Team:UC Davis/Protein Engineering Design

The aldehyde dehydrogenase family of enzymes (EC: 1.2.1.3, 1.2.1.5) was selected for use with our electrochemical biosensor. This family of enzymes catalyze the reaction of aliphatic, straight chain aldehydes and the oxidized form of beta-nicotinamide adenine dinucleotide (NAD+) to produce the corresponding carboxylic acid and the reduced form of beta-nicotinamide adenine dinucleotide (NADH).

SCHEME OF REACTION

The aldehyde dehydrogenase enzyme family was perfect our engineering and electrochemical applications:

1. This enzyme uses NAD+ as a coenzyme and produces NADH in a 1:1 molar ratio with the amount of aldehyde catalyzed. The concentration of NADH can be readily measured with a spectrophotometer reading absorbance at 340nm, allowing us to easily measure the rate of the reaction catalyzed by an aldehyde dehydrogenase enzyme.
2. The active site of aldehyde dehydrogenase is in the center of a long tunnel, where NAD+ enters from one side and the aldehyde substrate enters from the other side. This tunnel gives us a large amount of flexibility in engineering amino acid residues which will affect the catalytic efficiency of this enzyme toward certain aldehyde species.
3. We identified several commercial electrodes which oxidize NADH back to NAD+ and produce a current. Using a potentiostat, we can convert this current into a voltage and use a computer to determine the concentration of aldehydes in a sample. This cyclic conversion of NAD+/NADH allow us to use an electrochemical approach to quantify the concentration of aldehydes in a sample.

With aldehyde dehydrogenases in mind, we used two approaches to identify enzymes with the desired specificities we would use in our biosensor: bioprospecting and engineering.