The ternary complex of Pseudomonas aeruginosa alcohol dehydrogenase with NADH and ethylene glycol

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 (k cat ‫ס‬ 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 ADHs are oxidoreductases that catalyze the reversible oxidation of a wide range of primary and secondary alcohols into the corresponding carbonyl compounds, using NAD + or NADP + as coenzymes. These ubiquitous enzymes are present virtually in all life forms, from bacteria to vertebrates, and can be classified by size into three major classes (Jornvall 1994) et al. 1989) and Escherichia coli ADH (Goodlove et al. 1989). Several representative enzymes of the medium-chain zinccontaining ADH family have been characterized structurally, for example, bacteria ), archaea (Esposito et al. 2002Guy et al. 2003), cod (Ramaswamy et al. 1996), and human (Hurly et al. 1994;Xie et al. 1997;Niederhut et al. 2001 These authors contributed equally to this work.

Microbial metabolisms in a 2.5-km-deep ecosystem created by hydraulic fracturing in shales

“…Ethylene glycol is consumed over time, and is not detected at the last time point. This substrate is probably aerobically oxidized by Marinobacter and Vibrio at the early time points 18 (alcohol dehydrogenase), and fermented by Halanaerobium (propanediol dehydratase, acetalaldehyde dehydrogenase) later to yield ethanol, hydrogen and acetate. Candidatus Frackibacter also has the capacity to produce acetate via GB fermentation, homoacetogenesis (H 2 /CO 2 ) and sugar fermentation.…”

Biochemical characterization of ethanol-dependent reduction of furfural by alcohol dehydrogenases

“…and ethylene glycol has been determined and the ethanol-dependent reduction follows the established scheme for Zn-dependent alcohol dehydrogenases (Fig. 3, steps 1-5) (Levin et al 2004). Since NADH is not accumulated during the ethanol-dependent reduction of furfural, the dissociation of E.NADH (Fig.…”

Biosynthesis of 2-Deoxystreptamine by Three Crucial Enzymes in Streptomyces fradiae NBRC 12773

“…Sequence alignment of NeoA, BtrE and the structurally elucidated similar zinc-containing NAD(P) ϩ dependent alcohol dehydrogenases (ADHs), BaKR (Ketose reductase, Bemisia argentifolii, 1E3J) [27], PaADH (ADH, Pseudomonas aeruginosa, 1LLU) [28], htADH (ADH, B. stearothermophilus, 1RJW) [29], is shown in Fig. 3.…”