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Article

A Novel NADP(H)-Dependent 7alpha-HSDH: Discovery and Construction of Substrate Selectivity Mutant by C-Terminal Truncation

by
Yinping Pan
1,†,
Shijin Tang
1,†,
Minghai Zhou
1,
Fanglin Ao
1,
Zhuozhou Tang
1,
Liancai Zhu
1,2,*,
Deshuai Lou
3,
Jun Tan
3 and
Bochu Wang
1,*
1
Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400030, China
2
Modern Life Science Experiment Teaching Center, College of Bioengineering, Chongqing University, Chongqing 400030, China
3
Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological & Chemical Engineering, Chongqing University of Education, Chongqing 400067, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Catalysts 2022, 12(7), 781; https://doi.org/10.3390/catal12070781
Submission received: 16 June 2022 / Revised: 11 July 2022 / Accepted: 11 July 2022 / Published: 14 July 2022
(This article belongs to the Topic Advances in Enzymes and Protein Engineering)
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Abstract

7α-Hydroxysteroid dehydrogenase (7α-HSDH) plays an important role in the biosynthesis of tauroursodeoxycholic acid (TUDCA) using complex substrate chicken bile powder as raw material. However, chicken bile powder contains 4.74% taurocholic acid (TCA), and a new by-product tauroursocholic acid (TUCA) will be produced, having the risk of causing colorectal cancer. Here, we obtained a novel NADP(H)-dependent 7α-HSDH with good thermostability from Ursus thibetanus gut microbiota (named St-2-2). St-2-2 could catalyze taurochenodeoxycholic acid (TCDCA) and TCA with the catalytic activity of 128.13 and 269.39 U/mg, respectively. Interestingly, by a structure-based C-terminal truncation strategy, St-2-2△C10 only remained catalytic activity on TCDCA (14.19 U/mg) and had no activity on TCA. As a result, it can selectively catalyze TCDCA in waste chicken bile powder. MD simulation and structural analysis indicated that enhanced surface hydrophilicity and improved C-terminal rigidity affected the entry and exit of substrates. Hydrogen bond interactions between different subunits and interaction changes in Phe249 of the C-terminal loop inverted the substrate catalytic activity. This is the first report on substrate selectivity of 7α-HSDH by C-terminal truncation strategy and it can be extended to other 7α-HSDHs (J-1-1, S1-a-1).
Keywords: 7α-hydroxysteroid dehydrogenase (7α-HSDH; St-2-2); St-2-2∆C10; C-terminal truncation; substrate selectivity; tauroursodeoxycholic acid 7α-hydroxysteroid dehydrogenase (7α-HSDH; St-2-2); St-2-2∆C10; C-terminal truncation; substrate selectivity; tauroursodeoxycholic acid

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MDPI and ACS Style

Pan, Y.; Tang, S.; Zhou, M.; Ao, F.; Tang, Z.; Zhu, L.; Lou, D.; Tan, J.; Wang, B. A Novel NADP(H)-Dependent 7alpha-HSDH: Discovery and Construction of Substrate Selectivity Mutant by C-Terminal Truncation. Catalysts 2022, 12, 781. https://doi.org/10.3390/catal12070781

AMA Style

Pan Y, Tang S, Zhou M, Ao F, Tang Z, Zhu L, Lou D, Tan J, Wang B. A Novel NADP(H)-Dependent 7alpha-HSDH: Discovery and Construction of Substrate Selectivity Mutant by C-Terminal Truncation. Catalysts. 2022; 12(7):781. https://doi.org/10.3390/catal12070781

Chicago/Turabian Style

Pan, Yinping, Shijin Tang, Minghai Zhou, Fanglin Ao, Zhuozhou Tang, Liancai Zhu, Deshuai Lou, Jun Tan, and Bochu Wang. 2022. "A Novel NADP(H)-Dependent 7alpha-HSDH: Discovery and Construction of Substrate Selectivity Mutant by C-Terminal Truncation" Catalysts 12, no. 7: 781. https://doi.org/10.3390/catal12070781

APA Style

Pan, Y., Tang, S., Zhou, M., Ao, F., Tang, Z., Zhu, L., Lou, D., Tan, J., & Wang, B. (2022). A Novel NADP(H)-Dependent 7alpha-HSDH: Discovery and Construction of Substrate Selectivity Mutant by C-Terminal Truncation. Catalysts, 12(7), 781. https://doi.org/10.3390/catal12070781

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