X-ray analysis of yeast lipoamide dehydrogenase complexed with NAD⁺

Wataru Adachi,^a Kaoru Suzuki,^b Masaru Tsunoda,^c Takeshi Sekiguchi,^b Lester J. Reed^d and Akio Takénaka^a

^aGraduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan; ^bDepartment of Environmental Science, College of Science and Engineering, Iwaki-Meisei University, Chuodai-iino, Iwaki 970-8551, Japan; ^cSchool of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa, Tokyo 142-8555, Japan; ^dDepartment of Chemistry and Biochemistry, The University of Texas at Austin, Austin, TX78712, USA (wadachi@bio.titech.ac.jp).

Lipoamide dehydrogenase (E₃) is one of the components of 2-oxoacid dehydrogenase complex. This enzyme catalyses the oxidization of a dihydrolipoyl group of E₂ with the help of the cofactors, NAD⁺ and FAD. E₃ belongs to the pyridine nucleotide-disulphide oxidoreductase family of glutathione reductase, trypanothione reductase, mercuric ion reductase, etc. We already reported the native E₃ structure from Saccharomyces cerevisiae [1]. In this study, we co-crystallized yeast E₃ with NAD⁺ to elucidate the mechanism of substrate binding.

Diffraction data were collected at 100K and processed at 2.2 Å resolution. The space group and cell parameters are P2₁2₁2₁, a=66.6, b=96.4, and c=160.0Å, respectively. Initial phases were derived by molecular replacement using the native structure. The atomic parameters were refined (the final R=20.3% and R_free=24.6%).

The overall structure of yeast E₃-NAD⁺ complex is shown in Fig. 1. When the structure is superimposed on the native one, the overall root-mean-square deviation is 0.62 Å, suggesting no significant difference on NAD⁺ binding. The final electron density map clearly indicates that the adenosine moiety and the pyrophosphate group of NAD⁺ are bound to the enzyme, but the remaining nicotinamide moiety is disordered (see Fig. 2).

It is interesting to note that the binding mode of the nicotinamide moiety is different from those of the related enzymes in the same family. Contrary in the structures of those enzymes complexed with NAD(P)H (reduced form), the nicotinamide moiety is stacked on the isoalloxazine ring of FAD for electron transfer, and the side chain of a Tyr residue near the binding pocket is flip out. In the present enzyme, however, the nicotinamide does not enter the binding site, despite that the Tyr residue is replaced with Ile. It is concluded that the exact positioning of NAD⁺ on FAD depends on the redox state of the cofactor rather than on the existence of the Tyr side chain.

References

1 (1998) J. Biochem. 123, 668-674