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Protein Science (2004), 13:1547-1556. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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The ternary complex of Pseudomonas aeruginosa alcohol dehydrogenase with NADH and ethylene glycol

Inna Levin1,3, Gal Meiri1,3, Moshe Peretz1, Yigal Burstein1 and Felix Frolow2

1 Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
2 Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel

(RECEIVED November 24, 2003; FINAL REVISION March 11, 2004; ACCEPTED March 11, 2004)



Abstract

Pseudomonas aeruginosa alcohol dehydrogenase (PaADH; ADH, EC 1.1.1.1) catalyzes the reversible oxidation of primary and secondary alcohols to the corresponding aldehydes and ketones, using NAD as coenzyme. We crystallized the ternary complex of PaADH with its coenzyme and a substrate molecule and determined its structure at a resolution of 2.3 Å, using the molecular replacement method. The PaADH tetramer comprises four identical chains of 342 amino acid residues each and obeys ~222-point symmetry. The PaADH monomer is structurally similar to alcohol dehydrogenase monomers from vertebrates, archaea, and bacteria. The stabilization of the ternary complex of PaADH, the coenzyme, and the poor substrate ethylene glycol (kcat = 4.5 sec–1; Km > 200 mM) was due to the blocked exit of the coenzyme in the crystalline state, combined with a high (2.5 M) concentration of the substrate. The structure of the ternary complex presents the precise geometry of the Zn coordination complex, the proton-shuttling system, and the hydride transfer path. The ternary complex structure also suggests that the low efficiency of ethylene glycol as a substrate results from the presence of a second hydroxyl group in this molecule.

Keywords: alcohol dehydrogenase; Pseudomonas aeruginosa; ternary complex; NADH; ethylene glycol; ion-pair network; proton shuttling; 3D structure

Abbreviations: ADH, alcohol dehydrogenase • CbADH, Clostridium beijerinckii ADH • EG, ethylene glycol • HLADH, horse liver ADH • NCS, noncrystallographic symmetry • PaADH, Pseudomonas aeruginosa ADH • PEG, polyethylene glycol • TbADH, Thermoanaerobacter brockii ADH

Reprint requests to: Yigal Burstein, Department of Organic Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel; e-mail: yigal. burstein{at}weizmann.ac.il; fax: (972) 8-9342501.

3 These authors contributed equally to this work.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03531404.


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