A novel 7α-hydroxysteroid dehydrogenase: Magnesium ion significantly enhances its activity and thermostability.

7α-Hydroxysteroid dehydrogenase (7α-HSDH) plays an important role in the efficient biotransformation of taurochenodeoxycholic acid (TCDCA) to tauroursodeoxycholic acid (TUDCA). In this paper, a novel NADP(H)-dependent 7α-HSDH (named J-1-1) was discovered, heterologously expressed in Escherichia coli and biochemically characterized. J-1-1 exhibited high enzymatic activities. The specific activities of J-1-1 toward TCDCA, glycochenodeoxycholic acid (GCDCA) and ethyl benzoylacetate (EBA) were 188.3 ± 0.2, 217.6 ± 0.4, and 20.0 ± 0.2 U·mg-1, respectively, in 50 mM Glycine-NaOH, pH 10.5. Simultaneously, J-1-1 showed high thermostability; 73% of its activity maintained after heat treatment at 40 °C for 100 h. Particularly noteworthy is that magnesium ion could stabilize the structure of J-1-1, resulting in the enhancement of its enzymatic activity and thermostability. The enzymatic activity of J-1-1 increased 40-fold in the presence of 50 mM Mg2+, and T0.5 increased by approximately 6 °C. Furthermore, after heat treatment at 40 °C for 20 min, the control group only retained 52% of the residual enzyme activity, while the residual enzyme activity of the experimental group was still 77% of the J-1-1 enzyme activity with Mg2+ and without heat treatment. These properties of 7α-HSDH would be expected to contribute to more extensive applications in the biotransformation of related substrates.

Structural and functional characterization of a novel acidophilic 7α-hydroxysteroid dehydrogenase.

7α-Hydroxysteroid dehydrogenase (7α-HSDH) is an NAD(P)H-dependent oxidoreductase belonging to the short-chain dehydrogenases/reductases. In vitro, 7α-HSDH is involved in the efficient biotransformation of taurochenodeoxycholic acid (TCDCA) to tauroursodeoxycholic acid (TUDCA). In this study, a gene encoding novel 7α-HSDH (named as St-2-1) from fecal samples of black bear was cloned and heterologously expressed in Escherichia coli. The protein has subunits of 28.3 kDa and a native size of 56.6 kDa, which suggested a homodimer. We studied the relevant properties of the enzyme, including the optimum pH, optimum temperature, thermal stability, activators, and inhibitors. Interestingly, the data showed that St-2-1 differs from the 7α-HSDHs reported in the literature, as it functions under acidic conditions. The enzyme displayed its optimal activity at pH 5.5 (TCDCA). The acidophilic nature of 7α-HSDH expands its application environment and the natural enzyme bank of HSDHs, providing a promising candidate enzyme for the biosynthesis of TUDCA or other related chemical entities.

Engineering Clostridium absonum 7α-hydroxysteroid Dehydrogenase for Enhancing Thermostability Based on Flexible Site and ΔΔG Prediction.

BACKGROUND: Enhancing thermostability of the 7α-Hydroxysteroid dehydrogenases (7α-HSDHs) is beneficial to its industrial application broadly. For protein engineering to enhance thermostability the nonrational strategy, directed evolution, has been applied in obtaining more stable proteins through error-prone PCR or DNA rearrangement generating random mutations. However, the successful application of directed evolution needs to build a large mutant library. Site-directed mutations of CA 7α-HSDH had been performed to probe the relationship between the compactness increasing and thermostability enhancing. Although most of the mutations in ß-sheet core predicted by MAESTRO became more stable than wild type, unfortunately, all the mutations suffered dramatic activity loss. OBJECTIVE: The main objective of this study was to verify effects of the mutations in helices selected from the predicting results through MAESTRO on thermostability improving of CA 7α-HSDH. METHODS: Seven mutants, S22L, P124L, A125L, N171L, A195Q, L197E and Y259E were synthesized and verified through DNA sequencing in Sangon Biotech (Sangon, Shanghai, China). The two mutants, A104P and G105P were prepared by over-lapping PCR. The GST-fusion expression vector, pGEX-6p-1 (GE Healthcare), was used for protein expression with restriction sites BamH I and Not I. Thermostability was measured by circular dichroism (CD) spectrometer (MOS-450, BioLogic Inc). All the enzymes were diluted in PBS (pH 7.3, 10 mM) to OD222 value between 0.8 and 1, and temperature varied from 20°C to 95°C. Activity of enzyme was assayed by measuring the production of NADPH by UV-visible spectrophotometer at 340 nm. The activity assay was performed in 2 mL reaction mixture which contained PBS (pH 7.3, 10 mM), NADP+ (0.5 mM) and taurocholic acid (TCA) at 25°C. RESULTS: Based on unfolding free energy changes (ΔΔG) prediction seven mutations of Clostridium absonum (CA) 7α-HSDH were selected and experimentally verified, and these mutants fitted three-state denaturation model well, among which S22L located in the αA possessed the greatest Tm N→I increase (> 8°C). Mutants P124L, L197E, N171L and Y259E also became more stable than wild type CA 7α-HSDH with different ranges. Meanwhile, thermostability of the two mutants, A104P and G105P (in the coil between ßD and αD) resulting from the proline substitution method decreased significantly. Enzyme activity assays indicated that mutant L197E located in αF maximally maintained 28.7% of catalytic efficiency, and activity of the five mutants, P124L, A125L, N171L, A104P and G105P cannot be detected. CONCLUSION: Although all the mutants' activities decreased, the mutant L197E with the maximum activity retain suggested that the loop structure (residues 194 to 211) may be the favored candidate sites to enhance thermostability. In addition, CA 7α-HSDH may suffer structural destruction resulting from the proline substitution in A104 and G105.