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Investigation of the Mechanism and Application of Carboxylic Acid Reductases
[Thesis]. Manchester, UK: The University of Manchester; 2018.
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Abstract
This thesis reports the investigation of mechanism and the use of carboxylic acid reductases (CARs) for biotechnological applications and biocatalytic synthesis of industrially relevant chemical compounds. Chapter 3 focuses on understanding mechanistic properties of the CAR enzymes. It was demonstrated that phosphopantetheine group is essential for the functions of Mycobacterium marinum CAR (MCAR) and Nocardia sp. strain NRRL 5646 CAR (NCAR). Recent findings on the structure of these proteins allowed investigation of enzyme modularity by swapping the reductase domains of MCAR and NCAR with generation of biocatalytically active chimeric enzymes. By using stopped-flow techniques it was possible to determine that CARs are highly dynamic and involve many conformational changes. In chapter 4 the use of CARs was investigated for synthesis of anti-tuberculosis drug ethambutol from (S)-2-aminobutyric acid. Demonstrating that one of the intermediates – (S)-2-aminobutanal is unstable the whole cascade was redesigned. N-BOC and N-Cbz protected (S)-2-aminobutyric acid were accepted by MCAR and CAR 11 respectively. The cascade could not be fully established due to problems faced during removal of protective groups after enzymatic scale up reactions producing the required alcohol. Chapter 5 describes the investigation of CARs in biocatalytic production of cinnamyl alcohol. The optimized reactions involving lyophilized cells with MCAR, lyophilised cells containing Anabaena variabilis (AvPAL) and purified Saccharomyces cerevisiae (ScADH) could be scaled up for 100 mg L-phenylalanine substrate. Further studies revealed that all three enzymes can be co-expressed together for in vivo fermentation. Chapter 6 is about the exploitation of modularity of CAR enzymes to create novel biocatalysts with double reduction activity of acid substrates to the corresponding alcohol. Chimeric CAR enzymes were made by replacing reduction domains with double reduction domains known as GPL (from Mycobacterium smegmatis) and NRP (from Mycobacterium tuberculosis) . The newly formed enzymes were soluble, however they retained little to no activity and the results of reduction of acid substrates to alcohols were inconclusive.