Universal capability of 3-ketosteroid Δ1-dehydrogenases to catalyze Δ1-dehydrogenation of C17-substituted steroids

Wójcik P., Glanowski M., Wojtkiewicz A.M., Rohman A., Szaleniec M.

Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences, Niezapominajek 8, Krakow, 30239, Poland; Department of Chemistry, Faculty of Science and Technology, Universitas Airlangga, Surabaya, 60115, Indonesia; Laboratory of Proteomics, Research Center for Bio-Molecule Engineering (BIOME), Universitas Airlangga, Surabaya, 60115, Indonesia; Laboratory of Biophysical Chemistry, University of Groningen, Groningen, 9747 AG, Netherlands


Abstract

Background: 3-Ketosteroid Δ1-dehydrogenases (KSTDs) are the enzymes involved in microbial cholesterol degradation and modification of steroids. They catalyze dehydrogenation between C1 and C2 atoms in ring A of the polycyclic structure of 3-ketosteroids. KSTDs substrate spectrum is broad, even though most of them prefer steroids with small substituents at the C17 atom. The investigation of the KSTD’s substrate specificity is hindered by the poor solubility of the hydrophobic steroids in aqueous solutions. In this paper, we used 2-hydroxpropyl-β-cyclodextrin (HBC) as a solubilizing agent in a study of the KSTDs steady-state kinetics and demonstrated that substrate bioavailability has a pivotal impact on enzyme specificity. Results: Molecular dynamics simulations on KSTD1 from Rhodococcus erythropolis indicated no difference in ΔGbind between the native substrate, androst-4-en-3,17-dione (AD; − 8.02 kcal/mol), and more complex steroids such as cholest-4-en-3-one (− 8.40 kcal/mol) or diosgenone (− 6.17 kcal/mol). No structural obstacle for binding of the extended substrates was also observed. Following this observation, our kinetic studies conducted in the presence of HBC confirmed KSTD1 activity towards both types of steroids. We have compared the substrate specificity of KSTD1 to the other enzyme known for its activity with cholest-4-en-3-one, KSTD from Sterolibacterium denitrificans (AcmB). The addition of solubilizing agent caused AcmB to exhibit a higher affinity to cholest-4-en-3-one (Ping-Pong bi bi KmA = 23.7 μM) than to AD (KmA = 529.2 μM), a supposedly native substrate of the enzyme. Moreover, we have isolated AcmB isoenzyme (AcmB2) and showed that conversion of AD and cholest-4-en-3-one proceeds at a similar rate. We demonstrated also that the apparent specificity constant of AcmB for cholest-4-en-3-one (kcat/KmA = 9.25∙106 M−1 s−1) is almost 20 times higher than measured for KSTD1 (kcat/KmA = 4.71∙105 M−1 s−1). Conclusions: We confirmed the existence of AcmB preference for a substrate with an undegraded isooctyl chain. However, we showed that KSTD1 which was reported to be inactive with such substrates can catalyze the reaction if the solubility problem is addressed. © 2021, The Author(s).

1,2-dehydrogenation; 3-ketosteroid dehydrogenase; 3-ketosteroids; Cholest-4-en-3-one; Cholest-4-en-3-one Δ1-dehydrogenase; Cholesterol metabolism; Diosgenone; KSTD; Δ1-dehydrogenation


Journal

Microbial Cell Factories

Publisher: BioMed Central Ltd

Volume 20, Issue 1, Art No 119, Page – , Page Count


Journal Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85108862691&doi=10.1186%2fs12934-021-01611-5&partnerID=40&md5=410b1b6eafa2f9179209c59c2590fd7e

doi: 10.1186/s12934-021-01611-5

Issn: 14752859

Type: All Open Access, Gold, Green


References

Urich, K., Sterols and steroids (1994) Comp Anim Biochem.; Rohman, A., Dijkstra, B.W., The role and mechanism of microbial 3-ketosteroid Delta(1)-dehydrogenases in steroid breakdown (2019) J Steroid Biochem Mol Biol, 191, p. 105366. , COI: 1:CAS:528:DC%2BC1MXhtFSqu73E, PID: 30991094; García, J.L., Uhía, I., Galán, B., Catabolism and biotechnological applications of cholesterol degrading bacteria (2012) Microb Biotechnol, 5, pp. 679-699. , PID: 22309478, COI: 1:CAS:528:DC%2BC38Xhs1yrsbnO; Wang, P.H., Lee, T.H., Ismail, W., Tsai, C.Y., Lin, C.W., Tsai, Y.W., An oxygenase-independent cholesterol catabolic pathway operates under oxic conditions (2013) PLoS ONE, 8. , COI: 1:CAS:528:DC%2BC3sXhtVyht7zI, PID: 23826110; Kreit, J., Aerobic catabolism of sterols by microorganisms: key enzymes that open the 3-ketosteroid nucleus (2019) FEMS Microbiol Lett Oxford University Press, 366, pp. 1-13; Ringold, H.J., Hayano, M., Stefanovic, V., Concerning the stereochemistry and bacterial of the of steroids (1963) J Biol Chem; Zhang, R., Liu, X., Wang, Y., Han, Y., Sun, J., Shi, J., Identification, function, and application of 3-ketosteroid Δ1-dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons (2018) Microb Cell Fact BioMed Central, 17, pp. 1-16. , COI: 1:CAS:528:DC%2BC1MXjtFKhurs%3D; Wojtkiewicz, A.M., Wójcik, P., Procner, M., Flejszar, M., Oszajca, M., Hochołowski, M., The efficient Δ1-dehydrogenation of a wide spectrum of 3-ketosteroids in a broad pH range by 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans (2020) J Steroid Biochem Mol Biol, 202, p. 105731. , COI: 1:CAS:528:DC%2BB3cXhs1Cku7vK, PID: 32777354; Kondo, E., Steroid 1-dehydrogenation by a crude enzyme preparation from Arthrobacter simplex (1963) Agric Biol Chem, 27, pp. 69-70. , COI: 1:CAS:528:DyaF3sXotlCluw%3D%3D; Li, J., Guan, Y., Wang, H., Yao, S., 17-epoxyprogesterone by encapsulated Arthrobacter simplex cells in an aqueous/organic solvent two-liquid-phase system (2009) J Chem Technol Biotechnol, 84, pp. 208-214. , COI: 1:CAS:528:DC%2BD1MXhslektrY%3D; Liu, X., Zhang, R., Bao, Z., Yuan, C., Cao, H., Shi, J., Biotransformation of phytosterols to androst-1,4-diene-3,17-dione by Mycobacterium sp. ZFZ expressing 3-ketosteroid-δ1-dehydrogenase (2020) Catalysts, 10, pp. 1-12; Wang, Z.F., Huang, Y.L., Rathman, J.F., Yang, S., Lecithin-enhanced biotransformation of cholesterol to androsta-1,4-diene-3,17-dione and androsta-4-ene-3,17-dione (2002) J Chem Technol Biotechnol, 77, pp. 1349-1357. , COI: 1:CAS:528:DC%2BD38XpsVOnurk%3D; Santos, R.A., Caldeira, J.C.O., Steroid bioconversion in a novel aqueous two-phase system (1991) Biotechnol Lett, 13, pp. 349-354. , COI: 1:CAS:528:DyaK3MXksVOksr4%3D; Flygare, S., Larsson, P., Steroid transformation in aqueous two-phase systems: side-chain degradation of cholesterol by Mycobacterium sp (1989) Enzyme Microb Technol, 11, pp. 752-759. , COI: 1:CAS:528:DyaL1MXmtlSlt70%3D; Rohman, A., Dijkstra, B.W., Application of microbial 3-ketosteroid Δ1-dehydrogenases in biotechnology (2021) Biotechnol Adv, 49, p. 107751. , PID: 33823268; Hesselink, P.G.M., Van Vliet, S., De Vries, H., Witholt, B., Optimization of steroid side chain deavage by Mycobacterium sp. in the presence of cydodextrins (1989) Enzyme Microb Technol, 11, pp. 398-404. , COI: 1:CAS:528:DyaL1MXkslKnsbo%3D; Manosroi, A., Saowakhon, S., Manosroi, J., Enhancement of androstadienedione production from progesterone by biotransformation using the hydroxypropyl-beta-cyclodextrin complexation technique (2008) J Steroid Biochem Mol Biol, 108, pp. 132-136. , COI: 1:CAS:528:DC%2BD1cXhsFynsbc%3D, PID: 17936614; Penasse, L., Peyre, M., Studies of 3-oxo steroid delta-1-oxydo reductase of Arthrobacter simplex (1968) Steroids, 12, pp. 525-544. , COI: 1:CAS:528:DyaF1MXks1c%3D, PID: 5687830; Aries, V.C., Goddard, P., Hill, M.J., Degradation of steroids by intestinal bacteria. III. 3-Oxo-5β-steroid Δ1-dehydrogenase and 3-oxo-5β-steroid Δ4-dehydrogenase (1971) Biochim Biophys Acta, 248, pp. 482-488. , COI: 1:CAS:528:DyaE38XmvFCgsg%3D%3D; Zhang, Q., Ren, Y., He, J., Cheng, S., Multiplicity of 3-ketosteroid Δ1-dehydrogenase enzymes in Gordonia neofelifaecis NRRL B-59395 with preferences for different steroids (2015) Ann Microbiol, 65, pp. 1961-1971. , COI: 1:CAS:528:DC%2BC2MXisVWju74%3D; Chiang, Y.R., Ismail, W., Gallien, S., Heintz, D., Van Dorsselaer, A., Fuchs, G., Cholest-4-en-3-one-Δ1-dehydrogenase, a flavoprotein catalyzing the second step in anoxic cholesterol metabolism (2008) Appl Environ Microbiol, 74, pp. 107-113. , COI: 1:CAS:528:DC%2BD1cXnsVGksg%3D%3D, PID: 17993555; Wang, X., Feng, J., Zhang, D., Wu, Q., Zhu, D., Ma, Y., Characterization of new recombinant 3-ketosteroid-Δ1-dehydrogenases for the biotransformation of steroids (2017) Appl Microbiol Biotechnol, 101, pp. 6049-6060. , COI: 1:CAS:528:DC%2BC2sXhtVarsrrL, PID: 28634849; Mao, S., Wang, J.W., Liu, F., Zhu, Z., Gao, D., Guo, Q., Engineering of 3-ketosteroid ∆1-dehydrogenase based site – directed saturation mutagenesis for efficient biotransformation of steroidal substrates (2018) Microb Cell Fact BioMed Central, 17, pp. 1-13. , COI: 1:CAS:528:DC%2BC1MXjtFKhurs%3D; Sludge, D., Wei, S.T., Wu, Y., Lee, T., Huang, Y., Yang, C., Microbial functional responses to cholesterol catabolism in responses to cholesterol catabolism in (2018) MSystems., 3, pp. 1-19; Chiang, Y.R., Ismail, W., Müller, M., Fuchs, G., Initial steps in the anoxic metabolism of cholesterol by the denitrifying Sterolibacterium denitrificans (2007) J Biol Chem, 282, pp. 13240-13249. , COI: 1:CAS:528:DC%2BD2sXks1Ghu7k%3D, PID: 17307741; Chiang, Y., Ismail, W., Heintz, D., Schaeffer, C., Van, D.A., Fuchs, G., Study of anoxic and oxic cholesterol metabolism by Sterolibacterium denitrificans (2008) J Bacteriol, 190, pp. 905-914. , COI: 1:CAS:528:DC%2BD1cXhsFSgsrs%3D, PID: 18039763; Lin, C.W., Wang, P.H., Ismail, W., Tsai, Y.W., El, N.A., Yang, C.Y., Substrate uptake and subcellular compartmentation of anoxic cholesterol catabolism in Sterolibacterium denitrificans (2015) J Biol Chem, 290, pp. 1155-1169. , COI: 1:CAS:528:DC%2BC2MXpvFSjtA%3D%3D, PID: 25418128; Chiang, Y., Ismail, W., Anaerobic biodegradation of steroids (2017) Anaerobic utilization of hydrocarbons, oils, and lipids, pp. 1-32. , Boll M, (ed), Springer, Cham; Knol, J., Bodewits, K., Hessels, G.I., Dijkhuizen, L., Van Der Geize, R., 3-Keto-5α-steroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1 and its orthologue in Mycobacterium tuberculosis H37Rv are highly specific enzymes that function in cholesterol catabolism (2008) Biochem J, 410, pp. 339-346. , COI: 1:CAS:528:DC%2BD1cXhvVymtrg%3D, PID: 18031290; Rohman, A., Van Oosterwijk, N., Dijkstra, B.W., Purification, crystallization and preliminary X-ray crystallographic analysis of 3-ketosteroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1 (2012) Acta Crystallogr Sect F Struct Biol Cryst Commun, 68, pp. 551-556. , COI: 1:CAS:528:DC%2BC38XosVWqtb0%3D, PID: 22691786; Ma, Y.H., Wang, M., Fan, Z., Shen, Y.B., Zhang, L.T., The influence of host-guest inclusion complex formation on the biotransformation of cortisone acetate Δ1-dehydrogenation (2009) J Steroid Biochem Mol Biol, 117, pp. 146-151. , COI: 1:CAS:528:DC%2BD1MXhtlCku7vN, PID: 19744560; Martin del Valle, E.M., Cyclodextrins and their uses: a review (2004) Process Biochem, 39, pp. 1033-1046. , COI: 1:CAS:528:DC%2BD2cXjtl2lurg%3D; Loftsson, T., Magnúsdóttir, A., Másson, M., Sigurjónsdóttir, J.F., Self-association and cyclodextrin solubilization of drugs (2002) J Pharm Sci, 91, pp. 2307-2316. , COI: 1:CAS:528:DC%2BD38XosF2htLo%3D, PID: 12379916; Rohman, A., Van Oosterwijk, N., Thunnissen, A.M.W.H., Dijkstra, B.W., Crystal structure and site-directed mutagenesis of 3-ketosteroid δ1-dehydrogenase from Rhodococcus erythropolis SQ1 explain its catalytic mechanism (2013) J Biol Chem, 288, pp. 35559-35568. , COI: 1:CAS:528:DC%2BC3sXhvFWrt7bP, PID: 24165124; Kabsch, W., A discussion of the solution for the best rotation to relate two sets of vectors (1978) Acta Crystallogr Sect A, 34, pp. 827-828; Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R., (2016) Gaussian 16, Revision B.01, , Wallingford, CT: Gaussian, Inc; Søndergaard, C.R., Olsson, M.H.M., Rostkowski, M., Jensen, J.H., Improved treatment of ligands and coupling effects in empirical calculation and rationalization of pKa values (2011) J Chem Theory Comput, 7, pp. 2284-2295. , PID: 26606496, COI: 1:CAS:528:DC%2BC3MXnt1Gnsrs%3D; Olsson, M.H.M., SØndergaard, C.R., Rostkowski, M., Jensen, J.H., PROPKA3: consistent treatment of internal and surface residues in empirical pKa predictions (2011) J Chem Theory Comput, 7, pp. 525-537. , COI: 1:CAS:528:DC%2BC3MXit1aqsA%3D%3D, PID: 26596171; Anandakrishnan, R., Aguilar, B., Onufriev, A.V., H++ 3.0: Automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations (2012) Nucleic Acids Res; Salomon-Ferrer, R., Case, D.A., Walker, R.C., An overview of the Amber biomolecular simulation package (2012) WIREs Comput Mol Sci, 3, pp. 198-210. , COI: 1:CAS:528:DC%2BC3sXmtFaiu70%3D; Dupradeau, F.Y., Cézard, C., Lelong, R., Stanislawiak, É., Pêcher, J., Delepine, J.C., R.E.DD.B.: a database for RESP and ESP atomic charges, and force field libraries (2008) Nucleic Acids Res, 36, pp. 360-367. , COI: 1:CAS:528:DC%2BD1cXhtVWisr0%3D; Jorgensen, W.L., Chandrasekhar, J., Madura, J.D., Impey, R.W., Klein, M.L., Comparison of simple potential functions for simulating liquid water (1983) J Chem Phys, 79, pp. 926-935. , COI: 1:CAS:528:DyaL3sXksF2htL4%3D; Salomon-Ferrer, R., Case, D.A., Walker, R.C., An overview of the Amber biomolecular simulation package (2013) Wiley Interdiscip Rev Comput Mol Sci, 3, pp. 198-210. , COI: 1:CAS:528:DC%2BC3sXmtFaiu70%3D; Duan, Y., Wu, C., Chowdhury, S., Lee, M.C., Xiong, G., Zhang, W., A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations (2003) J Comput Chem, 24, pp. 1999-2012. , COI: 1:CAS:528:DC%2BD3sXovVygsbc%3D, PID: 14531054; Miller, B.R., Mcgee, T.D., Swails, J.M., Homeyer, N., Gohlke, H., Roitberg, A.E., MMPBSA py: an efficient program for end-state free energy calculations (2012) J Chem Theory Comput, 8, pp. 3314-3321. , COI: 1:CAS:528:DC%2BC38XhtV2gtrzP, PID: 26605738; Itagaki, E., Wakabayashi, T., Hatta, T., Purification and characterization of 3-ketosteroid-delta 1-dehydrogenase from Nocardia corallina (1990) Biochim Biophys Acta Rev Cancer, 1038, pp. 60-67. , COI: 1:CAS:528:DyaK3cXitlWltbc%3D; Sofińska, K., Wojtkiewicz, A.M., Wójcik, P., Zastawny, O., Guzik, M., Winiarska, A., Investigation of quaternary structure of aggregating 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans: In the pursuit of consensus of various biophysical techniques (2019) Biochim Biophys Acta Gen Subj; Zhang, H., Tian, Y., Wang, J., Li, Y., Wang, H., Mao, S., Construction of engineered Arthrobacter simplex with improved performance for cortisone acetate biotransformation (2013) Appl Microbiol Biotechnol, 97, pp. 9503-9514. , COI: 1:CAS:528:DC%2BC3sXhsVCnu73F, PID: 24037307; D’Souza, V.T., Lipkowitz, K.B., Cyclodextrins: introduction (1998) Chem Rev, 98, pp. 1741-1742. , PID: 11848946; Williams, R.O., III, Mahaguna, V., Sriwongjanya, M., Characterization of an inclusion complex of cholesterol and hydroxypropyl-b-cyclodextrin (1998) Eur J Pharm Biopharm, 46, pp. 355-360. , COI: 1:CAS:528:DyaK1MXks1eruw%3D%3D, PID: 9885309; Luo, J., Cui, H., Jia, H., Li, F., Cheng, H., Shen, Y., Identification, biological characteristics, and active site residues of 3-ketosteroid δ1-dehydrogenase homologues from arthrobacter simplex (2020) J Agric Food Chem, 69, pp. 9496-9512. , COI: 1:CAS:528:DC%2BB3cXhsFOis7vI; Sofińska, K., Wojtkiewicz, A.M., Wójcik, P., Zastawny, O., Guzik, M., Winiarska, A., Investigation of quaternary structure of aggregating 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans: In the pursuit of consensus of various biophysical techniques (2019) Biochim Biophys Acta – Gen Subj, 1863, pp. 1027-1039. , PID: 30876874, COI: 1:CAS:528:DC%2BC1MXmtVylsb4%3D; Van Der Geize, R., Hessels, G.I., Van Gerwen, R., Vrijbloed, J.W., Van Der Meijden, P., Dijkhuizen, L., Targeted disruption of the kstD gene encoding a 3-ketosteroid δ1- dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1 (2000) Appl Environ Microbiol, 66, pp. 2029-2036; van der Geize, R., Hessels, G.I., Dijkhuizen, L., Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid Δ1-dehydrogenase isoenzyme (2002) Microbiology, 148, pp. 3285-3292. , PID: 12368462

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