The reaction and inhibition mechanisms of

Drosophila alcohol dehydrogenase

J. Benach, R. Meijers , S. Atrian , R. Gonzlez-Duarte , V. Lamzin & R. Ladenstein Center for Structural Biochemistry, Karolinska Institute, S-141 57 Huddinge, Sweden EMBL c/o DESY, Notkestrasse 85 Geb. 25a, D-22603 Hamburg, Germany Department of Genetics, University of Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain Project number: PX-99-362 Beamline: X11
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Drosophila alcohol dehydrogenase (DADH; EC 1.1.1.1) is an NAD(H)-dependent oxidoreductase that catalyzes the oxidation of alcohols to aldehydes/ketones. This enzyme is one of the most well characterized member of the superfamily of the short-chain dehydrogenases/reductases (SDR) [1]. DADH subunits show an / single domain structure with a characteristic NAD(H) binding motif (Rossmann fold) [2]. The active site pocket comprises a hydrophobic and bifurcated cavity that explains why the enzyme is more efficient in oxidizing secondary aliphatic alcohols -preferably R enantiomers- than primary ones. High resolution studies on the 3-pentanone/NAD+/enzyme complex showed that the ketone inhibitor molecule has undergone a covalent reaction with the coenzyme in the ternary complex [3,4].

Figure 1: (a) Stereoview of the 3-pentanone-NAD+ adduct observed at 1.4 resolution and (b) zoomed section showing the covalent interaction between the ketone and the coenzyme.

Due to the presence of the positively charged ring of the coenzyme (NAD+) and the residue Lys155, the amino acid Tyr151 is in its deprotonated state at physiological pH. Tyr151 can abstract a proton from the enolic form of the ketone and catalyze a nucleophilic attack of the C atom to the C4 position of the coenzyme creating an NAD-ketone adduct. The binding of these NAD-ketone adducts to DADH accounts for the inactivation of the enzyme. In order to further understand the catalytic reaction mechanism of DADH crystals of a ternary complex of this enzyme and NAD+ and trifluoroethanol were obtained. Data was measured at the X11 beamline at the Hamburg outstation and diffracted beyond 1.1 resolution. The crystal structure of the ternary complex is still undergoing crystallographic refinement.

Figure 2: Zoomed section showing the active site of the ternary complex of Drosophila alcohol dehydrogenase at 1.1 resolution showing the main amino acids involved in catalytic activity (Ser138, Tyr151, Lys155), the NAD+ and the trifluoroethanol molecules. 3Fo-2Fc electron density map contoured at 1.7 and 4.4.

Table 1. X-ray data collection statistics for DADH trifluoroethanol NAD+ ternary complex
Cell parameters Space group Total reflections Unique reflections Resolution range Completeness R-merge I/sigma > 2 Multiplicity >2 Source Wavelength Temperature a=66.30 , b=52.73 , c=69.92 , =107.38 deg. P21 3758910 195445 50-1.05 (1.14 -1.12 ) 92.0% (85.2 %) 3.7% (32.7%) 74.6% (55.5%) 76% (72%) X11 EMBL, Hamburg 0.9092 100 K

References
[1] H. Jrnvall, B. Persson, M. Krook, S. Atrian, R. Gonzlez-Duarte, J. Jeffery, and D. Ghosh, Biochemistry, 34, 6003 (1995) [2] J. Benach, S. Atrian, R. Gonzlez-Duarte, and R. Ladenstein, J. Mol. Biol. 282, 383 (1998) [3] J. Benach, S. Atrian, R. Gonzlez-Duarte, and R. Ladenstein, J. Mol. Biol. 289, 335 (1999) [4] J. Benach. Doctoral thesis. Karolinska Institute. Stockholm, Sweden. (1999)