Alcohol dehydrogenases belong to the class of enzymes called oxidoreductases, which metabolize various xenobiotic compounds. Human alcohol dehydrogenases, specifically ADH1, oxidize primary alcohols to aldehydes in the skin, and human carbonyl reductase CBR1 reduces carbonyl groups in drug molecules, especially anticancer drugs, resulting in altered pharmacological effects. For the preliminary screening of inhibitors, a very simple and efficient system that uses enzymes isolated from yeast to mimic human alcohol dehydrogenase has been established. In addition, the advantage of efficient and simple methods of overexpressing the protein of yeast in E. coli as compared to the human protein makes it a viable industrial process. This study presents two such models using the recombinantly purified stereospecific enzymes from Candida parapsilosis ATCC 7330, Candida parapsilosis carbonyl reductase (CpCR) (PDB ID: 4OAQ) and (S)–specific carbonyl reductase (SRED) (PDB ID: 3CTM crystal structure of another protein with 100% identity with SRED) showing structural similarity with human alcohol dehydrogenases.
The first model where CpCR has 23.31% structural similarity with human alcohol dehydrogenase ADH1B1. Reaction conditions for oxidation of primary alcohols by CpCR with cinnamyl alcohol as the model substrate was optimized to 98.2 ± 0.23 % conversion. p-Nitro phenyl glyoxal (PNG) was found to be a novel inhibitor for CpCR, which showed competitive inhibitory activity at lower concentration (IC50,100 ± 1.27 mM) as compared to the standard inhibitor 4-methyl pyrazole (IC50, 400 ± 2.05 mM).
The second model, (S)-specific carbonyl reductase from Candida parapsilosis ATCC 7330 (SRED), was employed whose crystal structure, PDB ID: 3CTM (100% sequence identity with SRED), showed 27% similarity with CBR1 (PDB ID: 1WMA). Furthermore, it was found to have a similarity for reaction with isatin and inhibition with quercetin (non-competitive) as in CBR1. Isatin as substrate showed a Km of 5.3 ± 1.08 mM and Ki of 15.2 ± 1.64 µM for quercetin as an inhibitor. Using (S)-specific carbonyl reductase model enzyme, a novel inhibitor, ethyl (E)-4-(4-chlorophenyl)-2-oxobut-3-enoate (ECOB) was identified. ECOB showed mixed inhibition for isatin reduction, with a Ki of 14.87 ± 0.78 µM and KI of 5.02 ± 0.02 µM, respectively. Additionally, Circular dichroism helped in understanding the inhibitory pattern. ECOB showed a lower IC50 value of 7.2 µM compared to the standard inhibitor quercetin (IC50; 11.7 µM), a better inhibitor than quercetin.
Molecular modelling methods were studied to validate the two models designed for human enzymes.
Publications:
1) Leena, K., Gummadi, S. N., & Chadha, A. (2023). Candida parapsilosis carbonyl reductase as a tool for preliminary screening of inhibitors for alcohol dehydrogenase induced skin sensitization. Process Biochemistry, 126, 147–156. https://doi.org/10.1016/j.procbio.2023.01.006
2) Leena, K., Gummadi, S. N., & Chadha, A. (2023). (S)-specific carbonyl reductase from Candida parapsilosis ATCC 7330 as a model for the initial screening of inhibitors for human carbonyl reductase. Biochemical Engineering Journal, 109205. https://doi.org/10.1016/j.bej.2023.109205