Part:BBa_K1674001
3alpha-hydroxysteroid dehydrogenase
Introduction
The 3α-hydroxysteroid dehydrogenase (3α-HSD) is an enzyme which converts dihydrotestosterone (DHT), a potent derivative of the male hormone testosterone, to 3α-androstanediol, in a reduction reaction described in figure 1. The isoform that has been chosen to create this part is AKR1C9 originated from rat liver. Its advantage is the greater specificity to DHT compared to the human isoforms, found naturally in the prostate[1].
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Figure 1 The reaction catalized by 3-\(\alpha \)-HSD enzyme |
Design considerations
The AKR1C9
The nucleotide sequence for this isoform was taken from an article [2]. Making it compatible with the iGEM standards required changing base pairs in three restriction sites: EcoRI and two PstI.
Experiments and results
We over-expressed the 3α-HSD enzyme using T7 promoter in E.coli BL21 strain, which expresses the T7 polymerase, using the plasmid construct seen below. This composite part was also submitted it as BBa_K1674002.
The cloning was confirmed by sequencing and the over-expression by performing SDS-PAGE (below).
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Figure 3 The SDS-PAGE gel display the enzyme 3α-HSD at the right size 34kDa. It is over-produced in BL21 under T7 promoter. |
After a successful overexpression of the 3α-HSD enzyme under pT7 promoter, we conducted a series of experiments based on the 3α-HSD activity measurement protocol , where we measured NADPH fluorescence over time added to E.coli lysates. To get a basic idea of the kinetics of 3α-HSD enzymatic reaction, we first wanted to examine the effect of increase in initial substrate concentration (DHT). We sonicated BL21 cells after two hours induction with IPTG, added 150uM NADPH to the lysates in a 96-well plate and inserted into plate reader at 37℃ for 30 minutes for stabilization. Afterwards we added DHT in different concentration to each well and measured NADPH fluorescence during 5.5 hours. In a logarithmic time scale we can see a linear behavior in NADPH degradation, where the slope represents the degradation rate (Figure 4). When comparing the different BL21 strains, with and without the 3α-HSD gene, we can see clearly the graph slope is steeper in presence of 3α-HSD, implying faster NADPH degradation rate due to the specific enzymatic activity.
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Figure 4 NADPH degradation rate over time in logarithmic scale with initial concentration of 40uM DHT. |
If we take the slope from each DHT concentration graph, we can describe the reaction rate dependency of the substrate (Figure 5). It seems reaction rate in the absence of 3α-HSD enzyme stays relatively constant with increase in DHT concentration, while it rises logarithmically in the presence of the enzyme. We assume the enzymatic reaction rate reaches saturation at a DHT concentration between 40-60uM. This behavior is compatible with the Michaelis-Menten enzyme kinetics model, which we described in details on our modeling section.
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Figure 5 Reaction rate as function of DHT concentration. |
references
- Penning TM, Jina Y, Heredia VV, Lewis M, Structure–function relationships in 3-hydroxysteroid dehydrogenases: a comparison of the rat and human isoforms, Journal of Steroid Biochemistry & Molecular Biology 85 (2003) 247–255.
- Cheng KC, White PC, Qin KN, Molecular cloning and expression of rat liver 3 alpha-hydroxysteroid dehydrogenase, Mol Endocrinol. 1991 Jun;5(6):823-8.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |