Structure of rat BCKD kinase: Nucleotide-induced domain communication in a mitochondrial protein kinase

Machius, Mischa; Chuang, Jacinta L.; Wynn, R. Max; Tomchick, Diana R.; Chuang, David T.

doi:10.1073/pnas.201220098

Cited by 82 publications

(115 citation statements)

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“…In 12 of these cases, the structures represented arginine-processing enzymes, and in three other cases, arginine derivatives were used for crystallization. Only in the case of the structures of a branched-chain a-ketoacid dehydrogenase (BCKD) (PDB codes: 1GJV, 1GKX, and 1GKZ), was a relatively high concentration of arginine (300 mM) used during purification and crystallization (Machius et al 2001), in order to achieve better protein stability. During the crystallization process, the protein has to be stable for a prolonged period of time in highly concentrated solution, which may not be possible for proteins that easily aggregate.…”

Section: Resultsmentioning

confidence: 99%

Crystal structures of TM0549 and NE1324—two orthologs of E. coli AHAS isozyme III small regulatory subunit

et al. 2007

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Crystal structures of two orthologs of the regulatory subunit of acetohydroxyacid synthase III (AHAS, EC 2.2.1.6) from Thermotoga maritima (TM0549) and Nitrosomonas europea (NE1324) were determined by single-wavelength anomalous diffraction methods with the use of selenomethionine derivatives at 2.3 Å and 2.5 Å , respectively. TM0549 and NE1324 share the same fold, and in both proteins the polypeptide chain contains two separate domains of a similar size. Each protein contains a C-terminal domain with ferredoxin-type fold and an N-terminal ACT domain, of which the latter is characteristic for several proteins involved in amino acid metabolism. The ferredoxin domain is stabilized by a calcium ion in the crystal structure of NE1324 and by a Mg(H 2 O) 6 2+ ion in TM0549. Both TM0549 and NE1324 form dimeric assemblies in the crystal lattice.Keywords: acetohydroxyacid synthase; actolactate synthase; regulatory subunit; ACT domain; AHAS; protein refolding Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) catalyzes two physiologically significant reactions in the synthesis of isoleucine, valine, and leucine. In the first reaction, which is an initial step in the synthesis of isoleucine, 2-aceto-2-hydroxy butyrate is produced by condensation of 2-ketobutyrate with pyruvate. In the second reaction, 2-acetolactate is synthesized from two pyruvate molecules, and this provides substrates for synthesis of valine and leucine (Umbarger 1978(Umbarger , 1987Chipman et al. 1998). This pathway of branched-chain amino acid biosynthesis is characteristic for bacteria, fungi, algae, and higher plants, but not for animals. Accordingly, AHAS inhibitors have been developed as herbicides which have found broad application (Short and Colborn 1999). The inhibitors of branched-chain amino acids synthesis are also being tested as potential antituberculosis agents (Grandoni et al. 1998;Zohar et al. 2003;Choi et al. 2005).Three different FAD-dependent AHAS isozymes (I, II, and III) are found in enterobacteria (e.g., Escherichia ), but most other organisms from the bacteria encode only a single AHAS enzyme, similar to isozyme III from E. coli. The acetohydroxyacid synthases are multimeric proteins, and most frequently their biological unit is composed of two catalytic subunits (CSU) and two small regulatory subunits (SSU). In the absence of the SSU subunit, the CSU dimer is unstable (Vyazmensky et al. 1996), suggesting that in the holoenzyme the AHAS dimer is stabilized by a SSU dimer. The large catalytic subunits also show very weak activity without their associated regulatory subunits (Weinstock et al. 1992;Pang and Duggleby 1999). The E. coli AHAS enzyme can be reconstituted with SSU subunits from other organisms (Porat et al. 2004), and the reconstituted enzymes form stable heterotetramers. The properly associated holoenzymes show full catalytic activity, and are also susceptible to valine inhibition.Previous mutagenesis studies (Mendel et al. 2001;Kaplun et al. 2006) revealed that the valine binding sites are located at the dimerizatio...

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Section: Resultsmentioning

confidence: 99%

Crystal structures of TM0549 and NE1324—two orthologs of E. coli AHAS isozyme III small regulatory subunit

et al. 2007

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“…Other Methods-The construction and expression of His 6 -tagged BCK and MBP-BCK by co-transformation with the pGroESL plasmid overproducing chaperonins GroEL and GroES were described previously (7,11).…”

Section: Construction Of Expression Plasmids For E2bmentioning

confidence: 99%

“…The N-terminally His 6 -tagged BCK is soluble to the extent of 2-3 mg/ml only in the presence of 300 mM arginine (7). However, the requirement of high arginine concentration for solubility was circumvented when N-terminally His 6 -tagged BCK was allowed to bind to lip-DD.…”

Section: Interactions Of His 6 -Tagged Bck With Lipoylated Di-domainmentioning

confidence: 99%

“…The structures of rat BCK in the apo-, ADP-bound, and ATP␥S-bound forms were recently determined (7). The BCK structures feature a nucleotide-binding (K) domain and a fourhelix bundle (B) domain, which are similar to modules found in bacterial protein-histidine kinases (8) and the related pyruvate dehydrogenase kinase 2 (PDK2) of the mitochondrial pyruvate dehydrogenase complex (PDC) (9).…”

mentioning

confidence: 99%

“…Both ATPase (7) and phosphotransfer/kinase (11) activities of BCK are stimulated by lipoylated E2b. We have speculated that the E2b core binds to the B domain of BCK to promote the interaction between the B and K domains, resulting in the activation of BCK (7).…”

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The C-terminal Hinge Region of Lipoic Acid-bearing Domain of E2b Is Essential for Domain Interaction with Branched-chain α-Keto Acid Dehydrogenase Kinase

Chuang

Wynn

Chuang

2002

Journal of Biological Chemistry

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The branched-chain ␣-keto acid dehydrogenase (BCKD) kinase (abbreviated as BCK) down-regulates activity of the mammalian mitochondrial BCKD complex by reversible phosphorylation of the decarboxylase (E1b) component of the complex. The binding of BCK to the holotransacylase (E2b) core of the BCKD complex results in the stimulation of BCK activity. Here we show that the lipoylated lipoic acid-bearing domain (lip-LBD) (residues 1-84) of E2b alone does not interact with BCK. However, lip-LBD constructs containing various lengths of the C-terminal hinge region of LBD are able to bind to BCK as measured by a newly developed solubility-based binding assay. Isothermal titration calorimetry measurements produced a dissociation constant of 8.06 ؋ 10 ؊6 M and binding enthalpy of -3.68 kcal/mol for the interaction of BCK with a construct containing lip-LBD and the Glu-Glu-Asp-Xaa-Xaa-Glu sequence of the C-terminal hinge region of LBD. These thermodynamic parameters are similar to those obtained for binding of BCK to a lipoylated di-domain construct, which harbors LBD, the entire hinge region, and the downstream subunit-binding domain of E2b. Our data establish that the C-terminal hinge region of LBD containing the above negatively charged residues is essential for the interaction between the lip-LBD construct and BCK.The mammalian mitochondrial branched-chain ␣-keto acid dehydrogenase (BCKD) 1 complex catalyzes the rate-limiting step in the oxidation of branched-chain amino acids leucine, isoleucine, and valine (1). Genetic defects in this macromolecular multienzyme complex result in the accumulation of branched-chain amino acids leading to inheritable maple syrup urine disease. The clinical phenotype includes early onset and often fatal acidosis, neurological derangement, and mental retardation among survivors (1). The mammalian BCKD complex is organized around a cubic core of 24-meric dihydrolipoyl transacylase (E2b), to which multiple copies of branched-chain ␣-keto acid decarboxylase (E1b), dihydrolipoamide dehydrogenase (E3), BCKD kinase (abbreviated as BCK), and BCKD phosphatase are attached through ionic interactions (2-4). The activity of the BCKD complex is tightly regulated by a phosphorylation/dephosphorylation cycle in response to dietary and hormonal signals (5). Phosphorylation of Ser-292 and Ser-302 in the ␣ subunit of E1b by BCK results in the inactivation of the BCKD complex (6).The structures of rat BCK in the apo-, ADP-bound, and ATP␥S-bound forms were recently determined (7). The BCK structures feature a nucleotide-binding (K) domain and a fourhelix bundle (B) domain, which are similar to modules found in bacterial protein-histidine kinases (8) and the related pyruvate dehydrogenase kinase 2 (PDK2) of the mitochondrial pyruvate dehydrogenase complex (PDC) (9). The K domain is highly conserved between BCK and members of the GHL ATPase family (10), with the presence of characteristic N1, G1, F, and G2 boxes and an "ATP lid." In BCK, the direct back-to-back interaction of two opposing K domains produces a d...

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Two Novel Mutations in theBCKDK(Branched-Chain Keto-Acid Dehydrogenase Kinase) Gene Are Responsible for a Neurobehavioral Deficit in Two Pediatric Unrelated Patients

et al. 2014

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Inactivating mutations in the BCKDK gene, which codes for the kinase responsible for the negative regulation of the branched-chain α-keto acid dehydrogenase complex (BCKD), have recently been associated with a form of autism in three families. In this work, two novel exonic BCKDK mutations, c.520C>G/p.R174G and c.1166T>C/p.L389P, were identified at the homozygous state in two unrelated children with persistently reduced body fluid levels of branched-chain amino acids (BCAAs), developmental delay, microcephaly, and neurobehavioral abnormalities. Functional analysis of the mutations confirmed the missense character of the c.1166T>C change and showed a splicing defect r.[520c>g;521_543del]/p.R174Gfs1*, for c.520C>G due to the presence of a new donor splice site. Mutation p.L389P showed total loss of kinase activity. Moreover, patient-derived fibroblasts showed undetectable (p.R174Gfs1*) or barely detectable (p.L389P) levels of BCKDK protein and its phosphorylated substrate (phospho-E1α), resulting in increased BCKD activity and the very rapid BCAA catabolism manifested by the patients' clinical phenotype. Based on these results, a protein-rich diet plus oral BCAA supplementation was implemented in the patient homozygous for p.R174Gfs1*. This treatment normalized plasma BCAA levels and improved growth, developmental and behavioral variables. Our results demonstrate that BCKDK mutations can result in neurobehavioral deficits in humans and support the rationale for dietary intervention.

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Structure of rat BCKD kinase: Nucleotide-induced domain communication in a mitochondrial protein kinase

Cited by 82 publications

References 46 publications

Crystal structures of TM0549 and NE1324—two orthologs of E. coli AHAS isozyme III small regulatory subunit

Crystal structures of TM0549 and NE1324—two orthologs of E. coli AHAS isozyme III small regulatory subunit

The C-terminal Hinge Region of Lipoic Acid-bearing Domain of E2b Is Essential for Domain Interaction with Branched-chain α-Keto Acid Dehydrogenase Kinase

Two Novel Mutations in theBCKDK(Branched-Chain Keto-Acid Dehydrogenase Kinase) Gene Are Responsible for a Neurobehavioral Deficit in Two Pediatric Unrelated Patients

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