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Authors: Schelle, Luca S.
Stockinger, Peter
Pleiss, Jürgen
Nestl, Bettina M.
Title: The triple variant K170D/N174L/D239A compensates the destabilizing effect of variant K170D/N174L in β-hydroxyacid dehydrogenase (βHAD) from Arabidopsis thaliana
Issue Date: 2020 Verschiedenartige Texte Editorial modified version 6
Abstract: Chiral amines are essential building blocks in biologically active compounds, fine chemicals, agrochemicals and pharmaceuticals. In the last ten years, various enzymes were identified as new biocatalysts for chiral amine synthesis. Promising enzymes for the synthesis of primary, secondary, and tertiary amines are NADPH-dependent imine reductases (IREDs). Bioinformatics analysis revealed that IREDs are closely related to β-hydroxyacid dehydrogenases (βHADs). In recent work, we engineered the βHAD from Arabidopsis thaliana (βHAD_At) into imine-reducing enzymes by a single amino acid exchange.[7] The exchange of the proton-donor described lysine (K170) in βHAD_At by aspartic acid, the most common amino acid at this position in R-selective IREDs, led to a 12-fold increase in activity for the model substrate 2-methylpyrroline. At the same time, the activity for the natural substrate glyoxylic acid is reduced 885-fold, resulting in a total of 8200-fold change in catalytic activity through the exchange of an amino acid. At the same time, highly decreased soluble expression has been observed by exchanging asparagine at position 174 (N174) with leucine. We thus hypothesized, that the aspartic acid residue (D239) in near proximity to N174 will stabilize the underlying α-helix. Consequently, replacement of D239 with alanine should result in soluble expression of variants containing the N174 mutations. We generated variants K170D/D239A, N174L/D239A and K170D/N174L/D239A, and tested them on imine reduction of test substrates 2-methylpyrroline, 3,4-dihydroisoquinoline and 6-phenyl-2,3,4,5-tetrahydropyridine. Due to loss of essential cofactor and precipitation of purified proteins during purification procedure, activities of variants were determined using cell lysates. Notably, variants N174L/D239A and K170D/N174L/D239A demonstrated soluble expression and imine-reducing activities of up to 98 mU per mg of variant.
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