Engineering Isopropanol Dehydrogenase for Efficient Regeneration of Nicotinamide Cofactors

Abstract

Isopropanol dehydrogenase (IPADH) is one of the most attractive options for nicotinamide cofactor regeneration due to its low cost and simple downstream processing. However, poor thermostability and strict cofactor dependency hinder its practical application for bioconversions. In this study, we simultaneously improved the thermostability (433-fold) and catalytic activity (3.3-fold) of IPADH from Brucella suis via a flexible segment engineering strategy. Meanwhile, the cofactor preference of IPADH was successfully switched from NAD(H) to NADP(H) by 1.23 × 10⁶-fold. When these variants were employed in three typical bioredox reactions to drive the synthesis of important chiral pharmaceutical building blocks, they outperformed the commonly used cofactor regeneration systems (glucose dehydrogenase [GDH], formate dehydrogenase [FDH], and lactate dehydrogenase [LDH]) with respect to efficiency of cofactor regeneration. Overall, our study provides two promising IPADH variants with complementary cofactor specificities that have great potential for wide applications. IMPORTANCE Oxidoreductases represent one group of the most important biocatalysts for synthesis of various chiral synthons. However, their practical application was hindered by the expensive nicotinamide cofactors used. Isopropanol dehydrogenase (IPADH) is one of the most attractive biocatalysts for nicotinamide cofactor regeneration. However, poor thermostability and strict cofactor dependency hinder its practical application. In this work, the thermostability and catalytic activity of an IPADH were simultaneously improved via a flexible segment engineering strategy. Meanwhile, the cofactor preference of IPADH was successfully switched from NAD(H) to NADP(H). The resultant variants show great potential for regeneration of nicotinamide cofactors, and the engineering strategy might serve as a useful approach for future engineering of other oxidoreductases.

Keywords: cofactor regeneration; cofactor specificity reversal; isopropanol dehydrogenase; protein engineering; thermostability evolution.

FIG 1

Typical substrate-coupled approaches to regenerate reductive cofactor NAD(P)H or oxidative cofactor NAD(P)⁺, taking the alcohol dehydrogenase-catalyzed carbonyl reduction or hydroxyl oxidation as examples for the target bioreactions.

FIG 2

Analysis of the flexible regions of IPADH. Representative structures of holo-IPADH during MD simulations are shown (A). The green arrows indicate the movement of the flexible regions. (B and C) Average RMSF values of all residues calculated from MD simulations for the apo-IPADH_WT (B) and holo-IPADH_WT (C) at 300, 330, and 373 K, respectively. The RMSF value calculated [RMSF = (3B/8π²)^1/2] from the apo-IPADH_WT crystal structure was also compared. The local sequence alignment of Flexi-Zones 1 and 3 of IPADH with other thermostable enzymes is shown (D).

FIG 3

Laboratory evolution route of stability and activity in IPADH. The flexible zones in IPADH_WT are represented by light blue rectangles. The hot spot residues in IPADH_M1 for site-directed mutagenesis are represented by gray lines, and a positive hit for R48L is indicated in dark red. The substituted segments from TtADH and ScCR are represented by orange and yellow rectangles, respectively. The subscript number represents the position of the residue located in IPADH.

FIG 4

Performance of intermediate mutants in deconvolution analysis, with the activity of IPADH_M2 toward isopropanol as 100% and the T₅₀⁶⁰ of IPADH_M2 as the baseline.

FIG 5

Conformational population analysis of Flexi-Zone 3 (residues from Thr₁₈₆ to Lys₁₉₈) of IPADH_WT and IPADH_M3. The analysis was associated with the two most important principal components (PC1 and PC2), which are based on Cα contacts for IPADH_WT and IPADH_M3. The crystal structures of IPADH_WT (PDB ID 4NI5) and modeled IPADH_M3 prior to MD simulations are indicated by gray arrows. Representative conformations of Flexi-Zone 2 from the main conformation cluster κ of IPADH_WT and IPADH_M3 are shown as blue/gray cartoon and stick models, respectively. Hydrogen bonding interactions are presented as black dashes.

FIG 6

Reaction progress of representative bioconversions employing IPADH mutants to regenerate different kinds of nicotinamide cofactors. (A) Production of l-tert-Leu from TMP by EsLeuDH was coupled with IPADH_M3 or BmGDH to regenerate NADH. (B) Production of 12-oxo-CDCA from CA by Rr12α-HSDH was coupled with IPADH_M3 or LdLDH to regenerate NAD⁺. (C) Production of (S)-omeprazole from pyrmetazole by BVMO_CDX was coupled with IPADH_M4 or BstFDH to regenerate NADPH.