Random Mutagenesis-Driven Improvement of Carboxylate Reductase Activity Using an Amino Benzamidoxime-Mediated High-Throughput Assay

Daniel Schwendenwein, Anna K. Ressmann, Mark Dörr, Matthias Höhne, Marko D. Mihovilovic, Uwe T. Bornscheuer, Florian Rudroff, Margit Winkler

Research output: Contribution to journalArticleResearchpeer-review

Abstract

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 NiQ283P 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. (Figure presented.).

Original languageEnglish
Pages (from-to)2544-2549
Number of pages6
JournalAdvanced Synthesis & Catalysis
Volume361
Issue number11
Early online dateMar 2019
DOIs
Publication statusPublished - 6 Jun 2019

Fingerprint

Mutagenesis
Carboxylic acids
Assays
Oxidoreductases
Throughput
Aldehydes
Benzoic acid
Benzoic Acid
Benzoates
Substrates
Carboxylic Acids
Glutamine
NADP
Proline
Acids
Adenosine Triphosphate
carboxylic acid reductase
benzamidoxime
Phosphates
Derivatives

Keywords

  • aldehyde
  • amino benzamidoxime
  • carboxylic acid reductase (CAR)
  • carboxylic acids
  • high-throughput screening
  • mutagenesis

ASJC Scopus subject areas

  • Catalysis
  • Organic Chemistry

Fields of Expertise

  • Human- & Biotechnology

Cite this

Random Mutagenesis-Driven Improvement of Carboxylate Reductase Activity Using an Amino Benzamidoxime-Mediated High-Throughput Assay. / Schwendenwein, Daniel; Ressmann, Anna K.; Dörr, Mark; Höhne, Matthias; Mihovilovic, Marko D.; Bornscheuer, Uwe T.; Rudroff, Florian; Winkler, Margit.

In: Advanced Synthesis & Catalysis, Vol. 361, No. 11, 06.06.2019, p. 2544-2549.

Research output: Contribution to journalArticleResearchpeer-review

Schwendenwein, D, Ressmann, AK, Dörr, M, Höhne, M, Mihovilovic, MD, Bornscheuer, UT, Rudroff, F & Winkler, M 2019, 'Random Mutagenesis-Driven Improvement of Carboxylate Reductase Activity Using an Amino Benzamidoxime-Mediated High-Throughput Assay' Advanced Synthesis & Catalysis, vol. 361, no. 11, pp. 2544-2549. https://doi.org/10.1002/adsc.201900155
Schwendenwein, Daniel ; Ressmann, Anna K. ; Dörr, Mark ; Höhne, Matthias ; Mihovilovic, Marko D. ; Bornscheuer, Uwe T. ; Rudroff, Florian ; Winkler, Margit. / Random Mutagenesis-Driven Improvement of Carboxylate Reductase Activity Using an Amino Benzamidoxime-Mediated High-Throughput Assay. In: Advanced Synthesis & Catalysis. 2019 ; Vol. 361, No. 11. pp. 2544-2549.
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