Structural and Biochemical Characterization of Human Mitochondrial Branched-chain α-Ketoacid Dehydrogenase Phosphatase

• Published in: The Journal of biological chemistry Vol. 287; no. 12; pp. 9178 - 9192
• Main Authors: Wynn, R. Max, Li, Jun, Brautigam, Chad A., Chuang, Jacinta L., Chuang, David T.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.03.2012; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Branched-chain α-Ketoacid Dehydrogenase Complex; Catalytic Domain; Central β-Sandwich Fold; Crystallography, X-Ray; Humans; Kinetics; Magnesium - metabolism; Maple Syrup Urine Disease; Metabolic Diseases; Mitochondria - chemistry; Mitochondria - enzymology; Mitochondria - genetics; Mitochondrial Protein Phosphatases; Mn2+-dependent Phosphatases; Models, Molecular; Molecular Sequence Data; Phosphatase; Phosphoprotein Phosphatases - chemistry; Phosphoprotein Phosphatases - genetics; Phosphoprotein Phosphatases - metabolism; PP2A; PP2Cm Mitochondrial Phosphatase; Protein Phosphatase; Protein Phosphatase 2C; Protein Structure and Folding; X-ray Crystallography; Protein Phosphatase; Central β-Sandwich Fold; X-ray Crystallography; Maple Syrup Urine Disease; Metabolic Diseases; Mn2+-dependent Phosphatases; PP2Cm Mitochondrial Phosphatase; Mitochondrial Protein Phosphatases; PP2A; Phosphatase; Branched-chain α-Ketoacid Dehydrogenase Complex
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.314963

The branched-chain α-ketoacid dehydrogenase phosphatase (BDP) component of the human branched-chain α-ketoacid dehydrogenase complex (BCKDC) has been expressed in Escherichia coli and purified in the soluble form. The monomeric BDP shows a strict dependence on Mn2+ ions for phosphatase activity, whereas Mg2+ and Ca2+ ions do not support catalysis. Metal binding constants for BDP, determined by competition isothermal titration calorimetry, are 2.4 nm and 10 μm for Mn2+ and Mg2+ ions, respectively. Using the phosphorylated decarboxylase component (p-E1b) of BCKDC as a substrate, BDP shows a specific activity of 68 nmol/min/mg. The Ca2+-independent binding of BDP to the 24-meric transacylase (dihydrolipoyl transacylase; E2b) core of BCKDC results in a 3-fold increase in the dephosphorylation rate of p-E1b. However, the lipoyl prosthetic group on E2b is not essential for BDP binding or E2b-stimulated phosphatase activity. Acidic residues in the C-terminal linker of the E2b lipoyl domain are essential for the interaction between BDP and E2b. The BDP structure was determined by x-ray crystallography to 2.4 Å resolution. The BDP structure is dominated by a central β-sandwich. There are two protrusions forming a narrow cleft ∼10 Å wide, which constitutes the active site. The carboxylate moieties of acidic residues Asp-109, Asp-207, Asp-298, and Asp-337 in the active-site cleft participate in binding two metal ions. Substitutions of these residues with alanine nullify BDP phosphatase activity. Alteration of the nearby Arg-104 increases the Km for p-E1b peptide by 60-fold, suggesting that this residue is critical for the recognition of the native p-E1b protein. PP2Cm phosphatase was recently identified as the branched-chain α-ketoacid dehydrogenase complex (BCKDC) phosphatase (BDP). The 2.4 Å BDP structure reveals a central β-sandwich with two bound metal ions in the active-site cleft. Unlike related pyruvate dehydrogenase phosphatases, BDP strictly depends on Mn2+, and not Mg2+, for phosphatase activity. The structural/biochemical results validate PP2Cm as the BCKDC phosphatase.