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).

The aldehyde dehydrogenase enzyme family is perfect for 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 (highlighted in orange) gives us a large amount of flexibility in engineering amino acid residues which will alter 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. This will allow us to measure the amount of NADH produced over time using an electrochemical approach.

4. Preferred: Crystal structure available on the Protein Data Bank (PDB)

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.

We used several online databases to search for aldehyde dehydrogenases with unique specificity profiles. Keeping the context of the aldehyde dehydrogenase within our electrochemical sensor in mind, we focused exclusively on aldehyde dehydrogenases which used NAD(H) as a coenzyme (EC: 1.2.1.3) as opposed to aldehyde dehydrogenases which used NADP(H) (EC: 1.2.1.5). However, we did identify several aldehyde dehydrogenases which could use either coenzyme. BRENDA was our main resource for identifying aldehyde dehydrogenases which catalyzed the oxidation reaction on specific substrates. The specificity data provided within the literature cited for each entry on BRENDA helped guide our decisions for the set of enzymes we chose to characterize.

Selection Criteria:

1. DNA sequence readily available on Genbank or UniProtKB

2. Aldehyde dehydrogenase possesses unique specificity for one or more of the aldehyde species known to occur in olive oil

3. Aldehyde dehydrogenase uses NAD⁺ as a coenzyme for the dehydrogenase reaction

4. Aldehyde dehydrogenase is documented to express in Escherichia coli and is easily purified in high yield (>1 mg/mL)