Abstract
Dihydrolipoamide dehydrogenase (DLD) is a multifunctional protein well characterized as the E3 component of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes. Previously, conditions predicted to destabilize the DLD dimer revealed that DLD could also function as a diaphorase and serine protease. However, the relevance of these cryptic activities remained undefined. We analyzed human DLD mutations linked to strikingly different clinical phenotypes, including E340K, D444V, R447G, and R460G in the dimer interface domain that are responsible for severe multisystem disorders of infancy and G194C in the NAD(+)-binding domain that is typically associated with milder presentations. In vitro, all of these mutations decreased to various degrees dihydrolipoamide dehydrogenase activity, whereas dimer interface mutations also enhanced proteolytic and/or diaphorase activity. Human DLD proteins carrying each individual mutation complemented fully the respiratory-deficient phenotype of yeast cells lacking endogenous DLD even when residual dihydrolipoamide dehydrogenase activity was as low as 21% of controls. However, under elevated oxidative stress, expression of DLD proteins with dimer interface mutations greatly accelerated the loss of respiratory function, resulting from enhanced oxidative damage to the lipoic acid cofactor of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase and other mitochondrial targets. This effect was not observed with the G194C mutation or a mutation that disrupts the proteolytic active site of DLD. As in yeast, lipoic acid cofactor was damaged in human D444V-homozygous fibroblasts after exposure to oxidative stress. We conclude that the cryptic activities of DLD promote oxidative damage to neighboring molecules and thus contribute to the clinical severity of DLD mutations.
Highlights
Dihydrolipoamide dehydrogenase (DLD) is an enzyme involved in energy metabolism known to possess two cryptic enzymatic activities of as yet undefined significance
Mutations in Dimer Interface Domain of Human DLD Have Mild Effects on Dihydrolipoamide Dehydrogenase Activity in Vitro—We expressed in Escherichia coli the mature form of human DLD, including wild type (WT), four interface domain mutants (E340K, D444V, R447G, and R460G), and an NADϩ-binding domain mutant (G194C) (Fig. 2, A and B; numbering starts from the N terminus of the mature protein after removal of the 35-residue mitochondrion-targeting signal)
Human fibroblasts expressing D444V DLD revealed a pattern of damage similar to that of the corresponding yeast strain whereby the integrity of pyruvate dehydrogenase (PDH) and ␣-KGDH lipoic acid was largely unaffected during glycolytic growth but was progressively lost during respiratory growth upon exposure to pro-oxidant compounds that have been shown to serve as substrates for DLD diaphorase activity in vitro [7, 51, 52] (Fig. 6, C and D). These findings indicate that damage to the lipoic acid cofactor covalently bound to the E2 protein of PDH and ␣-KGDH probably represents an important mechanism associated with DLD dimer interface mutations
Summary
Dihydrolipoamide dehydrogenase (DLD) is an enzyme involved in energy metabolism known to possess two cryptic enzymatic activities of as yet undefined significance. Results: DLD mutations that enhance these cryptic activities promote oxidative damage to specific mitochondrial targets. Under elevated oxidative stress, expression of DLD proteins with dimer interface mutations greatly accelerated the loss of respiratory function, resulting from enhanced oxidative damage to the lipoic acid cofactor of pyruvate dehydrogenase and ␣-ketoglutarate dehydrogenase and other mitochondrial targets. This effect was not observed with the G194C mutation or a mutation that disrupts the proteolytic active site of DLD. We conclude that the cryptic activities of DLD promote oxidative damage to neighboring molecules and contribute to the clinical severity of DLD mutations
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