Download emboj7601444-sup

Supplementary data: Materials and methods
Materials
CoA, IB-CoA, IV-CoA, NADH, ATP, dihydrolipoamide and malic dehydrogenase were
purchased from Sigma-Aldrich. ATP-citrate lyase was purified from rat liver as described
previously (Linn and Srere, 1979). Dihydrolipoamide was prepared according to a previously
described method (Reed et al., 1958). Lip-LBD (residues 1-99 of human E2b) was expressed
and purified as described previously (Chuang et al., 2002; Wynn et al., 2004). Human E2pCD
was expressed and purified as also described earlier (Harris et al., 1997).
Purification of E2bCD
Wild-type bovine E2bCD (residues 162-421) fused at their amino-termini to maltosebinding protein (MBP), with the tobacco-etch virus (TEV) recognition sequence inserted in
between the two moieties, was expressed in E. coli BL-21 (DE3) cells, similar to the method
described previously (Wynn et al., 1994). Mutations were introduced using the QuickChange
site-directed mutagenesis system from Stratagene (La Jolla, CA). The fusion proteins were
purified with amylose resin, followed by digestion with the TEV protease to remove the MBP
portion. The resulting E2bCD’s were further purified with Superdex S-200 column
equilibrated with a buffer containing 50 mM K-phosphate (pH 7.5), 50 mM KCl and 5%
(v/v) glycerol. The purified proteins were concentrated to 28 mg/ml, and DTT was added to a
final 20 mM concentration. The concentrations of MBP-E2bCD and E2bCD were determined
using the calculated extinction coefficients at 280 nm of 1.108 mg-1 ml and 0.575 mg-1 ml,
respectively.
Activity assay
The rate of the reverse acyltransfer reaction (Reaction 2) catalyzed by bovine E2bCD was
measured by the method developed previously (Angier et al., 1987). The reaction mixture in
0.5 ml contained 50 mM Tris-HCl (pH 8.3), 20 mM Na-citrate, 10 mM MgCl2, 5 mM ATP,
0.2 mM NADH, 10 units (mol/min) of malic dehydrogenase, 0.1 unit of ATP-citrate lyase,
and varying concentrations of E2bCD, IV-CoA, and dihydrolipoamide. The reactions were
initiated by the addition of IV-CoA. The rate of the acyltransfer reaction at 30°C was
determined by monitoring the decline of absorbance at 340 nm. All kinetic data were fitted by
nonlinear regression analysis using KaleidaGraph (Synergy Software, Essex Junction, VT).
References
Angier, S.J., Miles, J.S., Srere, P.A., Engel, P.C. and Guest, J.R. (1987) The effects of
deletion mutagenesis on the pyruvate dehydrogenase complex of Escherichia coli.
Biochem. Soc. Trans., 15, 832-833.
Chuang, J.L., Wynn, R.M. and Chuang, D.T. (2002) The C-terminal hinge region of lipoic
acid-bearing domain of E2b is essential for domain interaction with branched-chain
alpha-keto acid dehydrogenase kinase. J Biol Chem, 277, 36905-36908.
Harris, R.A., Bowker-Kinley, M.M., Wu, P., Jeng, J. and Popov, K.M. (1997)
Dihydrolipoamide dehydrogenase-binding protein of the human pyruvate
dehydrogenase complex. DNA-derived amino acid sequence, expression, and
reconstitution of the pyruvate dehydrogenase complex. J Biol Chem, 272, 1974619751.
Linn, T.C. and Srere, P.A. (1979) Identification of ATP citrate lyase as a phosphoprotein. J
Biol Chem, 254, 1691-1698.
Reed, L.J., Leach, F.R. and Koike, M. (1958) Studies on a lipoic acid-activating system. J
Biol Chem, 232, 123-142.
Wynn, R.M., Davie, J.R., Zhi, W., Cox, R.P. and Chuang, D.T. (1994) In vitro reconstitution
of the 24-meric E2 inner core of bovine mitochondrial branched-chain alpha-keto acid
dehydrogenase complex: requirement for chaperonins GroEL and GroES.
Biochemistry, 33, 8962-8968.
Wynn, R.M., Kato, M., Machius, M., Chuang, J.L., Li, J., Tomchick, D.R. and Chuang, D.T.
(2004) Molecular mechanism for regulation of the human mitochondrial branchedchain alpha-ketoacid dehydrogenase complex by phosphorylation. Structure, 12,
2185-2196.