The branched-chain alpha-keto acid dehydrogenase (BCKDH) complex is a crucial enzyme complex involved in the catabolism of branched-chain amino acids (BCAAs). BCAAs, including leucine, isoleucine, and valine, are essential amino acids that the human body cannot synthesize and must obtain from dietary sources. The BCKDH complex catalyzes an irreversible, rate-limiting step in the breakdown of these amino acids, playing a vital role in energy production, nutrient sensing, and cellular signaling.
Metabolic Roles of α-Keto Acid Dehydrogenase Complexes
Mitochondrial α-keto acid dehydrogenase complexes, including BCKDHc, are multi-subunit enzymes with crucial metabolic roles. Besides BCKDHc, the enzyme family comprises the pyruvate dehydrogenase complex (PDHc), α-ketoglutarate dehydrogenase complex (KGDHc), and α-ketoadipate dehydrogenase complex (KADHc). These complexes catalyze the oxidative decarboxylation of their specific α-keto acid substrates, generating the respective acyl-CoA products and reducing NAD+ to NADH (+H+). α-ketoadipate and α-ketoglutarate are α-keto acids with ω-carboxylic end groups, while pyruvate and the implicated branched-chain α-keto acids are α-keto acids with aliphatic end groups.
The PDHc connects glycolysis to the citric acid cycle and the biosynthetic pathways of certain fatty acids and steroids by producing acetyl-CoA from pyruvate. The BCKDHc and KADHc participate in the catabolism of selected amino acids, channeling acetyl-CoA and succinyl-CoA into the Krebs cycle. The substrates of the BCKDHc are the oxoacids α-ketoisocaproate, α-keto-β-methylvalerate, and α-ketoisovalerate, which form from the branched-chain amino acids Leu, Ile, and Val, respectively, by transamination. The KADHc converts α-ketoadipate, a common metabolic intermediate in the Lys, hydroxy-Lys, and Trp degradation pathways, to glutaryl-CoA. The BCKDHc also accepts α-ketobutyrate as a substrate and converts it to propionyl-CoA, a degradation product of Met.
The fate of acetyl-CoA produced by the above three enzyme complexes (PDHc, KADHc, BCKDHc), e.g., mitochondrial oxidation or feeding lipid synthesis pathways in the cytosol, depends on the activities of the KGDHc and isocitrate dehydrogenase, rate-limiting enzymes in the Krebs cycle; the KGDHc generates succinyl-CoA from α-ketoglutarate in the Krebs cycle. Succinyl-CoA is also a precursor for porphyrin synthesis.
A hybrid complex could also form between the hKGDHc and hKADHc both in vitro and in vivo, which suggests a link between the Krebs cycle and the Lys/Trp catabolic pathways.
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Catalyzed Reactions
The overall reactions take place in a series of steps that require the concerted actions of multiple components with distinct catalytic activities, as well as three prosthetic groups and two coenzymes/co-substrates. The first step is the decarboxylation of the specific α-keto acid substrate, catalyzed by the thiamine diphosphate (ThDP)-dependent E1 components (pyruvate dehydrogenase, E1p; α-ketoglutarate dehydrogenase, E1k; α-ketoadipate dehydrogenase, E1a or more commonly referred to as dehydrogenase E1 and transketolase domain-containing 1, DHTKD1; and branched-chain α-keto acid dehydrogenase, E1b). The emerging reactive intermediate is an enamine (and a C2α-carbanion, in equilibrium), which then reductively acylates the lipoyllysyl arm of the respective E2 component ([dihydro]lipoamide/lipoyl acetyltransferase/transacetylase, E2p; [dihydro]lipoamide/lipoyl succinyltransferase/transsuccinylase, E2k; [dihydro]lipoamide/lipoyl acyltransferase/transacylase, E2b), again by E1. Subsequently, a transthiolesterification reaction with CoA, catalyzed by E2, will generate the respective acyl-CoA product. In order to regenerate the catalytic power of the complexes, the reduced lipoate prosthetic group is reoxidized by the FAD-dependent E3 component ([dihydro]lipoamide/lipoyl dehydrogenase, [DH]LADH) producing NADH (+H+).
In the above-discussed series of reactions, the rate-limiting steps differ among the cognate complexes. For instance, based on kinetic studies using recombinant E. coli enzymes, the rate-limiting step is the formation of the first covalently attached pre-decarboxylation intermediate on the E1p (lactyl-ThDP) for the PDHc, while it is the E2k-catalyzed succinyl transfer to CoA for the KGDHc. For the hKADHc and hBCKDHc, the reductive acylations of the respective hE2-lipoates by the corresponding hE1 components are the rate-limiting steps.
Substrate specificity is primarily provided by the E1 and to a lesser degree by the E2 components, while the E3 component with the regenerative function is shared among all the complexes. The E1b component has evidently the broadest substrate specificity as it accepts all the implicated branched-chain α-keto acids and also α-ketobutyrate, among a few other compounds, as substrates, e.g., the bovine E1b also converts pyruvate and 4-methylthio-α-ketobutyrate, an alternative degradation product of Met. Observed maximal reaction rates were 70, 50, 40, 30, and 20% for α-ketoisocaproate, α-ketobutyrate, α-keto-β-methylvalerate, 4-methylthio-α-ketobutyrate, and pyruvate, respectively, relative to α-ketoisovalerate. In terms of substrate specificity, there are overlaps, although to much smaller degrees, in the other complexes, as well. For instance, hE1a is also capable of converting α-ketoglutarate and α-ketopimelate, hE1k can use α-ketoadipate, and ecE1k can also operate with pyruvate and α-ketovalerate. Acceptance of non-traditional/physiological substrates in an in vitro subunit-specific enzyme assay likely plays little significance in vivo, since subsequently, the relevant E2 component also has to be able to accept the emerging intermediates as substrates. For example, although ecE1k could use pyruvate or α-ketovalerate, no overall ecKGDHc activity could be observed with these substrates, implying discrimination at the E2k level. Similarly, benzoylformate could readily be decarboxylated by hE1b; however, the resulting intermediate could not be further processed by hE2b, leading to the (irreversible [suicidal]) inhibition of the hBCKDHc. The hE2k component, however, possesses comparable succinyl- and glutaryltransferase activities (see above that hE1k can also use α-ketoadipate and that there also exists a functional hybrid hKADHc/hKGDHc complex). Interestingly, the lipoate-carrying (or lipoyl) domains (LDs) of the ecE2p and ecE2k subunits could only serve as proper substrates for the ecElp and ecElk components, respectively.
Overall Structures of the Complexes
The α-keto acid dehydrogenase complexes possess fundamentally similar structural architectures inasmuch as they all comprise multiple copies of three catalytically active components (E1, E2, E3); the respective subunits display high structural similarity over different species and cognate complexes. The E2 components are of particular structural importance since these also form unique scaffolds to which the relevant E1 and E3 subunits can directly or indirectly and usually selectively tether as peripheral components. The active sites of the three catalytic components are interconnected by the flexible, lipoylated arms of the E2 core structure. In certain members of the family, these “mandatory” components are supplemented with additional structurally important (E3-binding protein, E3BP, in most eukaryotic PDHc) or regulatory (kinases and phosphatases in most eukaryotic PDHc and BCKDHc) proteins/enzymes. Stability and activity of the BCKDHc isolated from rat liver proved also to depend on the presence of K+.
There are a few exceptions to the above rather general structural architecture (individual/unique E1-3 subunits/components) in nature. For instance, catalytic domains of the E1k and E2k subunits are expressed as a single protein chain with no characteristic lipoyl prosthetic group in Corynebacteriales (actinobacteria), therefore, hybrid complexes form where an E2p subunit provides the lipoate moiety. Or the Pseudomonas putida (Gram-negative soil bacterium) expresses two different E3 isoforms, and E3-Val is specifically expressed for the assembly of the BCKDHc, while E3-Glc is part of the PDHc and KGDHc. In the diplonemid Paradiplonema papillatum, for the PDHc activity, E1p is replaced by the archaeal protein AceE that partners with the E2 and E3 subunits from the BCKDHc and/or KGDHc. Furthermore, as was already mentioned above, the hKGDHc and hKADHc form a hybrid complex, where they in fact also share the hE2k component, while the hE1a and hE1k components exhibit activities of the same order of magnitude [in the muscle, heart, and brain (Artiukhov et al. 2020)]; interestingly, no E1a protein has been yet identified in prokaryotes.
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Mitochondrial supercomplexes were shown to exist in in vitro studies of mitochondria. These clearly included the electron transport system protein complexes (CI, CIII, CIV or CII, CIII, CIV), which were the focus of most studies, but unknown associated proteins were also visible on cryo-electron tomography images. Earlier however, Lyubarev and Kurganov, based on enzyme interaction data, proposed a supercomplex-like organization of the TCA cycle enzymes with succinate dehydrogenase being one of the anchor points to the mitochondrial membrane. Novel results from Plokhikh et al., where presumably E1/E3 associates were visible near the CI and also in close proximity to the E2 core, point toward a broad inclusion of proteins in the mitochondrial supercomplexes, with PDHc and KGDHc being anchored near the membrane and CI.
Structural Characteristics of the Individual Components
Structures of the E1 Components
The E1 component catalyzes the first two steps in the catalytic cycle using a thiamine diphosphate (ThDP, or thiamine pyrophosphate, TPP) prosthetic group and Mg2+. The E1 proteins in the cognate complexes show surprisingly low sequence identity (<20%), yet they all adopt the characteristic α-keto acid dehydrogenase fold. The thiamine-containing active sites reside at the subunit interfaces; this was first described in the related transketolase enzyme. Even though these enzymes exist in either dimeric or tetrameric form, they always function as “dimers of active sites”; the hE1a, E1k in all species, E1b in the Gram-positive Lactococcus lactis bacteria, and E1p in Gram-negative bacteria form homodimers, whereas the E1b in most species and E1p in Gram-positive bacteria and eukaryotes form heterotetrameric assemblies (α2β2) encapsulating two active sites [here an αβ heterodimer is functionally equivalent to an E1k chain]. Each protein chain in the homodimeric E1p component comprises a flexible N-terminal stretch and three domains; the three domains-namely, the N-terminal, middle, and C-terminal domains-all adopt α/β-class folds. The pyrophosphate moiety in ThDP is coordinated by the N-terminal domain in one subunit, whereas the aminopyrimidine moiety is tethered by both the same N-terminal domain and the middle domain in the adjacent monomer; exact locations of the thiamine-binding residues/patches and the conformation of the prosthetic group may vary among species and/or ThDP-dependent enzymes. The E1k and hE1a components are more closely related to the heterotetrameric E1p and E1b components in terms of intrinsic structure while also adopting the above-described domain structure.
The N-terminal stretches in E1k in most species and E1p in Gram-negative bacteria participate in the assembly of the related complexes via interacting with the corresponding E2k catalytic core domain (CD) and E3 component and the peripheral subunit-binding domain (PSBD) of E2p, respectively. E1k has an extended N-terminal region with varying lengths in different species that usually carries two E2k-binding motifs, not present in hE1a.
Branched-Chain Amino Acids and BCKDH Complex
Branched-chain amino acids (BCAAs) constitute approximately 35% of essential amino acids in mammals, which cannot be synthesized by the body. These amino acids are vital components of most proteins. BCAAs, composed of leucine, isoleucine, and valine, are not synthesized in animals but in plants, bacteria, and fungi. They play important roles in nutrient sensing and cellular signaling, especially leucine, which activates the mammalian/mechanistic target of rapamycin complex 1 (mTORC1). BCAAs promote protein synthesis in muscle during physical training and under conditions of negative energy balance, such as syndromes of cachexia and aging.
In BCAA metabolism, the first step involves the transformation of BCAAs into branched-chain α-ketoacids (BCKAs) under the catalysis of branched-chain aminotransferases (BCATs). The corresponding BCKAs of leucine, isoleucine, and valine are α-ketoisocaproate (KIC), α-keto-β-methylvalerate (KMV), and α-ketoisovalerate (KIV). Two different genes encode BCATs: BCAT1, mostly expressed in the brain and encoding a cytoplasmic protein, and BCAT2, widely expressed and encoding a mitochondrial protein. This reversible transamination reaction largely occurs in skeletal muscle. After BCKAs are released back into circulation, they are oxidatively decarboxylated to acyl-CoA mainly in the liver.
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The branched-chain α-keto acid dehydrogenase (BCKDH) complex is an important enzyme that catalyzes this irreversible and rate-controlling process in BCAA catabolism. Although the liver and other tissues (such as adipose and muscle) all possess BCKDH and can carry out the catabolism of BCKAs, the liver is reported as the highest metabolic efficiency organ in the body in the oxidative decarboxylation of BCKAs. BCKAs covalently bind to a coenzyme A group and lose CO2 in an oxidative decarboxylation process catalyzed by BCKDH.
BCKDH Complex Components and Function
The BCKDH complex consists of three main components: E1, E2, and E3.
- E1 (Decarboxylase): Encoded by the BCKDHA and BCKDHB genes, E1 exists as an α2/β2 heterotetramer. It functions as a thiamin-dependent decarboxylase. The BCKDHA gene provides instructions for making one part, the alpha subunit, of a group of enzymes called the branched-chain alpha-keto acid dehydrogenase (BCKD) enzyme complex. Two alpha subunits connect with two beta subunits, which are produced from the BCKDHB gene, to form a critical piece of the enzyme complex called the E1 component.
- E2 (Transacylase): Encoded by the DBT gene, E2 acts as a lipoate-dependent dihydrolipoyl transacylase, transferring the acyl groups to coenzyme A.
- E3 (Dihydrolipoamide Dehydrogenase): Encoded by the DLD gene, E3 is a FAD-dependent dihydrolipoyl dehydrogenase and functions in transferring the released electrons to NAD+.
BCKDH converts KIC to isovaleryl-CoA (IV-CoA), KMV to α-methylbutyryl-CoA (MB-CoA), and KIV to isobutyryl-CoA (IB-CoA).
Regulation of BCKDH Complex
The activity of the BCKDH complex is tightly regulated by phosphorylation and dephosphorylation.
- BCKDH Kinase (BCKDK): BCKDK suppresses the activity of BCKDH by adding phosphate to three residues of BCKDHA. BCKAs, especially KIC, allosterically regulate BCKDK. High concentrations of BCKAs inhibit BCKDK activity, preventing BCAAs from running out when they are at low concentrations. BCKDK also seems to be regulated by the BCKDH complex.
- Mitochondrial Phosphatase 2C (PP2Cm): The dephosphorylation process, which activates the BCKDH complex, is catalyzed by mitochondrial phosphatase 2C (PP2Cm).
Downstream Catabolism
After the decarboxylation of BCKAs catalyzed by BCKDH, the subsequent catabolism step is similar to the oxidation of fatty acids. Each reaction is unique to the three BCAAs, and the mitochondrial matrix is the only site where these reactions take place. In the end, the carbons from BCAA catabolism are either released as CO2 or enter the tricarboxylic acid (TCA) cycle.
Detection Methods
Advancements in analytical techniques have enabled the detection of amino acids at very low concentrations in complex matrices.
- Liquid Chromatography-Mass Spectrometry (LC-MS) and Capillary Electrophoresis-Mass Spectrometry (CE-MS): These methods are widely used for detecting plasma BCAAs. Improved LC-MS methods include isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) and high-performance liquid chromatography with electrospray ionization mass spectrometry (LC-ESI-MS).
- Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (LC-ESI±-MS/MS): This method allows for the simultaneous investigation of BCAAs and BCKAs in human serum.
- Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Imaging (MALDI-MSI): This technique analyzes BCAAs localization in tissues, especially in the brain, by simultaneously locating and quantifying BCAAs in tissue sections.
- Detection of BCKDK: Real-time quantitative PCR, western blot, and immunohistochemical assays are used to analyze the expression of BCKDK. The activity of BCKDH, representing BCKDK function, can be measured by scintigraphy, and the phosphorylation rate of BCKDH also represents BCKDK function.
Role of Gut Microbiota
The gut microbiota plays an important role in modulating the utilization of numerous essential nutrients for the host. Gut microbiota can affect the circulating BCAA pool. The bacterial metabolism is considered to be the source of high levels of serum BCAAs in high-fat diets (HFD).
- In diet-induced obese (DIO) mice treated with luffa administration, the relative abundances of g-Enterortabdus, g-Eubacterium-xylanophilum-group, and g-Butyricicoccus were increased in 16S rRNA gene sequencing.
- In dietary luffa-treated DIO mice, the mRNA expression of the enzymes that catalyze BCAA catabolism to decrease BCAA levels is upregulated in tissues such as the liver, adipose, and colon.
- Patients with heart failure have lower plasma essential amino acid levels than healthy controls due to the lack of Eubacterium and Prevotella, leading to the decreased biosynthesis of essential amino acids (especially BCAAs and histidine).
- Gavage with Bacteroides spp. reduced the defects in BCAA catabolism of brown adipose tissue (BAT) caused by obesity and protected mice from obesity by altering gut microbiota composition and reducing the levels of BCAA and BCKA in BAT.
Implications in Metabolic Disorders
Dysregulation of BCAA metabolism and BCKDH complex activity are implicated in several metabolic disorders, including obesity, insulin resistance, and heart failure.
Insulin Resistance and Type 2 Diabetes Mellitus (T2DM)
Concentrations of plasma BCAAs, BCKAs, and carnitine esters are elevated in insulin resistance (IR), obesity, and type 2 diabetes mellitus (T2DM) patients and mice. Circulating levels of BCAA and BCKA show a correlation with body weight. Decreased metabolism of BCAAs contributes to obesity. The translation of BCAAs metabolism enzymes in adipose and liver is inhibited in obese mice, and the increased expression of BCKDK leads to the increased phosphorylation of BCKDH E1α and impaired activity of BCKDH in the liver. BCKDK inhibitor treatment of diet-induced obese mice can inhibit weight gain and reduce the BCAAs and BCKAs concentrations in plasma. BCKDK promotes the occurrence of obesity by inhibiting BCAAs metabolism in obese animals. Elevated BCAAs in plasma might be related to impaired insulin action since it increases protein catabolism in obese animals.
Elevated plasma BCAAs and BCKAs concentrations are early signs of insulin resistance in clinical studies. The increase of circulating BCKAs is better correlated with the severity of IR and T2DM, making BCKAs more effective and reliable biomarkers for IR. Accumulation of BCKAs is also an indicator of glucose intolerance and cardiac insufficiency and diabetes.
BCAAs and BCKAs are involved in multiple IR pathways, including mTOR, insulin receptor substrate 1 (IRS1) pathway, fatty acid oxidation, and c-Jun NH2 terminal kinase. Activation of the mTORC1 pathway by leucine via Rag GTPases is considered to be an important mechanism of the impaired insulin signaling pathway. Serum BCKAs inhibits insulin signaling by inhibiting AKT phosphorylation.
BCKDK can not only regulate BCAAs metabolism with BCAA as substrate but also phosphorylate ATP-Citrate Lyase (ACL), a critical enzyme in fatty acid synthesis from the beginning, and regulate the de novo formation pathway of fat, thus regulating the content of fatty acids.
Heart Failure
BCAAs are found to be increased in the failing heart, and cardiac BCAAs oxidation is decreased in insulin resistance. In a permanent myocardial infarction (MI) model, BCAAs catabolism was damaged seriously in the myocardium, leading to an elevation in BCAAs levels and activating mTOR signaling, exacerbating cardiac dysfunction and remodeling. BCAA levels were increased in dilated cardiomyopathy (DCM) hearts, accompanied by a decreased expression of mitochondrial BCAT2 and total expression of BCKDH compared to non-failing control. Phosphorylation of BCKDH and expression of cardiac PP2Cm were reduced in the DCM hearts, with an unchanged expression of BCKDK. KLF15 expression is inhibited through the TAK1/P38MAPK axis in heart failure patients, thus inhibiting BCAA catabolism, which leads to BCAAs accumulated in the heart. Increasing the oxidation rate of BCAAs has proved a practicable way to improve the contractile function of failing hearts in mice.
Maple Syrup Urine Disease (MSUD)
Mutations in genes encoding components of the BCKDH complex can cause maple syrup urine disease (MSUD), a rare inherited metabolic disorder characterized by the accumulation of BCAAs and their toxic byproducts in the body. The BCKDHA gene provides instructions for making one part, the alpha subunit, of a group of enzymes called the branched-chain alpha-keto acid dehydrogenase (BCKD) enzyme complex. Two alpha subunits connect with two beta subunits, which are produced from the BCKDHB gene, to form a critical piece of the enzyme complex called the E1 component.
Most BCKDHA mutations change single amino acids in the alpha subunit of the BCKD enzyme complex. In the Old Order Mennonite population, where maple syrup urine disease occurs frequently, the most common mutation replaces the amino acid tyrosine with the amino acid asparagine at position 438 (written as Tyr438Asn or Y438N). Mutations in the BCKDHA gene disrupt the normal function of the BCKD enzyme complex, preventing it from effectively breaking down leucine, isoleucine, and valine. As a result, these amino acids and their byproducts build up in the body, which is toxic to cells and tissues, particularly in the nervous system.
BCKDH Kinase and Exercise
BCKDH kinase is responsible for the inactivation of the BCKDH complex by phosphorylation. Acute exercise activates the complex in association with a decrease in the bound form of kinase in both liver and muscle. The free form of kinase in both tissues was slightly increased, but the total amount of the kinase was not affected by acute exercise. The protein amount ratio of bound kinase to E1beta component of the complex was much higher in muscle than in the liver of rats, reflecting the low activity state of the complex in muscle. These results suggest that the amount of the bound kinase plays an important role in regulation of the activity state of the complex.