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.

Functional contribution of coenzyme specificity-determining sites of 7α-hydroxysteroid dehydrogenase from Clostridium absonum.

Studies of the molecular determinants of coenzyme specificity help to reveal the structure-function relationship of enzymes, especially with regards to coenzyme specificity-determining sites (CSDSs) that usually mediate complex interactions. NADP(H)-dependent 7α-hydroxysteroid dehydrogenase from Clostridium absonum (CA 7α-HSDH), a member of the short-chain dehydrogenase/reductase superfamily (SDRs), possesses positively charged CSDSs that mainly contain T15, R16, R38, and R194, forming complicated polar interactions with the adenosine ribose C2 phosphate group of NADP(H). The R38 residue is crucial for coenzyme anchoring, but the influence of the other residues on coenzyme utilization is still not clear. Hence, we performed alanine scanning mutagenesis and molecular dynamic (MD) simulations. The results suggest that the natural CSDSs have the greatest NADP(H)-binding affinity, but not the best activity (kcat) toward NADP+. Compared with the wild type and other mutants, the mutant R194A showed the highest catalytic efficiency (kcat/Km), which was more than three-times that of the wild type. MD simulation and kinetics analysis suggested that the importance of the CSDSs of CA 7α-HSDH should be in accordance with the following order R38>T15>R16>R194, and S39 may have a supporting role in NADP(H) anchoring for mutants R16A/T194A and T15A/R16A/T194A.

The ß-Sheet Core is the Favored Candidate of Engineering SDR for Enhancing Thermostability but not for Activity.

BACKGROUND: 7α-Hydroxysteroid dehydrogenases (7α-HSDHs) can stereoselectively catalyze steroids, aromatic α-ketoesters, and benzaldehyde analogues playing a critical role in the biotransformation and poor thermostability that hinders their biomedical and industrial applications. OBJECTIVE: This study was to investigate how to enhance the thermostability of 7α-HSDH from Clostridium absonum (CA 7α-HSDH). METHOD: Based on the three-dimensional structure of CA 7α-HSDH, recently reported program MAESTRO was used to compute the ΔΔG and predict the single-point mutants that could enhance its thermostability. The selected mutants were verified experimentally. RESULTS: The results from the circular dichroism spectrum indicated that three of the mutants, N89L, N184I, and A185I, fitted a three-state model and the values for Tm N→I and Tm I→D increased with different ranges. In particular, the Tm N→I for the N184I mutant increased maximally by 9.93°C. Meanwhile, the denaturation process of the G189I mutant fitted the two-state model and it was more stable than the wild type, judging from the denaturation curves. Nevertheless, the enzyme catalytic activity analysis suggested that only the N89L mutant held a 2.28% catalytic efficiency, compared to the wild type, CA 7α-HSDH, and the activities of the other three mutants could not be detected. Molecular dynamics (MD) simulations were performed to determine the structural changes that occurred in the mutations and the results indicated that ß-sheet structures in the mutants without detectable activity had changed significantly. CONCLUSION: Judging from the locations of the mutated sites, residues in the ß-sheet core were considered as the favored candidates for SDR engineering to enhance the thermostability but not for activity holding.