A polyextremophilic alcohol dehydrogenase from the Atlantis II Deep Red Sea brine pool

Abstract

Enzymes originating from hostile environments offer exceptional stability under industrial conditions and are therefore highly in demand. Using single-cell genome data, we identified the alcohol dehydrogenase (ADH) gene, adh/a1a, from the Atlantis II Deep Red Sea brine pool. ADH/A1a is highly active at elevated temperatures and high salt concentrations (optima at 70 °C and 4 m KCl) and withstands organic solvents. The polyextremophilic ADH/A1a exhibits a broad substrate scope including aliphatic and aromatic alcohols and is able to reduce cinnamyl-methyl-ketone and raspberry ketone in the reverse reaction, making it a possible candidate for the production of chiral compounds. Here, we report the affiliation of ADH/A1a to a rare enzyme family of microbial cinnamyl alcohol dehydrogenases and explain unique structural features for halo- and thermoadaptation.

Keywords: alcohol dehydrogenase; extremophiles; extremozyme; halophiles; thermophiles.

Figure 1

Effect of various factors on the enzyme activity. (A) Salt concentration (saturation limit: 4 m for KCl, 5 m for NaCl), (B) temperature, (C) pH in oxidation, (D) pH in reduction reaction, and (E) metal ions (0.2 mm), protein without additive as control; and (F) cofactors for oxidation/reduction (oxidation: 100% = 0.49 μmol·min⁻¹·mg⁻¹; reduction: 100% = 0.31 μmol·min⁻¹·mg⁻¹). Error bars indicate SDs. *Protein precipitated during the reaction.

Figure 2

Effect of salt concentration and solvent on the stability of ADH/A1a. (A) Activity of ADH/A1a after 24 h in various salt concentrations. ADH/A1a was dialyzed against varying salt concentrations (10 mm HEPES/NaOH, pH: 7.5, 0.1–3 m NaCl) at 4 °C for 24 h, and the remaining activity was determined. (B) Activity of ADH/A1a over time in different organic solvents. ADH/A1a (in 10 mm HEPES/NaOH, pH 7.5, 2 m NaCl) was incubated with different amounts of organic solvents at room temperature, and the residual activity was assayed over time. ADH/A1a incubated without solvent was treated as the control. The activities of ADH/A1a were assayed in the oxidation reaction under standard conditions. (100% = 0.49 μmol·min⁻¹·mg⁻¹). Error bars indicate SDs.

Figure 3

Structure of the homology model of ADH/A1a. (A) Tetrameric structure of ADH/A1a. Monomers are indicated by different colors; Zn ions are dark gray. (B) Conserved catalytic Zn‐binding motif cys42‐his64‐cys151 of ADH/A1a.

Figure 4

Phylogenetic tree of ADH/A1a and selected ADHs of closest subfamilies. The branches are colored according to the subfamilies, which were annotated using CDD. The EC numbers indicate the functional annotation given by kegg. The calculation of the phylogenetic tree was performed using the maximum‐likelihood algorithm of FastTree2. *Experimentally characterized.