Purification, characterization, and application of a high activity 3-ketosteroid-Δ1-dehydrogenase from Mycobacterium neoaurum DSM 1381
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Δ1-Dehydrogenation is one of the most important reactions for steroid drug modification. Numerous 3-ketosteroid-Δ1-dehydrogenases (KstDs) catalyzing this reaction were observed in various organisms. However, only a few have been characterized and used for substrate conversion. In this study, a promising enzyme (KstD2) from Mycobacterium neoaurum DSM 1381 was purified and characterized. Interestingly, KstD2 displayed a high activity on a range of substrates, including 17α-hydroxypregn-4-ene-3,20-dione (17α-OH-P); androsta-4,9(11)-diene-3,17-dione (NSC 44826); and 4-androstene-3,17-dione (AD). These reactions were performed under optimal conditions at 40 °C and pH 8.0. Noteworthy, both the activity and stability of the enzyme were sensitive to various metal ions. After optimizing the expression and biocatalyst conditions, up to 1586 U mg−1 intracellular KstD activity on AD could be produced. Furthermore, the associated conversion rate was 99% with 30 g L−1 AD after 8 h. On the other hand, we obtained 99%, 90%, and over 80% of conversion with 20 g L−1 NSC 44826; 10 g L−1 16,17α-epoxyprogesterone; and 20 g L−1 17α-OH-P or canrenone, respectively, after 24 h. Sequence homology and structural analyses indicated that the residue R178 located in a unique short loop among cluster 2 is crucial for substrate recognition which was confirmed by mutagenesis. In summary, this study reports on the first purification and characterization of a KstD from cluster 2. Its remarkable properties deserve more attention to potentially lead to further industrial applications.
Keywords3-Ketosteroid-Δ1-dehydrogenase Mycobacterium neoaurum Biocatalysis Steroids Heterogeneous expression
Conflict of interest
The authors declare that they have no conflict of interest.
This research was funded by the grants from the State Key Project of Research and Development Plan (grant number, 2017YFE0112700).
Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by the authors.
- Guevara G, Fernandez de Las Heras L, Perera J, Navarro Llorens JM (2017) Functional differentiation of 3-ketosteroid Δ1-dehydrogenase isozymes in Rhodococcus ruber strain Chol-4. Microb Cell Factories 16(1):42. https://doi.org/10.1186/s12934-017-0657-1
- Knol J, Bodewits K, Hessels GI, Dijkhuizen L, Van der Geize R (2008) 3-Keto-5 alpha-steroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1 and its orthologue in Mycobacterium tuberculosis H37Rv are highly specific enzymes that function in cholesterol catabolism. Biochem J 410:339–346. https://doi.org/10.1042/bj20071130
- Mao S, Wang JW, Liu F, Zhu Z, Gao D, Guo Q, Xu P, Ma Z, Hou Y, Cheng X, Sun D, Lu F, Qin HM (2018) Engineering of 3-ketosteroid-(1)-dehydrogenase based site-directed saturation mutagenesis for efficient biotransformation of steroidal substrates. Microb Cell Factories 17(1):141. https://doi.org/10.1186/s12934-018-0981-0 CrossRefGoogle Scholar
- Qin N, Shen Y, Yang X, Su L, Tang R, Li W, Wang M (2017) Site-directed mutagenesis under the direction of in silico protein docking modeling reveals the active site residues of 3-ketosteroid-Delta(1)-dehydrogenase from Mycobacterium neoaurum. World J Microbiol Biotechnol 33(7):146. https://doi.org/10.1007/s11274-017-2310-x CrossRefGoogle Scholar
- Rohman A, van Oosterwijk N, Thunnissen A-MWH, Dijkstra BW (2013) Crystal structure and site-directed mutagenesis of 3-ketosteroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1 explain its catalytic mechanism. J Biol Chem 288(49):35559–35568. https://doi.org/10.1074/jbc.M113.522771
- Shao ML, Zhang X, Rao ZM, Xu MJ, Yang TW, Li H, Xu ZH, Yang ST (2016) A mutant form of 3-ketosteroid-Δ(1)-dehydrogenase gives altered androst-1,4-diene-3, 17-dione/androst-4-ene-3,17-dione molar ratios in steroid biotransformations by Mycobacterium neoaurum ST-095. J Ind Microbiol Biotechnol 43(5):691–701. https://doi.org/10.1007/s10295-016-1743-9 CrossRefGoogle Scholar
- Shao M, Chen Y, Zhang X, Rao Z, Xu M, Yang T, Li H, Xu Z, Yang S (2017a) Enhanced intracellular soluble production of 3-ketosteroid-Δ1-dehydrogenase from Mycobacterium neoaurum in Escherichia coli and its application in the androst-1,4-diene-3,17-dione production. J Chem Technol Biotechnol 92(2):350–357. https://doi.org/10.1002/jctb.5012 CrossRefGoogle Scholar
- Shao M, Sha Z, Zhang X, Rao Z, Xu M, Yang T, Xu Z, Yang S (2017b) Efficient androst-1,4-diene-3,17-dione production by co-expressing 3-ketosteroid-Δ1-dehydrogenase and catalase in Bacillus subtilis. J Appl Microbiol 122(1):119–128. https://doi.org/10.1111/jam.13336
- Wang X, Feng J, Zhang D, Wu Q, Zhu D, Ma Y (2017) Characterization of new recombinant 3-ketosteroid-Δ1-dehydrogenases for the biotransformation of steroids. Appl Microbiol Biotechnol 101(15):6049–6060. https://doi.org/10.1007/s00253-017-8378-2
- Yao K, Xu LQ, Wang FQ, Wei DZ (2014) Characterization and engineering of 3-ketosteroid- big up tri, open1-dehydrogenase and 3-ketosteroid-9alpha-hydroxylase in Mycobacterium neoaurum ATCC 25795 to produce 9alpha-hydroxy-4-androstene-3,17-dione through the catabolism of sterols. Metab Eng 24:181–191. https://doi.org/10.1016/j.ymben.2014.05.005 CrossRefGoogle Scholar
- Zhang W, Shao M, Rao Z, Xu M, Zhang X, Yang T, Li H, Xu Z (2013) Bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione by recombinant Bacillus subtilis expressing ksdd gene encoding 3-ketosteroid-Delta(1)-dehydrogenase from Mycobacterium neoaurum JC-12. J Steroid Biochem Mol Biol 135:36–42. https://doi.org/10.1016/j.jsbmb.2012.12.016 CrossRefGoogle Scholar
- Zhang Q, Ren Y, He J, Cheng S, Yuan J, Ge F, Li W, Zhang Y, Xie G (2015) Multiplicity of 3-ketosteroid Δ1-dehydrogenase enzymes in Gordonia neofelifaecis NRRL B-59395 with preferences for different steroids. Ann Microbiol 65(4):1961–1971. https://doi.org/10.1007/s13213-015-1034-0 CrossRefGoogle Scholar
- Zhang X, Wu D, Yang TW, Xu MJ, Rao ZM (2016) Over-expression of Mycobacterium neoaurum 3-ketosteroid-Δ1-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione. Electron J Biotechnol 24:84–90. https://doi.org/10.1016/j.ejbt.2016.10.004
- Zhang RJ, Liu XC, Wang YS, Han YC, Sun JS, Shi JP, Zhang BG (2018) Identification, function, and application of 3-ketosteroid Δl -dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons. Microb Cell Factories 17:16. https://doi.org/10.1186/s12934-018-0916-9