M.Machius, J.Li, and D.T.Chuang (2008).
Structural basis for inactivation of the human pyruvate dehydrogenase complex by phosphorylation: role of disordered phosphorylation loops.
  Structure, 16, 1849-1859.
PDB codes: 3exe 3exf 3exg 3exh 3exi
18004749 V.I.Bunik, and D.Degtyarev (2008).
Structure-function relationships in the 2-oxo acid dehydrogenase family: substrate-specific signatures and functional predictions for the 2-oxoglutarate dehydrogenase-like proteins.
  Proteins, 71, 874-890.  
16084384 R.A.Frank, J.V.Pratap, X.Y.Pei, R.N.Perham, and B.F.Luisi (2005).
The molecular origins of specificity in the assembly of a multienzyme complex.
  Structure, 13, 1119-1130.
PDB code: 2bp7
15576032 R.M.Wynn, M.Kato, M.Machius, J.L.Chuang, J.Li, D.R.Tomchick, and D.T.Chuang (2004).
Molecular mechanism for regulation of the human mitochondrial branched-chain alpha-ketoacid dehydrogenase complex by phosphorylation.
  Structure, 12, 2185-2196.
PDB codes: 1u5b 1x7w 1x7x 1x7y 1x7z 1x80
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