Cross-talk between Thiamin Diphosphate Binding and Phosphorylation Loop Conformation in Human Branched-chain α-Keto Acid Decarboxylase/Dehydrogenase

Li, Jun; Wynn, R. Max; Machius, Mischa; Chuang, Jacinta L.; Karthikeyan, Subramanian; Tomchick, Diana R.; Chuang, David T.

doi:10.1074/jbc.m403611200

Cited by 30 publications

(49 citation statements)

References 37 publications

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“…It is noteworthy that the order-disorder transformation in this ␣2 E1 dimer differs considerably from those that have been observed in certain ␣2␤2 E1 heterotetramers (14,32). In the latter cases, the addition of the cofactor alone to the disordered apoenzyme produced an ordered holoenzyme.…”

Section: Discussionmentioning

confidence: 75%

“…In the ␣2 homodimeric E1 protein, disorder occurs in both the apoenzymes and holoenzymes, and it is only the formation of the pre-decarboxylation reaction intermediate that triggers ordering. In addition, the loops in ␣2 E1 that exhibit transformation are not the same as those reported to undergo transformation in the ␣2␤2 cases (14,32,33).…”

Section: Discussionmentioning

confidence: 78%

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A Thiamin-bound, Pre-decarboxylation Reaction Intermediate Analogue in the Pyruvate Dehydrogenase E1 Subunit Induces Large Scale Disorder-to-Order Transformations in the Enzyme and Reveals Novel Structural Features in the Covalently Bound Adduct

Arjunan

Sax

Brunskill

et al. 2006

Journal of Biological Chemistry

168

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Thiamin-dependent enzymes play key roles in sugar metabolism, typically catalyzing the decarboxylation of ␣-keto acids and the transfer of an aldehyde or an acyl group (1-5). Examples include the E1 components in pyruvate dehydrogenase complexes (PDHc), 2 pyruvate decarboxylase, transketolase, etc. Crystallographic studies (6 -12) have elucidated many of the structural, stereochemical, and biochemical details in the mechanism of action of these enzymes and in the catalytic role of the cofactor ThDP (thiamin diphosphate, vitamin B1 diphosphate, Fig. 1, top left). Despite the enormous contributions made by these and other studies to our understanding of how such enzymes function, important details still remain obscure. There are, for example, no detailed structural data on the first ThDP-bound intermediate in the presence of any enzyme (for example, ␣-lactylthiamin diphosphate (␣-LThDP) in PDHc E1 and pyruvate decarboxylase), which is postulated to form in the currently accepted mechanism of thiamin catalysis (Fig. 1, top, third object from the right). In an effort to obtain structural information pertaining to this key intermediate, we have determined the crystal structure of PDHc E1 from Escherichia coli in complex with ␣-phosphonolactylthiamin diphosphate (PLThDP).PLThDP is the product of the reaction between ThDP and methylacetylphosphonate, with the latter being an analogue of the true substrate pyruvate and a potent inhibitor of PDHc. The complex formed with PLThDP instead of ThDP therefore mimics the structure of the enzyme-bound, reactive tetrahedral intermediate ␣-LThDP (13) in the decarboxylation step of the PDHc E1 reaction. It differs from the complex formed with the true substrate only in the replacement of the carboxylate group by a methyl phosphonate (PO 3 Me) group. However, unlike the C2␣-CO 2 bond normally cleaved in the reaction with pyruvate, the C2␣-PO 3 Me bond remains intact. The reaction is therefore trapped in a pre-CO 2 release-like state, and the structure represents a covalently bound, pre-decarboxylation reaction intermediate analogue.There have been three covalently bound reaction intermediate structures reported for ThDP-dependent enzymes (11,12,14), but they all represented the planar enamine intermediate (Fig. 1, top right object) that exists only after decarboxylation. The E1-PLThDP structure is thus the first structural example of a covalently bound, pre-decarboxylation reaction intermediate analogue in any ThDP-dependent enzyme.* This work was supported by a grant from the Veterans Affairs Merit Review Program and National Institutes of Health Grant GM-61791 (to W. F.) and by National Institutes of Health Grant GM-62330 (to F. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (codes 2G25 and 2G28)

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Section: Discussionmentioning

confidence: 75%

Section: Discussionmentioning

confidence: 78%

A Thiamin-bound, Pre-decarboxylation Reaction Intermediate Analogue in the Pyruvate Dehydrogenase E1 Subunit Induces Large Scale Disorder-to-Order Transformations in the Enzyme and Reveals Novel Structural Features in the Covalently Bound Adduct

Arjunan

Sax

Brunskill

et al. 2006

Journal of Biological Chemistry

168

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“…The mixture was preincubated at room temperature for 15 min. The phosphorylation reaction was started after addition of 0.4 mM ATP to the reaction mixture, and the reaction was terminated at different time points by addition of higher salt concentration (15). Overall BCKDC activity was measured as described above.…”

Section: Methodsmentioning

confidence: 99%

“…E1 was reconstituted with BCATm at a 1:1 molar ratio, and GDH1 was added at varied molar ratios to BCATm. The rate of decarboxylation at 30°C was measured by monitoring the reduction of the dye at 600 nm (15,33).…”

Section: Methodsmentioning

confidence: 99%

“…We have shown that the deaminated BCKA product of BCATm is directly transferred to the E1 decarboxylase of BCKDC through complex formation between the two enzymes and substrate channeling (15). The association between BCATm and E1 is specific for the PLP form of the BCATm isozyme, requires a reduced CXXC center, is inhibited by phosphorylation of E1, and is dissociated by the BCKDC reaction product NADH (5).…”

mentioning

confidence: 99%

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Branched-chain Amino Acid Metabolon

Islam

Nautiyal

Wynn

et al. 2010

Journal of Biological Chemistry

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The catabolic pathway for branched-chain amino acids includes deamination followed by oxidative decarboxylation of the deaminated product branched-chain ␣-keto acids, catalyzed by the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain ␣-keto acid dehydrogenase enzyme complex (BCKDC). We found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of branched-chain ␣-keto acids from BCATm to E1. The protein complex also contains glutamate dehydrogenase (GDH1), 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1, pyruvate carboxylase, and BCKDC kinase. GDH1 binds to the pyridoxamine 5-phosphate (PMP) form of BCATm (PMP-BCATm) but not to the pyridoxal 5-phosphateBCATm and other metabolon proteins. Leucine activates GDH1, and oxidative deamination of glutamate is increased further by addition of PMP-BCATm. Isoleucine and valine are not allosteric activators of GDH1, but in the presence of 5-phosphate-BCATm, they convert BCATm to PMP-BCATm, stimulating GDH1 activity. Sensitivity to ADP activation of GDH1 was unaffected by PMP-BCATm; however, addition of a 3 or higher molar ratio of PMP-BCATm to GDH1 protected GDH1 from GTP inhibition by 50%. Kinetic results suggest that GDH1 facilitates regeneration of the form of BCATm that binds to E1 decarboxylase of the BCKDC, promotes metabolon formation, branched-chain amino acid oxidation, and cycling of nitrogen through glutamate.It has been proposed that the enzymes that are responsible for catalyzing sequential reactions in several metabolic pathways are highly organized in supramolecular complexes termed metabolons (1). The concept of metabolic enzymes associating to form a supramolecular complex was hypothesized 50 years ago (2). Recently, Benkovic and co-workers (3) have shown the in vivo assembly of six proteins in the purine catabolic pathway to form a "purinosome." The formation of the purinosome appears to be dynamically regulated by stimulation of de novo purine biosynthesis in response to changes in purine levels. Another group (4) also has shown that glycolytic enzymes ("glycosome") are organized into complexes, and this assembly is regulated by the oxidation and phosphorylation states of the proteins. The advantages of such a supramolecular assembly include efficient channeling of substrates between enzymes in a pathway, regulating pathway flux by association and dissociation, and by targeting the assembly of the interacting proteins to the appropriate intracellular structures. Previously, using liver mitochondrial extracts and purified BCATm, 2 we showed that the first two enzymes in BCAA catabolism, mitochondrial branched-chain aminotransferase (BCATm) and branchedchain ␣-keto acid decarboxylase (E1) of the BCKDC, associate in vitro (5). It is not yet known if this BCAA metabolon forms in tissues expressing BCATm, and if other associated proteins are functionally involved in the metabolon.BCATs (mitochondrial and cytosolic isozymes) catalyze reversible transamination of BCAA...

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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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Cross-talk between Thiamin Diphosphate Binding and Phosphorylation Loop Conformation in Human Branched-chain α-Keto Acid Decarboxylase/Dehydrogenase

Cited by 30 publications

References 37 publications

A Thiamin-bound, Pre-decarboxylation Reaction Intermediate Analogue in the Pyruvate Dehydrogenase E1 Subunit Induces Large Scale Disorder-to-Order Transformations in the Enzyme and Reveals Novel Structural Features in the Covalently Bound Adduct

A Thiamin-bound, Pre-decarboxylation Reaction Intermediate Analogue in the Pyruvate Dehydrogenase E1 Subunit Induces Large Scale Disorder-to-Order Transformations in the Enzyme and Reveals Novel Structural Features in the Covalently Bound Adduct

Branched-chain Amino Acid Metabolon

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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