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

Abstract

The dihydrolipoamide acyltransferase (E2b) component of the branched-chain a-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-Å 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.

Analysis of gene mutations in Chinese patients with maple syrup urine disease

“…The locus of I256 is predicted to be in the C-terminal core domain [22]. Therefore, the p.I256IfsX15 mutation disrupts the stability of the natural cubic core [28]. The c.51+5G>C splicing mutation was predicted with GENSCAN to affect the normal splicing pattern of exon 1.…”

“…E239-β is located in the inner core of the E1β protein. The E239L mutation may affect the stability of the region [28]. Patient P10, carrying the c.951+1G>T and R285X allelic variants, presented the classic type and died at the age of 11 months.…”

Lipoic acid metabolism and mitochondrial redox regulation

“…The E2 subunit undergoes a conformational change upon CoA binding, which allows for the lipoyl-acyl intermediate to enter the acyltransferase catalytic site (79). This mechanism prevents the reduced lipoamide from entering the E2 catalytic site when free CoA levels are reduced.…”

A Novel Branched-chain Amino Acid Metabolon

“…There is considerable structural information on the mammalian BCKD complex proteins (31)(32)(33)(34)(35) and the BCAT isozymes (6,7,36). The BCAT isozymes are homodimers and belong to the fold type IV class of PLP (vitamin B 6 )-dependent enzymes.…”