(634e) Engineering the Active Site Microenvironment of a Thermostable Alcohol Dehydrogenase As a Means to Modulate Kinetic Activity | AIChE

(634e) Engineering the Active Site Microenvironment of a Thermostable Alcohol Dehydrogenase As a Means to Modulate Kinetic Activity


Abdallah, W. - Presenter, Columbia University
Lancaster, L., UC Riverside
Wheeldon, I., University of California, Riverside
Banta, S., Columbia University
Engineering the microenvironment around enzymatic active sites through chemical or physical modification using protein engineering techniques can lead to substantial benefits in catalytic performance (1). We have worked extensively with the thermostable alcohol dehydrogenase D (AdhD) from Pyrococcus furiosus (2) and we have begun to determine the effects of these types of modifications on activity and selectivity. In one approach, we performed domain insertion, where a large conformationally dynamic peptide was inserted into a labile loop region near the active site, and this introduced dynamic control over the catalytic activity (3). The insertion, the fifth block of the RTX domain from the adenylate cyclase toxin Bordetella pertussis, is disordered in the absence of calcium and folds into the β-roll secondary structure upon calcium addition. The chimeric fusion protein, β-AdhD, exhibited tunable control of cofactor selectivity through the addition or removal of calcium, which was not seen in the wild type enzyme. A second approach to modifying the local chemical environment focused on altering the local charge near the active site of AdhD without fundamentally altering the local structure. This was explored with three supercharged superfolder GFP (sfGFP) variants containing a net charge of -30, 0, and +36. The variants were covalently attached to AdhD using the SpyTag/SpyCatcher conjugation platform. AdhD catalyzes the conversion of NAD+ and 2,3-butanediol to NADH and acetoin. The localized charge of the sfGFP changes the local pH around the active site resulting in varying catalytic rates depending on the net charge of the protein and the pH of the local environment. These effects were not observed when the proteins were simply mixed together. Thus, the attachment of charged domains to enzymes serves as a means to modulate pH-dependent activities. Both of these techniques are modular and demonstrate the ability of protein engineering approaches to regulate activity through modifications made at locations outside of the active site.

1. Lancaster, L., Abdallah, W., Banta, S., Wheeldon, I. (2018) Chemical Society Reviews (Submitted)

2. Solanki, K., Abdallah, W., and Banta, S. (2016) Biotechnology Journal 11(12) 1483-1497.

3. Abdallah, W., Solanki, K., and Banta, S. (2018) ACS Catalysis 8(2) 1602-1613.