Rational Engineering of 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase for a Biomimetic Nicotinamide Mononucleotide Cofactor
Enzymes are powerful biological catalysts for natural substrates but they have low catalytic efficiency for non-natural substrates. Protein engineering can be used to optimize enzymes for catalysis and stability. 3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidoreduc...
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MDPI AG
2022-09-01
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| Online Access: | https://www.mdpi.com/2073-4344/12/10/1094 |
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| author | Yan-Liang Chen Yun-Hao Chou Chia-Lin Hsieh Shean-Jaw Chiou Tzu-Pin Wang Chi-Ching Hwang |
| author_facet | Yan-Liang Chen Yun-Hao Chou Chia-Lin Hsieh Shean-Jaw Chiou Tzu-Pin Wang Chi-Ching Hwang |
| author_sort | Yan-Liang Chen |
| collection | DOAJ |
| description | Enzymes are powerful biological catalysts for natural substrates but they have low catalytic efficiency for non-natural substrates. Protein engineering can be used to optimize enzymes for catalysis and stability. 3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidoreduction reaction of NAD<sup>+</sup> with androsterone. Based on the structure and catalytic mechanism, we mutated the residues of T11, I13, D41, A70, and I112 and they interacted with different portions of NAD<sup>+</sup> to switch cofactor specificity to biomimetic cofactor nicotinamide mononucleotide (NMN<sup>+</sup>). Compared to wild-type 3α-HSD/CR, the catalytic efficiency of these mutants for NAD<sup>+</sup> decreased significantly except for the T11 mutants but changed slightly for NMN<sup>+</sup> except for the A70K mutant. The A70K mutant increased the catalytic efficiency for NMN<sup>+</sup> by 8.7-fold, concomitant with a significant decrease in NAD<sup>+</sup> by 1.4 × 10<sup>4</sup>-fold, resulting in 9.6 × 10<sup>4</sup>-fold cofactor specificity switch toward NMN<sup>+</sup> over NAD<sup>+</sup>. Meanwhile, the I112K variant increased the thermal stability and changed to a three-state transition from a two-state transition of thermal unfolding of wild-type 3α-HSD/CR by differential scanning fluorimetry. Molecular docking analysis indicated that mutations on these residues affect the position and conformation of the docked NAD<sup>+</sup> and NMN<sup>+</sup>, thereby affecting their activity. A70K variant sterically blocks the binding with NAD<sup>+</sup>, restores the H-bonding interactions of catalytic residues of Y155 and K159 with NMN<sup>+</sup>, and enhances the catalytic efficiency for NMN<sup>+</sup>. |
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| language | English |
| last_indexed | 2024-03-09T20:31:07Z |
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| spelling | doaj.art-6f1be6357a8248abad432cb20d13c9612023-11-23T23:23:30ZengMDPI AGCatalysts2073-43442022-09-011210109410.3390/catal12101094Rational Engineering of 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase for a Biomimetic Nicotinamide Mononucleotide CofactorYan-Liang Chen0Yun-Hao Chou1Chia-Lin Hsieh2Shean-Jaw Chiou3Tzu-Pin Wang4Chi-Ching Hwang5Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, TaiwanGraduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, TaiwanGraduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, TaiwanDepartment of Biochemistry, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, TaiwanDepartment of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 80708, TaiwanGraduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, TaiwanEnzymes are powerful biological catalysts for natural substrates but they have low catalytic efficiency for non-natural substrates. Protein engineering can be used to optimize enzymes for catalysis and stability. 3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidoreduction reaction of NAD<sup>+</sup> with androsterone. Based on the structure and catalytic mechanism, we mutated the residues of T11, I13, D41, A70, and I112 and they interacted with different portions of NAD<sup>+</sup> to switch cofactor specificity to biomimetic cofactor nicotinamide mononucleotide (NMN<sup>+</sup>). Compared to wild-type 3α-HSD/CR, the catalytic efficiency of these mutants for NAD<sup>+</sup> decreased significantly except for the T11 mutants but changed slightly for NMN<sup>+</sup> except for the A70K mutant. The A70K mutant increased the catalytic efficiency for NMN<sup>+</sup> by 8.7-fold, concomitant with a significant decrease in NAD<sup>+</sup> by 1.4 × 10<sup>4</sup>-fold, resulting in 9.6 × 10<sup>4</sup>-fold cofactor specificity switch toward NMN<sup>+</sup> over NAD<sup>+</sup>. Meanwhile, the I112K variant increased the thermal stability and changed to a three-state transition from a two-state transition of thermal unfolding of wild-type 3α-HSD/CR by differential scanning fluorimetry. Molecular docking analysis indicated that mutations on these residues affect the position and conformation of the docked NAD<sup>+</sup> and NMN<sup>+</sup>, thereby affecting their activity. A70K variant sterically blocks the binding with NAD<sup>+</sup>, restores the H-bonding interactions of catalytic residues of Y155 and K159 with NMN<sup>+</sup>, and enhances the catalytic efficiency for NMN<sup>+</sup>.https://www.mdpi.com/2073-4344/12/10/1094protein engineeringenzyme catalysissteady-state kineticsrational designbiomimetic cofactorprotein thermal stability |
| spellingShingle | Yan-Liang Chen Yun-Hao Chou Chia-Lin Hsieh Shean-Jaw Chiou Tzu-Pin Wang Chi-Ching Hwang Rational Engineering of 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase for a Biomimetic Nicotinamide Mononucleotide Cofactor Catalysts protein engineering enzyme catalysis steady-state kinetics rational design biomimetic cofactor protein thermal stability |
| title | Rational Engineering of 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase for a Biomimetic Nicotinamide Mononucleotide Cofactor |
| title_full | Rational Engineering of 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase for a Biomimetic Nicotinamide Mononucleotide Cofactor |
| title_fullStr | Rational Engineering of 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase for a Biomimetic Nicotinamide Mononucleotide Cofactor |
| title_full_unstemmed | Rational Engineering of 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase for a Biomimetic Nicotinamide Mononucleotide Cofactor |
| title_short | Rational Engineering of 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase for a Biomimetic Nicotinamide Mononucleotide Cofactor |
| title_sort | rational engineering of 3α hydroxysteroid dehydrogenase carbonyl reductase for a biomimetic nicotinamide mononucleotide cofactor |
| topic | protein engineering enzyme catalysis steady-state kinetics rational design biomimetic cofactor protein thermal stability |
| url | https://www.mdpi.com/2073-4344/12/10/1094 |
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