<br/>Enzyme engineering is a quintessential field in biochemistry due to its potential in manipulating enzyme characteristics. Enzymes are cost-effective and sustainable alternatives for catalysts, making them beneficial for industrial applications. Old Yellow enzymes (OYE) have been experimentally studied for many years for its efficiency in reducing organic compounds. In the past, OYE 1 is known for its role as an alkene reductase and studies have been conducted to increase its product yield. Recently, mutations are performed in the active site of the enzyme to change the stereoselectivity of products. Our experimental collaborators found that the mutation of Trp 116, an active site residue led to the different orientation of substrate binding resulting in different stereoselective product for certain substrates. This diverse product formation has led to its usage in synthesizing intermediates for medicinal compounds like flurbiprofen and pregabalin etc. OYE 3, a member of the OYE family with 80.5% sequence identity with OYE 1 also showed some important stereochemical differences. Our goal is to understand why OYE 3 differs from OYE 1 and how to use this knowledge to extend the synthetic utility of OYE 3 and alkene reductases in general. The only significant structural difference between the active site is in loop 6(289-309) at position 296, with Phe for OYE 1 and Ser for OYE 3. Modeling the substrate orientation through crystal structure for OYE 1 and OYE 3 suggested the same product formation contrary to experimental findings. This indicated that the dynamic behavior of OYE 1 and OYE 3 needs to be studied to understand their divergent stereopreferences. For this, molecular dynamics simulations using the AMBER GPU suite is performed to study the mobility of loop 6. We found that loop 6 is much more flexible and solvent exposed, for OYE 3 than OYE 1, which could be the reason behind the differences in stereochemistry. To the best of our knowledge, this is the first study for OYE where loop dynamics has been found responsible for stereoselectivity of products. Further, mutational studies are conducted and binding energy difference of different orientations of the substrate is evaluated using umbrella sampling to predict the preferred product. The results obtained from our group are consistent with the experimental data, leading us to further investigate the catalytic properties of the active site.<br/>

Enzyme engineering to computationally predict stereoselective products for biocatalysts

2019 Fall National Meeting

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