TY - JOUR
T1 - Crystal structure of human dihydrolipoamide dehydrogenase
T2 - NAD +/NADH binding and the structural basis of disease-causing mutations
AU - Brautigam, Chad A
AU - Chuang, Jacinta L.
AU - Tomchick, Diana R
AU - Machius, Mischa
AU - Chuang, David T
N1 - Funding Information:
The X-ray diffraction data were collected at the Argonne National Laboratory Structural Biology Center at the Advanced Photon Source, which is supported by the US Department of Energy, Office of Energy Research, under contract no. W-31-109-ENG-38. We thank the beamline personnel for their assistance, Dr Richard M. Wynn for helpful discussions and a critical reading of the manuscript, and Drs Argyrides Argyrou and John Blanchard for supplying d , l -lipoylpentanoic acid. Funding for this project came from NIH grant DK26758 and Welch Foundation grant I-1286.
PY - 2005/7/15
Y1 - 2005/7/15
N2 - Human dihydrolipoamide dehydrogenase (hE3) is an enzymatic component common to the mitochondrial α-ketoacid dehydrogenase and glycine decarboxylase complexes. Mutations to this homodimeric flavoprotein cause the often-fatal human disease known as E3 deficiency. To catalyze the oxidation of dihydrolipoamide, hE3 uses two molecules: non-covalently bound FAD and a transiently bound substrate, NAD+. To address the catalytic mechanism of hE3 and the structural basis for E3 deficiency, the crystal structures of hE3 in the presence of NAD+ or NADH have been determined at resolutions of 2.5 Å and 2.1 Å, respectively. Although the overall fold of the enzyme is similar to that of yeast E3, these two structures differ at two loops that protrude from the proteins and at their FAD-binding sites. The structure of oxidized hE3 with NAD+ bound demonstrates that the nicotinamide moiety is not proximal to the FAD. When NADH is present, however, the nicotinamide base stacks directly on the isoalloxazine ring system of the FAD. This is the first time that this mechanistically requisite conformation of NAD+ or NADH has been observed in E3 from any species. Because E3 structures were previously available only from unicellular organisms, speculations regarding the molecular mechanisms of E3 deficiency were based on homology models. The current hE3 structures show directly that the disease-causing mutations occur at three locations in the human enzyme: the dimer interface, the active site, and the FAD and NAD+-binding sites. The mechanisms by which these mutations impede the function of hE3 are discussed.
AB - Human dihydrolipoamide dehydrogenase (hE3) is an enzymatic component common to the mitochondrial α-ketoacid dehydrogenase and glycine decarboxylase complexes. Mutations to this homodimeric flavoprotein cause the often-fatal human disease known as E3 deficiency. To catalyze the oxidation of dihydrolipoamide, hE3 uses two molecules: non-covalently bound FAD and a transiently bound substrate, NAD+. To address the catalytic mechanism of hE3 and the structural basis for E3 deficiency, the crystal structures of hE3 in the presence of NAD+ or NADH have been determined at resolutions of 2.5 Å and 2.1 Å, respectively. Although the overall fold of the enzyme is similar to that of yeast E3, these two structures differ at two loops that protrude from the proteins and at their FAD-binding sites. The structure of oxidized hE3 with NAD+ bound demonstrates that the nicotinamide moiety is not proximal to the FAD. When NADH is present, however, the nicotinamide base stacks directly on the isoalloxazine ring system of the FAD. This is the first time that this mechanistically requisite conformation of NAD+ or NADH has been observed in E3 from any species. Because E3 structures were previously available only from unicellular organisms, speculations regarding the molecular mechanisms of E3 deficiency were based on homology models. The current hE3 structures show directly that the disease-causing mutations occur at three locations in the human enzyme: the dimer interface, the active site, and the FAD and NAD+-binding sites. The mechanisms by which these mutations impede the function of hE3 are discussed.
KW - Dihydrolipoamide dehydrogenase
KW - E3
KW - Lipoamide dehydrogenase
KW - X-ray crystallography
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U2 - 10.1016/j.jmb.2005.05.014
DO - 10.1016/j.jmb.2005.05.014
M3 - Article
C2 - 15946682
AN - SCOPUS:20444475852
SN - 0022-2836
VL - 350
SP - 543
EP - 552
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 3
ER -