DOI: https://doi.org/10.14710/jksa.24.5.161-169

Molecular Cloning and Expression of Haloacid Dehalogenase Gene from a Local Pseudomonas aeruginosa ITB1 Strain and Tertiary Structure Prediction of the Produced Enzyme

Biochemistry Research Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Bandung, Indonesia

Received: 6 Jun 2021; Revised: 8 Jul 2021; Accepted: 13 Jul 2021; Published: 31 Jul 2021.
Open Access Copyright 2021 Jurnal Kimia Sains dan Aplikasi under http://creativecommons.org/licenses/by-sa/4.0.

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Abstract

Organohalogens are widely utilized as pesticides, herbicides, solvents, and for many other industrial purposes. However, the use of these compounds caused some negative impacts to the environment due to their toxicity and persistency. In the light of this, some microbes have been identified and employed to perform dehalogenation, converting halogenated organic compounds to non-toxic materials. In this research, we successfully cloned and sequenced the haloacid dehalogenase gene from a local Pseudomonas aeruginosa ITB1 strain, which is involved in the degradation of monochloroacetate. First, the haloacid dehalogenase gene was amplified by PCR using a pair of primers designed from the same gene sequences of other P. aeruginosa strains available in the GenBank. The cloned gene in pGEM-T in E. coli TOP10 was sequenced, analyzed, and then sub-cloned into pET-30a(+) for expression in E. coli BL21 (DE3). To facilitate direct sub-cloning, restriction sequences of EcoRI (G/AATTC) and HindIII (A/AGCTT) were added to the forward and reversed primers, respectively. The expressed protein in E. coli BL21 (DE3) appeared as a 26-kDa protein in SDS-PAGE analysis, which is in good agreement with the size predicted by ExPASy Protparam. We obtained that the best expression in LB liquid medium was achieved with 0.01 mM IPTG induction at 30°C incubation for 3 hours. We also found that the enzyme is more concentrated in the pellet cells as inclusion bodies. Furthermore, the in-silico analysis revealed that this enzyme consists of 233 amino acid residues. This enzyme’s predicted tertiary structure shows six β-sheets flanked by α-helixes and thus belongs to Group II haloacid dehalogenase. Based on the structural prediction, amino acid residues of Asp7, Ser121, and Asn122 are present in the active site and might play essential roles in catalysis. The presented study laid the foundation for recombinant haloacid dehalogenase production from P. aeruginosa local strains. It provided an insight into the utilization of recombinant local strains to remediate environmental problems caused by organohalogens.

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Keywords: cloning; haloacid dehalogenase; PCR; Pseudomonas aeruginosa
Funding: Institut Teknologi Bandung

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Language : EN
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  1. Susanne Fetzner, Franz Lingens, Bacterial dehalogenases: biochemistry, genetics, and biotechnological applications, Microbiological reviews, 58, 4, (1994), 641-685 https://doi.org/10.1128/mr.58.4.641-685.1994
  2. Benjamin Erable, Isabelle Goubet, Sylvain Lamare, Marie-Dominique Legoy, Thierry Maugard, Bioremediation of halogenated compounds: Comparison of dehalogenating bacteria and improvement of catalyst stability, Chemosphere, 65, 7, (2006), 1146-1152 https://doi.org/10.1016/j.chemosphere.2006.04.007
  3. Habeebat Adekilekun Oyewusi, Roswanira Abdul Wahab, Fahrul Huyop, Dehalogenase-producing halophiles and their potential role in bioremediation, Marine Pollution Bulletin, 160, (2020), 111603 https://doi.org/10.1016/j.marpolbul.2020.111603
  4. Thiau-Fu Ang, Jonathan Maiangwa, Abu Bakar Salleh, Yahaya M. Normi, Thean Chor Leow, Dehalogenases: From Improved Performance to Potential Microbial Dehalogenation Applications, Molecules, 23, 5, (2018), 1100 https://doi.org/10.3390/molecules23051100
  5. Vinayak Agarwal, Zachary D. Miles, Jaclyn M. Winter, Alessandra S. Eustáquio, Abrahim A. El Gamal, Bradley S. Moore, Enzymatic Halogenation and Dehalogenation Reactions: Pervasive and Mechanistically Diverse, Chemical Reviews, 117, 8, (2017), 5619-5674 https://doi.org/10.1021/acs.chemrev.6b00571
  6. Katja E. Hill, Julian R. Marchesi, Andrew J. Weightman, Investigation of Two Evolutionarily Unrelated Halocarboxylic Acid Dehalogenase Gene Families, Journal of Bacteriology, 181, 8, (1999), 2535-2547 https://doi.org/10.1128/JB.181.8.2535-2547.1999
  7. Tatsuo Kurihara, Nobuyoshi Esaki, Kenji Soda, Bacterial 2-haloacid dehalogenases: structures and reaction mechanisms, Journal of Molecular Catalysis B: Enzymatic, 10, 1, (2000), 57-65 https://doi.org/10.1016/S1381-1177(00)00108-9
  8. Jason W. Schmidberger, Jacqueline A. Wilce, Andrew J. Weightman, James C. Whisstock, Matthew C. J. Wilce, The Crystal Structure of DehI Reveals a New α-Haloacid Dehalogenase Fold and Active-Site Mechanism, Journal of Molecular Biology, 378, 1, (2008), 284-294 https://doi.org/10.1016/j.jmb.2008.02.035
  9. Tatsuo Kurihara, A mechanistic analysis of enzymatic degradation of organohalogen compounds, Bioscience, biotechnology, and biochemistry, 75, 2, (2011), 189-198 https://doi.org/10.1271/bbb.100746
  10. Halina Novak, Jennifer Littlechild, 4 - Marine enzymes with applications for biosynthesis of fine chemicals, in: A. Trincone (Ed.) Marine Enzymes for Biocatalysis, Woodhead Publishing, 2013, https://doi.org/10.1533/9781908818355.1.89
  11. Jeremy D. Selengut, MDP-1 Is a New and Distinct Member of the Haloacid Dehalogenase Family of Aspartate-Dependent Phosphohydrolases, Biochemistry, 40, 42, (2001), 12704-12711 https://doi.org/10.1021/bi011405e
  12. Ji-Quan Liu, Tatsuo Kurihara, Masaru Miyagi, Susumu Tsunasawa, Mitsuhiro Nishihara, Nobuyoshi Esaki, Kenji Soda, Paracatalytic inactivation of L-2-haloacid dehalogenase from Pseudomonas sp. YL by hydroxylamine: evidence for the formation of an ester intermediate, Journal of Biological Chemistry, 272, 6, (1997), 3363-3368 https://doi.org/10.1074/jbc.272.6.3363
  13. Yayue Wang, Yanbin Feng, Xupeng Cao, Yinghui Liu, Song Xue, Insights into the molecular mechanism of dehalogenation catalyzed by D-2-haloacid dehalogenase from crystal structures, Scientific Reports, 8, 1, (2018), 1454 https://doi.org/10.1038/s41598-017-19050-x
  14. Tamao Hisano, Yasuo Hata, Tomomi Fujii, Ji-Quan Liu, Tatsuo Kurihara, Nobuyoshi Esaki, Kenji Soda, Crystal Structure of L-2-Haloacid Dehalogenase from Pseudomonas sp. YL: an α/β Hydrolase Structure that is Different from the α/β Hydrolase Fold, Journal of Biological Chemistry, 271, 34, (1996), 20322-20330 https://doi.org/10.1074/jbc.271.34.20322
  15. H. C. Birnboim, J. Doly, A rapid alkaline extraction procedure for screening recombinant plasmid DNA, Nucleic Acids Research, 7, 6, (1979), 1513-1523 https://doi.org/10.1093/nar/7.6.1513
  16. Surinder Mohan Singh, Amulya Kumar Panda, Solubilization and refolding of bacterial inclusion body proteins, Journal of Bioscience and Bioengineering, 99, 4, (2005), 303-310 https://doi.org/10.1263/jbb.99.303
  17. Mitra Ashayeri-Panah, Fereshteh Eftekhar, Bahram Kazemi, Joan Joseph, Cloning, optimization of induction conditions and purification of Mycobacterium tuberculosis Rv1733c protein expressed in Escherichia coli, Iranian journal of microbiology, 9, 2, (2017), 64-73
  18. Martina Mühlmann, Eva Forsten, Saskia Noack, Jochen Büchs, Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures, Microbial Cell Factories, 16, 1, (2017), 220 https://doi.org/10.1186/s12934-017-0832-4
  19. René M. de Jong, Bauke W. Dijkstra, Structure and mechanism of bacterial dehalogenases: different ways to cleave a carbon–halogen bond, Current Opinion in Structural Biology, 13, 6, (2003), 722-730 https://doi.org/10.1016/j.sbi.2003.10.009
  20. Carrie A. Rye, Michail N. Isupov, Andrey A. Lebedev, Jennifer A. Littlechild, Biochemical and structural studies of a l-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii, Extremophiles, 13, 1, (2008), 179 https://doi.org/10.1007/s00792-008-0208-0
  21. Paola R. Beassoni, Lisandro H. Otero, Maria J. Massimelli, Angela T. Lisa, Carlos E. Domenech, Critical Active-Site Residues Identified by Site-Directed Mutagenesis in Pseudomonas aeruginosa Phosphorylcholine Phosphatase, A New Member of the Haloacid Dehalogenases Hydrolase Superfamily, Current Microbiology, 53, 6, (2006), 534-539 https://doi.org/10.1007/s00284-006-0365-2
  22. Muhammad Hasanuddin Zainal Abidin, Khairul Bariyyah Abd Halim, Fahrul Huyop, Tengku Haziyamin Tengku Abdul Hamid, Roswanira Abdul Wahab, Azzmer Azzar Abdul Hamid, The mechanistic role of active site residues in non-stereo haloacid dehalogenase E (DehE), Journal of Molecular Graphics and Modelling, 90, (2019), 219-225 https://doi.org/10.1016/j.jmgm.2019.05.003
  23. Aliyu Adamu, Roswanira Abdul Wahab, Firdausi Aliyu, Aminu Hamza Aminu, Mariya Musa Hamza, Fahrul Huyop, Haloacid dehalogenases of Rhizobium sp. and related enzymes: Catalytic properties and mechanistic analysis, Process Biochemistry, 92, (2020), 437-446 https://doi.org/10.1016/j.procbio.2020.02.002

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