Active site dynamics in the zinc-dependent medium chain alcohol dehydrogenase superfamily

Abstract

Despite being the subject of intensive investigations, many aspects of the mechanism of the zinc-dependent medium chain alcohol dehydrogenase (MDR) superfamily remain contentious. We have determined the high-resolution structures of a series of binary and ternary complexes of glucose dehydrogenase, an MDR enzyme from Haloferax mediterranei. In stark contrast to the textbook MDR mechanism in which the zinc ion is proposed to remain stationary and attached to a common set of protein ligands, analysis of these structures reveals that in each complex, there are dramatic differences in the nature of the zinc ligation. These changes arise as a direct consequence of linked movements of the zinc ion, a zinc-bound bound water molecule, and the substrate during progression through the reaction. These results provide evidence for the molecular basis of proton traffic during catalysis, a structural explanation for pentacoordinate zinc ion intermediates, a unifying view for the observed patterns of metal ligation in the MDR family, and highlight the importance of dynamic fluctuations at the metal center in changing the electrostatic potential in the active site, thereby influencing the proton traffic and hydride transfer events.

Fig. 1.

The active site of the glucose dehydrogenase binary and ternary complexes. (A–E) The protein is shown as a cartoon, with residues highlighted in the text in atom representation. The NADP(H) is shown as sticks (yellow carbons) with the catalytic zinc (magenta) and 2 water molecules (red) shown as spheres. The sigmaA mF_O-DF_C electron density map for the various substrates, contoured at 1σ, is shown as gray lines. (A) The NADPH/Zn structure (blue carbons), (B) the NADP⁺/Zn/glucose structure (orange carbons), (C) the NADP⁺/Zn/gluconolactone structure (green carbons), (D) an overlap of the NADP⁺/Zn/gluconolactone (green, protein and lactone; magenta, zinc; and red, water) and NADP⁺/Zn/glucose structures (orange; zinc large sphere, water small sphere), showing the movement of the zinc and its associated water between the 2 complexes, and (E) an overlap of the structures of the GlcDH lactone complex, (green magenta, zinc), T. brockii ADH (purple, 1ykf, ref. 38), human SDH (blue, 1pl6, ref. 11), and LADH (light brown, 1heu, ref. 17).

Fig. 2.

A schematic diagram, viewed down the His 63 Ne2–Zn bond, of the zinc ligands in the NADP(H)/Zn complex (Left), NADP⁺/Zn/glucose complex (Center), and NADP⁺/Zn/gluconolactone complex (Right). The arrows give the direction of the movement of the zinc and water between the complexes.