Structure-guided engineering of Lactococcus lactis alcohol dehydrogenase LlAdhA for improved conversion of isobutyraldehyde to isobutanol

Abstract

We have determined the X-ray crystal structures of the NADH-dependent alcohol dehydrogenase LlAdhA from Lactococcus lactis and its laboratory-evolved variant LlAdhA(RE1) at 1.9Å and 2.5Å resolution, respectively. LlAdhA(RE1), which contains three amino acid mutations (Y50F, I212T, and L264V), was engineered to increase the microbial production of isobutanol (2-methylpropan-1-ol) from isobutyraldehyde (2-methylpropanal). Structural comparison of LlAdhA and LlAdhA(RE1) indicates that the enhanced activity on isobutyraldehyde stems from increases in the protein's active site size, hydrophobicity, and substrate access. Further structure-guided mutagenesis generated a quadruple mutant (Y50F/N110S/I212T/L264V), whose KM for isobutyraldehyde is ∼17-fold lower and catalytic efficiency (kcat/KM) is ∼160-fold higher than wild-type LlAdhA. Combining detailed structural information and directed evolution, we have achieved significant improvements in non-native alcohol dehydrogenase activity that will facilitate the production of next-generation fuels such as isobutanol from renewable resources.

Fig. 1

The LlAdhA asymmetric unit dimer with catalytic domains (blue, light blue), Rossmann fold (orange, yellow), structural zinc atoms (white spheres), and catalytic zinc atoms (black spheres).

Fig. 2

The cofactor-binding site of LlAdhA (green), with cofactor coordinates adopted from the superimposed NADH (orange) co-crystal structure of PaADH (PDB ID: 1LLU). Hydrogen bonds to the main chain of residues Gly174, Leu175, Val262, Leu264, Ser286, and Val288 anchor NADH to the active site. The other hydroxyl groups are bound to the protein via a series of amino acids, Thr41, His40, His44 Asp195, Lys200, and Arg333.

Fig. 3

(A) The active site of LlAdhA (orange) superimposed with LlAdhA^RE1 (green). The catalytic zinc is shown as a blue sphere. Selected active site residues are shown in stick form. Thr41 and His44 are implicated in the proton conduction pathway from the active site to solvent. Isobutanol (white) and NAD⁺ (magenta) were modeled by alignment of htADH and PaADH (see Methods), respectively. (B) Surface representation of LlAdhA (orange) and LlAdhA^RE1 (green) active site cavities reveals an enlarged channel opening in LlAdhA^RE1’s that may result in improved access of larger non-native substrates to the active site.