Cryptic proteolytic activity of dihydrolipoamide dehydrogenase

Abstract

The mitochondrial enzyme, dihydrolipoamide dehydrogenase (DLD), is essential for energy metabolism across eukaryotes. Here, conditions known to destabilize the DLD homodimer enabled the mouse, pig, or human enzyme to function as a protease. A catalytic dyad (S456 -E431) buried at the homodimer interface was identified. Serine protease inhibitors and an S456A or an E431A point mutation abolished the proteolytic activity, whereas other point mutations at the homodimer interface domain enhanced the proteolytic activity, causing partial or complete loss of DLD activity. In humans, mutations in the DLD homodimer interface have been linked to an atypical form of DLD deficiency. These findings reveal a previously unrecognized mechanism by which certain DLD mutations can simultaneously induce the loss of a primary metabolic activity and the gain of a moonlighting proteolytic activity. The latter could contribute to the metabolic derangement associated with DLD deficiency and represent a target for therapies of this condition.is a f lavindependent oxidoreductase required for the complete reaction of at least five different multienzyme complexes. In the mitochondrial matrix, the DLD homodimer functions as the E3 component of the pyruvate, ␣-ketoglutarate, and branchedchain amino acid-dehydrogenase complexes and the glycine cleavage system. In the context of these four multienzyme complexes, DLD utilizes dihydrolipoic acid and NAD ϩ to generate lipoic acid and NADH (1). The opposite reaction, from lipoic acid and NADH to dihydrolipoic acid and NAD ϩ , is catalyzed by the DLD homodimer in the context of the NAD ϩ -dependent peroxidase-peroxinitrate reductase of Mycobacterium tuberculosis (2). In addition, DLD has diaphorase activity, being able to catalyze the oxidation of NADH to NAD ϩ by using different electron acceptors such as O 2 , labile ferric iron (3), nitric oxide (4), and ubiquinone (5, 6). In this capacity, DLD is believed to primarily have a prooxidant role, achieved by reducing O 2 to a superoxide radical or ferric to ferrous iron, which in turn catalyzes production of hydroxyl radical through Fenton chemistry (3). However, the ability to scavenge nitric oxide and to reduce ubiquinone to ubiquinol suggests that the diaphorase activity of DLD may also have an antioxidant role (4-6). The oligomeric state of DLD can change from dimeric to monomeric or tetrameric depending on the pH in the mitochondrial matrix, and changes in the oligomeric state of the protein have been shown to correlate with a shift from DDL activity (only present in dimeric and tetrameric DLD) to diaphorase activity (present in both oligomeric and monomeric forms of the protein) (7,8). Thus, DLD represents a highly versatile oxidoreductase with multiple critical roles in energy metabolism and redox balance (2,7,(9)(10)(11)(12).Here, we found that DLD can also function as a moonlighting protease. Moonlighting enzymes are a growing category of molecules that can accomplish multiple functions through a variety of mechanisms including, among other...

Molecular dynamics study of the structural basis of dysfunction and the modulation of reactive oxygen species generation by pathogenic mutants of human dihydrolipoamide dehydrogenase

“…As detailed in the Introduction, LADH is greatly susceptible to the ambient conditions, and as many studies suggest, changes in LADH function are based on specific structural (conformational) changes [3,8,10,11,27,29,58]. It is important to note that several pathogenic mutations [30] or the drop in pH alone [3] can facilitate the dissociation of LADH from multienzyme complexes.…”

“…FAD binds non-covalently and is thought to play a crucial role in ROS-generation [28]. Monomerization of LADH was proposed to occur due to mild acidification [8] or specific pathogenic mutations [27,29], which could potentially account for the transformation of LADH to a diaphorase [8] and/or a Ser-protease [29], respectively. Later studies partly also by our laboratory, disproved monomer formation under the above conditions and proposed that instead, specific conformational changes would be responsible for the observed phenomena [3,11,30].…”

Changes in dihydrolipoamide dehydrogenase expression and activity during postnatal development and aging in the rat brain

“…While DLDH itself may be a source of reactive oxygen species (Bando and Aki, 1991, Gazaryan, et al, 2002, Sreider, et al, 1990, Tahara, et al, 2007, it is also capable of scavenging nitric oxide (Igamberdiev, et al, 2004) and can serve as an antioxidant by protecting other proteins against oxidative inactivation by 4-hydroxyl-2-nonenal (Korotchkina, et al, 2001). Moreover, DLDH can also act as a proteolytic enzyme when the stability of its homodimer is altered (Babady, et al, 2007).…”

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

“…Therefore, to examine the question of whether the mode of wild-type hSBDb and hSBDb* binding to hE3 was significantly different, we turned to solution NMR studies. Intriguingly, we observed deterioration of the NMR spectrum with time, indicating damage to hSBDb presumably by the cryptic proteolytic activity of hE3 (29). These observations likely are the compounded effects of the high protein concentration and the long acquisition times typically used in the NMR studies and can be alleviated by using an hE3 S456A mutant, referred to here as hE3*.…”