Structural and Thermodynamic Basis for Weak Interactions between Dihydrolipoamide Dehydrogenase and Subunit-binding Domain of the Branched-chain α-Ketoacid Dehydrogenase Complex

Abstract

The purified mammalian branched-chain ␣-ketoacid dehydrogenase complex (BCKDC), which catalyzes the oxidative decarboxylation of branched-chain ␣-keto acids, is essentially devoid of the constituent dihydrolipoamide dehydrogenase component (E3). The absence of E3 is associated with the low affinity of the subunit-binding domain of human BCKDC (hSBDb) for hE3. In this work, sequence alignments of hSBDb with the E3-binding domain (E3BD) of the mammalian pyruvate dehydrogenase complex show that hSBDb has an arginine at position 118, where E3BD features an asparagine. Substitution of Arg-118 with an asparagine increases the binding affinity of the R118N hSBDb variant (designated hSBDb*) for hE3 by nearly 2 orders of magnitude. The enthalpy of the binding reaction changes from endothermic with the wild-type hSBDb to exothermic with the hSBDb* variant. This higher affinity interaction allowed the determination of the crystal structure of the hE3/hSBDb* complex to 2.4-Å resolution. The structure showed that the presence of Arg-118 poses a unique, possibly steric and/or electrostatic incompatibility that could impede E3 interactions with the wild-type hSBDb. Compared with the E3/E3BD structure, the hE3/hSBDb* structure has a smaller interfacial area. Solution NMR data corroborated the interactions of hE3 with Arg-118 and Asn-118 in wild-type hSBDb and mutant hSBDb*, respectively. The NMR results also showed that the interface between hSBDb and hE3 does not change significantly from hSBDb to hSBDb*. Taken together, our results represent a starting point for explaining the long standing enigma that the E2b core of the BCKDC binds E3 far more weakly relative to other ␣-ketoacid dehydrogenase complexes.The mitochondrial ␣-ketoacid dehydrogenase complexes include the pyruvate dehydrogenase complex (PDC), 4 the ␣-ketoglutarate dehydrogenase complex and the branched-chain ␣-ketoacid dehydrogenase complex (BCKDC) (1, 2). These macromolecular protein complexes catalyze the oxidative decarboxylation of ␣-keto acids according to Reaction 1.Each ␣-ketoacid dehydrogenase complex consists of three catalytic components: a thiamine diphosphate-dependent decarboxylase/dehydrogenase (E1), a lipoyl transacylase (E2), and dihydrolipoamide dehydrogenase (E3). The overall reaction is the sum of individual reactions catalyzed by the three enzyme components, which are linked by substrate channeling. The ␣-ketoacid dehydrogenase complexes are organized around a symmetrical oligomeric E2 core, to which the E1 and E3 components are attached via noncovalent interactions. The E1 and E2 components are specific for each ␣-ketoacid dehydrogenase complex, whereas the E3 component is common among the three multienzyme complexes. The E1p, E1k, and E1b components (with the lowercase letter corresponding to each complex), similarly the E2p, E2k, and E2b components, belong to PDC, ␣-ketoglutarate dehydrogenase complex, and BCKDC, respectively. Both E1 and E3 components bind in a mutually exclusive manner to the subunit-binding domains (SBD) from the 24-meri...

Urinary Branched-Chain 2-Oxo Acids as a Biomarker for Function of B-Group Vitamins in Humans

“…MSUD is a defect of BCKDC that consists of E1a1b/E2/E3 subunits. Dihydrolipoamide dehydrogenase (E3) subunit is the same protein subunit of pyruvate dehydrogenase complex and 2-oxoglutarate dehydrogenase complex (18,19). MSUD is often classified into five types by the pattern of signs and symptoms: the classic severe MSUD, intermediate MSUD, intermittent MSUD, dihydrolipoamide dehydrogenase (E3)-deficient MSUD with lactic acidosis, and thiamin-responsive MSUD (19).…”

Regulation of pyruvate metabolism and human disease

“…However, E3 binds PDH much more strongly compared to the core complex of the branch chain amino acid dehydrogenase complex [80, 81]. This difference is attributed to the presence of an arginine residue in the E3 binding protein of the branched chain amino acid dehydrogenase complex, whereas an asparagine is in the PDH complex.…”

“…E3 is associated not only with the PDH complex but also with the α-ketoglutarate dehydrogenase complex and the branched chain amino acid dehydrogenase complex, where it performs a similar catalytic function. However, E3 binds PDH much more strongly compared to the core complex of the branch chain amino acid dehydrogenase complex [ 80 , 81 ]. This difference is attributed to the presence of an arginine residue in the E3 binding protein of the branched chain amino acid dehydrogenase complex, whereas an asparagine is in the PDH complex.…”

Computational Study on New Natural Compound Inhibitors of Pyruvate Dehydrogenase Kinases

“…PDKs are non-covalently bound to the lipoyl domain (L2) of the E2 subunit of PDC. They phosphorylate the pyruvate dehydrogenase (PDH)-E1α and inactivate PDC [ 4 , 5 , 6 , 7 ]. In mammalians, there are four isoenzymes of PDKs (PDK 1 to 4) with different binding affinity, phosphorylation site specificity and tissue distribution [ 8 , 9 ].…”