Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bhatti HN, Khera RA (2012) Biological transformations of steroidal compounds: a review. Steroids 77(12):1267–1290. https://doi.org/10.1016/j.steroids.2012.07.018
Donova MV, Egorova OV (2012) Microbial steroid transformations: current state and prospects. Appl Microbiol Biotechnol 94(6):1423–1447. https://doi.org/10.1007/s00253-012-4078-0
Fernandes P, Cruz A, Angelova B, Pinheiro HM, Cabral JMS (2003) Microbial conversion of steroid compounds: recent developments. Enzym Microb Technol 32(6):688–705. https://doi.org/10.1016/s0141-0229(03)00029-2
Fernandez-Cabezon L, Galan B, Garcia JL (2018) New insights on steroid biotechnology. Front Microbiol 9:958. https://doi.org/10.3389/fmicb.2018.00958
García J, Uhía I, Galán B (2012) Catabolism and biotechnological applications of cholesterol degrading bacteria. Microb Biotechnol 5(6):679–699. https://doi.org/10.1111/j.1751-7915.2012.00331.x
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
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
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
Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13(11):2498–2504. https://doi.org/10.1101/gr.1239303
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
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
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
Song B, Zhou Q, Xue HJ, Liu JJ, Zheng YY, Shen YB, Wang M, Luo JM (2018) IrrE Improves organic solvent tolerance and Δ(1)-dehydrogenation productivity of Arthrobacter simplex. J Agric Food Chem 66(20):5210–5220. https://doi.org/10.1021/acs.jafc.8b01311
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
Wu Y, Li H, Zhang X-M, Gong J-S, Rao Z-M, Shi J-S, Zhang X-J, Xu Z-H (2015) Efficient hydroxylation of functionalized steroids by Colletotrichum lini ST-1. J Mol Catal B-Enzym 120:111–118. https://doi.org/10.1016/j.molcatb.2015.07.003
Xie R, Shen Y, Qin N, Wang Y, Su L, Wang M (2015) Genetic differences in ksdD influence on the ADD/AD ratio of Mycobacterium neoaurum. J Ind Microbiol Biotechnol 42(4):507–513. https://doi.org/10.1007/s10295-014-1577-2
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
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
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
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
Conflict of interest
The authors declare that they have no conflict of interest.
Funding
This research was funded by the grants from the State Key Project of Research and Development Plan (grant number, 2017YFE0112700).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
This article does not contain any studies with human participants or animals performed by the authors.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
ESM 1
(PDF 690 kb)
Rights and permissions
About this article
Cite this article
Zhang, R., Xu, X., Cao, H. et al. Purification, characterization, and application of a high activity 3-ketosteroid-Δ1-dehydrogenase from Mycobacterium neoaurum DSM 1381. Appl Microbiol Biotechnol 103, 6605–6616 (2019). https://doi.org/10.1007/s00253-019-09988-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-019-09988-5