Our best new basic part is BBa_K2539150: the protein-coding gene ALDH2*1. This gene codes for human mitochondrial acetaldehyde dehydrogenase (ALDH2), ALDH2*1 is the wild type form, which is responsible for oxidizing acetaldehyde, a toxic intermediate of ethanol metabolism, into harmless acetic acid (Larson et al., 2005; Farrés et al., 1994). We were able to test and validate ALDH2*1’s ability to metabolize acetaldehyde. The design and characterization of BBa_K2539150 are documented below.
Part Design
We modified the ALDH2*1 sequence (NCBI: NM_001204889.1) to remove an internal PstI site.
PCR Check Results
The part was confirmed by PCR using the primers VF2 and VR, as well as sequencing by Tri-I Biotech.
BBa_K2539150 contains just the sequence of ALDH2*1. PCR check using VF2 and VR primers produced a band at the expected size of 1.8 kb (yellow box).(Cloning & Figures: Catherine C)
Characterization
We tested the enzyme function of ALDH2*1 (BBa_K2539150) by placing it under the control of a constitutive promoter (BBa_K2539100). We compared ALDH2*1 (the wild type) activity to ALDH2*2 (the mutant) activity. ALDH2*1 should convert acetaldehyde to acetate faster than the mutant form.
Testing enzyme function in cell lysates:
When ALDH2 converts acetaldehyde into acetate, NADH is produced. To test the ability of recombinant ALDH2*1 to metabolize acetaldehyde, we used reagents from a kit (Megazyme, K-ACHYD) to quantify the amount of NADH produced by taking absorbance readings at 340 nm. This wavelength is highly absorbed by the reduced form, NADH, but not the oxidized form, NAD+ (Harimech et al., 2015; McComb et al., 1976). High absorbance values would indicate more conversion of acetaldehyde into acetate.
The wild type ALDH2*1 converts acetaldehyde at a faster rate than mutant ALDH2*2. (A) The conversion of acetaldehyde to acetate by ALDH2 uses NAD+ and produces NADH. (B) Experimental setup. The supernatant from ALDH2-expressing E. coli cell lysates was mixed with acetaldehyde and NAD+ to initiate the reaction at 25°C. NADH concentration was measured by taking absorbance readings at 340 nm. (C) Relative activity of lysates containing either ALDH2*1, ALDH2*2, or inactive ALDH2*1 (boiled to denature proteins; negative control). Error bars represent standard error. (Experiment & Figure: Justin W, Yvonne W, Catherine C)
Over a 40 minute period, E. coli carrying the ALDH2*1-expressing construct (BBa_K2539100) produced more NADH than the mutant form (BBa_K2539200), while the negative control (boiled BBa_K2539100) did not change significantly. This shows that wild type ALDH2*1 is more efficient at metabolizing acetaldehyde compared to the mutant ALDH2*2.
Testing enzyme function of purified samples:
To purify ALDH2*1 and ALDH2*2 enzymes, we added HIS-tags to the DNA designs (BBa_K2539101 and BBa_K2539201). We repeated our functional test using purified enzymes (results shown below), and saw a much clearer difference between ALDH2*1 (BBa_K2539150) and ALDH2*2 (BBa_K2539250), compared to when we used bacterial cell extracts. In our tests, purified ALDH2*2 did not have any effect on NADH, while purified ALDH2*1 significantly increased NADH levels.
Purified ALDH2*1 has a much higher activity level compared to purified ALDH2*2. Using the same procedure, we tested the enzymatic activity of purified ALDH2*1 and ALDH2*2 at 25°C. A negative control containing only elution buffer (from the protein purification process) was also included (gray). ALDH2*1 steadily metabolized more acetaldehyde compared to both ALDH2*2 and the negative control, both of which did not seem to have any effect. The error bars represent standard error, and are bolded for ALDH2*1 and ALDH2*2 to highlight the difference between the two groups. (Experiment & Figure: Justin W)
References
Farrés J, Wang X, Takahashi K, Cunningham SJ, Wang TT, Weiner H. (1994). Effects of changing glutamate 487 to lysine in rat and human liver mitochondrial aldehyde dehydrogenase. A model to study human (Oriental type) class 2 aldehyde dehydrogenase. J Biol Chem. 13;269(19):13854-60.
Harimech PK, Hartmann R, Rejman R, del Pino P, Rivera-Gila P, Parak WJ. (2015). Encapsulated enzymes with integrated fluorescence-control of enzymatic activity. J. Mater. Chem. B. 3, 2801-2807.
Larson HN, Weiner H, Hurley TD. (2005). Disruption of the Coenzyme Binding Site and Dimer Interface Revealed in the Crystal Structure of Mitochondrial Aldehyde Dehydrogenase “Asian” Variant. J Biol Chem. 280(34):30550-6.
McComb RB, Bond LW, Burnett RW, Keech RC, Bowers, GN Jr. (1976). Determination of the molar absorptivity of NADH. Clin Chem. 22(2): 141–150.
