The first part of the project employs the production of AOR enzyme in already developed A. evansii as well as optimization of the expression system for cascade enzymes (aldolases, amine reductases, transhydrogenase, etc.)  in E. coli hosts. The project's international partners have already developed expression of those enzymes. 

The enzyme production will be up-scaled from flask to fermentors and they will be purified using affinity chromatography. 

This task will encompass an investigation of the catalytic mechanism of AOR. The research in the task will combine experimental techniques to test the mechanistic hypotheses. We will use kinetics with isotope labelling substrates and stopped-flow and steady-state kinetics to determine half-reaction rates and obtain apparent kinetic parameters for mutant variants . We will also extensively investigate the substrate spectrum, testing the limits of AOR specificity and investigating, if the enzyme can discriminately select enantiomers of acid for reduction. Such an experiment will extensively rely on UHPLC-MS detection due to the lack of a UV-vis-based assay. We will also employ chiral chromatography for the investigation of reaction enantiospecificity.

In this task, we will also conduct modeling of the reaction mechanism using MD, QM-only and QM:MM approaches. 

PROJECT OPUS 26 "Tungsten Aldehyde Oxidoreductase - a novel hydrogenase. Studies of reaction mechanism and potential biocatalytic applications" is sponsored by the National Science Centre Poland 

In this task, we will apply cutting-edge spectroscopic and structural techniques to study the structural characteristics of the enzyme, such as high-resolution cryo-EM, EXAFS, and EPR.

We will use genetically modified AOR that has no capability to oligomerize for the structural cryo-EM characterization of enzyme at different stages of reaction. The EXAFS spectroscopy will be supported by QM:MM modeling of the active site. Finally, we will conduct EPR investigations using an as-isolated AOR frozen in different stages of the oxidation and reduction processes via a freeze-quench procedure (in the hope of
catching of the oxidation transitions in W atom)

In that task we will establish several working cascade systems that utilize AOR at the first step. We will start with the already tested system with BaDH and concentrate on the expansion of the substrate range (mostly in the direction of substitution at the phenyl
ring) as well as testing the AOR-BaDH coexpression system which is developed by our partners in Marburg. 

We will also test the cascade system utilizing other enzymes that can convert aldehydes such as aldolases, imine reductases, amine reductases or amine transferases.