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Random Mutagenesis‐Driven Improvement of Carboxylate Reductase Activity using an Amino Benzamidoxime‐Mediated High‐Throughput Assay
Author(s) -
Schwendenwein Daniel,
Ressmann Anna K.,
Doerr Mark,
Höhne Matthias,
Bornscheuer Uwe T.,
Mihovilovic Marko D.,
Rudroff Florian,
Winkler Margit
Publication year - 2019
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.201900155
Subject(s) - chemistry , benzoic acid , mutagenesis , carboxylic acid , high throughput screening , carboxylate , amino acid , steric effects , substrate (aquarium) , stereochemistry , biochemistry , combinatorial chemistry , mutation , oceanography , gene , geology
Carboxylic acid reductases (CARs) catalyze the direct adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH) dependent reduction of carboxylic acids to their corresponding aldehydes. The identification and improvement of CARs by protein engineering is, however, severely limited by the lack of fast and generic methods to quantify aldehydes. Within this study, we applied a convenient high‐throughput assay (HTA) based on amino benzamidoxime (ABAO) that allows the substrate‐independent and chemoselective quantification of aldehydes. Random mutagenesis of the well‐known CAR from Nocardia iowensis (CAR Ni ) to improve its activity for sterically demanding 2‐substituted benzoic acid derivatives was conducted in a K M ‐dependent fashion, and the HTA applied in the presence of microbial cells. The study identified a hot spot in the active site of CAR Ni that increased the affinity to 2‐methoxybenzoic acid 9‐fold upon mutation from glutamine to proline (Q283P). The catalytic performance of CAR Ni Q283P appeared to be significantly improved also for other substrates such as 2‐substituted (2‐Cl, 2‐Br) as well as 3‐ and 4‐substituted benzoic acids (3‐OMe, 4‐OMe), and even aliphatic octanoic acid.