4EEZ

Crystal Structure of Lactococcus lactis Alcohol Dehydrogenase variant RE1

Summary for 4EEZ

• Entry DOI: 10.2210/pdb4eez/pdb
• Related: 4EEX
• Descriptor: Alcohol dehydrogenase 1, ZINC ION, TETRAETHYLENE GLYCOL, ... (4 entities in total)
• Functional Keywords: alcohol dehydrogenase, site-saturation mutagenesis, directed evolution, isobutyraldehyde, biofuel, oxidoreductase
• Biological source: Lactococcus lactis subsp. lactis
• Total number of polymer chains: 2
• Total formula weight: 74142.16

Authors

Liu, X.,Bastian, S.,Snow, C.D.,Brustad, E.M.,Saleski, T.,Xu, J.H.,Meinhold, P.,Arnold, F.H. (deposition date: 2012-03-28, release date: 2012-09-26, Last modification date: 2024-02-28)

Primary citation

Liu, X.,Bastian, S.,Snow, C.D.,Brustad, E.M.,Saleski, T.E.,Xu, J.H.,Meinhold, P.,Arnold, F.H.
Structure-guided engineering of Lactococcus lactis alcohol dehydrogenase LlAdhA for improved conversion of isobutyraldehyde to isobutanol.
J.Biotechnol., 164:188-195, 2012

PubMed 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.

PubMed: 22974724
DOI: 10.1016/j.jbiotec.2012.08.008

Experimental method

X-RAY DIFFRACTION (1.9 Å)