1OLX: Roles Of His291-alpha And His146-beta' In The Reductive Acylation Reaction Catalyzed By Human Branched-chain Alpha-ketoacid Dehydrogenase

Citation:
Abstract
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.
PDB ID: 1OLXDownload
MMDB ID: 23966
PDB Deposition Date: 2003/8/18
Updated in MMDB: 2003/11
Experimental Method:
x-ray diffraction
Resolution: 2.25  Å
Source Organism:
Similar Structures:
Biological Unit for 1OLX: tetrameric; determined by author and by software (PQS)
Molecular Components in 1OLX
Label Count Molecule
Proteins (4 molecules)
2
2-oxoisovalerate Dehydrogenase Alpha Subunit(Gene symbol: BCKDHA)
Molecule annotation
2
2-oxoisovalerate Dehydrogenase Beta Subunit(Gene symbol: BCKDHB)
Molecule annotation
Chemicals (12 molecules)
1
4
2
2
3
2
4
4
* Click molecule labels to explore molecular sequence information.

Citing MMDB
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