Steroid catabolism in bacteria: Genetic and functional analyses of stdH and stdJ in Pseudomonas putida DOC21

Authors Affiliation(s)

  • Departamento de Biología Molecular, Facultades de Veterinaria y de Biología, Universidad de León, Campus de Vegazana, León 24007, ESPAÑA

Can J Biotech, Volume 2Issue 1,  Pages 88-99,  DOI: https://doi.org/10.24870/cjb.2018-000119

Received: Feb 21, 2018; Revised: Apr 4, 2018; Accepted: Apr 16, 2018

Abstract

Pseudomonas putida DOC21 assimilates a large variety of steroids, including bile acids, via a single 9, 10-seco pathway. Two specific mutants knocked down in stdH and stdJ were obtained by deletion (strains P. putida DOC21ΔstdH and P. putida DOC21ΔstdJ). Analysis of these mutants revealed that both had lost the ability to fully degrade bile acids and that the genes stdH and stdJ are involved in oxidation of the A and B rings of the polycyclic steroid structure. Moreover, whereas P. putida DOC21ΔstdH and P. putida DOC21ΔstdJ were unable to degrade testosterone or 4-androstene-3,17-dione (AD), P. putida DOC21ΔstdJ was also unable to assimilate androsta-1,4-diene-3,17-dione (ADD). When cultured in medium containing lithocholate and succinate, P. putida DOC21ΔstdH and P. putida DOC21ΔstdJ accumulated AD and ADD, respectively. Genetic and bioinformatics analyses revealed that: (i) stdH encodes a 3-ketosteroid-Δ1-dehydrogenase; (ii) StdJ is the reductase component of a 3-ketosteroid 9α-hydroxylase; (iii) the trans-expression of stdH and stdJ in the corresponding mutant restored the lost catabolic function(s), and (iv) full steroid metabolism by P. putida DOC21ΔstdH was restored by its expression of kstD2, but not kstD1 or kstD3, of Rhodococcus ruber Chol-4. Our results shed light on the systems used by bacteria to oxidize the A and B rings of steroid compounds. In addition, as the mutants described herein were able to synthesize two pharmaceutically important synthons, AD and ADD, they may be of value in industrial applications.

References

  1. Oppermann, U.C.T. and Maser, E. (1996) Characterization of a 3α-hydroxysteroid dehydrogenase/carbonyl reductase from the Gram- Negative bacterium Comamonas testosteroni. Eur J Biochem 241: 744-749.Crossref
  2. García, J.L., Uhía, I. and Galán, B. (2012) Catabolism and    biotechnological applications of cholesterol degrading bacteria. Microb Biotechnol 5: 679-699.Crossref
  3. Kreit, J. (2017) Microbial catabolism of sterols: focus on the enzymes that transform the sterol 3β-hydroxy-5-en into 3-keto-4-en. FEMS Microbiol Lett 364: fnx007.Crossref
  4. Barrientos, A., Merino, E., Casabon, I., Rodríguez, J., Crowe, A.M., Holert, J., Philipp, B., Eltis, L.D., Olivera, E.R. and Luengo, J.M. (2015) Functional analyses of three acyl-CoA synthetases involved in bile acid degradation in Pseudomonas putida DOC21. Environ Microbiol 17: 47-63.Crossref
  5. Capyk, J.K., Casabon, I., Gruninger, R., Strynadka, N.C. and Eltis, L.D. (2011) Activity of 3-ketosteroid-9alpha- hydroxylase (KshAB) indicates cholesterol side chain and ring degradation occur simultaneously in Mycobacterium tuberculosis. J Biol Chem 286: 40717-40724.Crossref
  6. Holert, J., Jagmann, N. and Philipp, B. (2013) The essential function of genes for a hydratase and an aldehyde dehydrogenase for growth of Pseudomonas sp. strain Chol1 with the steroid compound cholate indicates an aldolytic reaction step for deacetylation of the side chain. J Bacteriol 195: 3371-3380.Crossref
  7. Holert, J., Kulić, Ž., Yücel, O., Suvekbala, V., Suter, M.J.F., Möller, H.M. and Philipp, B. (2013) Degradation of the acyl side chain of the steroid compound cholate in Pseudomonas sp. strain Chol1 proceeds via an aldehyde intermediate. J Bacteriol 195: 585-595.Crossref
  8. Itagaki, E., Matushita, H. and Hatta, T. (1990) Steroid transhydrogenase activity of 3-ketosteroid-delta 1- dehydrogenase from Nocardia corallina. J Biochem 108: 122-127.
  9. Rohman, A., van Oosterwijk, N., Thunnissen, A.M.W.H. and Dijkstra, B.W. (2013) Crystal structure and site- directed mutagenesis of 3-ketosteroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1 explain its catalytic mechanism. J Biol Chem 288: 35559-35568.Crossref
  10. Fernández de las Heras, L., van der Geize, R., Drzyzga, O., Perera, J. and Maria Navarro Llorens, J. (2012) Molecular characterization of three 3-ketosteroid-Δ1- dehydrogenase isoenzymes of Rhodococcus ruber strain Chol-4. J Steroid Biochem Mol Biol 132: 271-281.Crossref
  11. Hatta, T., Wakabayashi, T. and Itagaki, E. (1991) 3- Keto-5α-steroid-Δ4-dehydrogenase from Nocardia corallina: purification and characterization. J Biochem 109: 581-586.
  12. van Oosterwijk, N., Knol, J., Dijkhuizen, L., van der Geize, R. and Dijkstra, B.W. (2012) Structure and catalytic mechanism of 3-ketosteroid-Δ4-(5α)- dehydrogenase from Rhodococcus jostii RHA1 genome. J Biol Chem 287: 30975-30983.Crossref
  13. Galán, B., Uhía, I., García-Fernández, E., Martínez, I., Bahillo, E., de la Fuente, J.L., Barredo, J.L., Fernández- Cabezón, L. and García, J.L. (2017) Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons. Microb Biotechnol 10: 138-150.Crossref
  14. Gibson, D.T., Wang, K.C., Sih, C.J. and Whitlock Jr., H. (1966) Mechanisms of steroid oxidation by microorganisms. IX. On the mechanism of ring A cleavage in the degradation of 9,10-seco steroids by microorganisms. J Biol Chem 241: 551-559.
  15. van der Geize, R., Hessels, G.I., van Gerwen, R., van der Meijden, P. and Dijkhuizen, L. (2002) Molecular and functional characterization of kshA and kshB, encoding two components of 3-ketosteroid 9α-hydroxylase, a class IA monooxygenase, in Rhodococcus erythropolis strain SQ1. Mol Microbiol 45: 1007-1018.Crossref
  16. Petrusma, M., Dijkhuizen, L. and van der Geize, R. (2012) Structural features in the KshA terminal oxygenase protein that determine substrate preference of 3-ketosteroid 9α-hydroxylase enzymes. J Bacteriol 194: 115-121. Crossref
  17. Petrusma, M., Hessels, G., Dijkhuizen, L. and van der Geize, R. (2011) Multiplicity of 3-ketosteroid-9α- hydroxylase enzymes in Rhodococcus rhodochrous DSM43269 for specific degradation of different classes of steroids. J Bacteriol 193: 3931-3940.Crossref
  18. Uhía, I., Galán, B., Kendall, S.L., Stoker, N.G. and García, J.L. (2012) Cholesterol metabolism in Mycobacterium smegmatis. Environ Microbiol Rep 4: 168-182. Crossref
  19. Kavanagh, K.L., Jornvall, H., Persson, B. and Oppermann, U. (2008) Medium- and short-chain dehydrogenase/reductase gene and protein families: the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes. Cell Mol Life Sci 65: 3895-3906.Crossref
  20. Kallberg, Y., Oppermann, U. and Persson, B. (2010) Classification of the short-chain dehydrogenase/reductase superfamily using hidden Markov models. FEBS J 277: 2375-2386. Crossref
  21. Kisiela, M., Skarka, A., Ebert, B. and Maser, E. (2012) Hydroxysteroid dehydrogenases (HSDs) in bacteria: a bioinformatic perspective. J Steroid Biochem Mol Biol 129: 31-46. Crossref
  22. Horinouchi, M., Kurita, T., Hayashi, T. and Kudo, T. (2010) Steroid degradation genes in Comamonas testosteroni TA441: Isolation of genes encoding a Δ4(5)- isomerase and 3α- and 3β-dehydrogenases and evidence for a 100 kb steroid degradation gene hot spot. J Steroid Biochem Mol Biol 122: 253-263.Crossref
  23. Knol, J., Bodewits, K., Hessels, G.I., Dijkhuizen, L. and van der Geize, R. (2008) 3-Keto-5alpha-steroid delta(1)- 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.Crossref
  24. Bragin, E.Y., Shtratnikova, V.Y., Dovbnya, D.V., Schelkunov, M.I., Pekov, Y.A., Malakho, S.G., Egorova, O.V., Ivashina, T.V., Sokolov, S.L., Ashapkin, V.V. and Donova, M.V. (2013) Comparative analysis of genes encoding key steroid core oxidation enzymes in fast- growing Mycobacterium spp. strains. J Steroid Biochem Mol Biol 138: 41-53.Crossref
  25. Guevara, G., Fernández de las Heras, L., Perera, J. and Navarro Llorens, J.M. (2017) Functional differentiation of 3-ketosteroid Δ1-dehydrogenase isozymes in Rhodococcus ruber strain Chol-4. Microb Cell Fact 16: 42. Crossref
  26. Merino, E., Barrientos, A., Rodríguez, J., Naharro, G., Luengo, J.M. and Olivera, E.R. (2013) Isolation of cholesterol- and deoxycholate-degrading bacteria from soil samples: evidence of a common pathway. Appl Microbiol Biotechnol 97: 891-904.Crossref
  27. Durfee, T., Nelson, R., Baldwin, S., Plunkett, G. 3rd, Burland, V., Mau, B., Petrosino, J.F., Qin, X., Muzny, D.M., Ayele, M., Gibbs, R.A., Csörgo, B., Pósfai, G., Weinstock, G.M. and Blattner, F.R. (2008) The  complete genome sequence of Escherichia coli DH10B: insights into the biology of a laboratory workhorse.  J Bacteriol 190: 2597-2606.Crossref
  28. Herrero, M., de Lorenzo, V. and Timmis, K.N. (1990) Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol 172: 6557-6567.
  29. Quandt, J. and Hynes, M.F. (1993) Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria. Gene 127: 15-21.Crossref
  30. Kovach, M.E., Elzer, P.H., Hill, D.S., Robertson, G.T., Farris, M.A., Roop, R.M. 2nd, and Peterson, K.M. (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic- resistance cassettes. Gene 166: 175-176.Crossref
  31. Martínez-Blanco, H., Reglero, A., Rodríguez-Aparicio, L.B. and Luengo, J.M. (1990) Purification and biochemical characterization of phenylacetyl-CoA ligase from Pseudomonas putida. A specific enzyme for the catabolism of phenylacetic acid. J Biol Chem 265: 7084-7090.
  32. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors.  Proc Natl Acad Sci USA 74: 5463-5467.
  33. Sambrook, J.F. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, New York, NY, USA. ISBN- 13 978-0-87969-577-4
  34. Delcher, A.L., Bratke, K.A., Powers, E.C. and Salzberg, S.L. (2007) Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23: 673-679. Crossref
  35. Lukashin, A. and Borodovsky, M. (1998) GeneMark.hmm: new solutions for gene finding. Nucleic Acids Res 26: 1107-1115.
  36. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.  Nucleic Acids Res 25: 3389-3402.
  37. Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792-1797.Crossref
  38. Kelley, L.A., Mezulis, S., Yates, C.M., Wass, M.N. and Sternberg, M.J. (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10: 845-858.Crossref 
  39. Kumar, S., Stecher, G. and Tamura, K. (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33: 1870-1874.Crossref
  40. Saitou, N. and Nei, M. (1987) The Neighbor-Joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406-425.Crossref
  41. Zuckerkandl, E. and Pauling, L. (1965) 'Evolutionary divergence and convergence in proteins'. In Evolving Genes and Proteins (Bryson V, Vogel, HJ, Eds). Academic Press, New York, USA. 97-166.
  42. Jones, D.T., Taylor, W.R. and Thornton J.M. (1992) The rapid generation of mutation data matrices from protein sequences. Bioinformatics 8: 275-282. Crossref
  43. Crooks, G.E., Hon, G., Chandonia, J.M. and Brenner, S.E. (2004) WebLogo: A sequence logo generator. Genome Res 14: 1188-1190.Crossref
  44. Donnenberg, M.S. and Kaper, J.B. (1991) Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect Immun 59: 4310-4317.
  45. Horinouchi, M., Hayashi, T., Yamamoto, T. and Kudo, T. (2003) A new bacterial steroid degradation gene cluster in Comamonas testosteroni TA441 which consists of aromatic-compound degradation genes for seco-steroids and 3-ketosteroid dehydrogenase genes. Appl Environ Microbiol 69: 4421-4430.Crossref
  46. Yao, K., Xu, L.Q., Wang, F.Q. and Wei, D.Z. (2014) Characterization and engineering of 3-ketosteroid-Δ1- dehydrogenase and 3-ketosteroid-9α-hydroxylase in Mycobacterium neoaurum ATCC 25795 to produce 9α- hydroxy-4-androstene-3,17-dione through the catabolism of sterols. Metab Eng 24: 181-191.Crossref
  47. Brzostek, A., Sliwinski, T., Rumijowska-Galewicz, A., Korycka-Machala, M. and Dziadek, J. (2005) Identification and targeted disruption of the gene encoding the main 3-ketosteroid dehydrogenase in Mycobacterium smegmatis. Microbiology 151: 2393-2402. Crossref
  48. Dym, O. and Eisenberg, D. (2001) Sequence-structure analysis of FAD-containing proteins. Protein Sci 10: 1712-1728. Crossref
  49. Capyk, J.K., D'Angelo, I., Strynadka, N.C. and Eltis, L.D. (2009) Characterization of 3-ketosteroid 9{alpha}- hydroxylase, a Rieske oxygenase in the cholesterol degradation pathway of Mycobacterium tuberculosis .J Biol Chem 284: 9937-9946.Crossref
  50. Petrusma, M., Dijkhuizen, L. and van der Geize, R. (2009) Rhodococcus rhodochrous DSM 43269 3- ketosteroid 9α-hydroxylase, a two-component iron- sulfur-containing monooxygenase with subtle steroid substrate specificity.  Appl Environ Microbiol 75: 5300-5307.Crossref 
  51. Hu, Y., van der Geize, R., Besra, G.S., Gurcha, S.S.,  Liu, A., Rohde, M., Singh, M. and Coates, A. (2010) 3- ketosteroid 9α-hydroxylase is an essential factor in the pathogenesis of Mycobacterium tuberculosis. Mol Microbiol 75: 107-121.Crossref
  52. Dodson, R.M. and Muir, R.D. (1961) Microbiological transformations. VI. The microbiological aromatization of steroids. J Am Chem Soc 83: 4627-4631.Crossref