Abstract
Thermostability improvement of enzymes used industrially or commercially would develop their capacity and commercial potential due to increased enzymatic competence and cost-effectiveness. Several stabilizing factors have been suggested to be the base of thermal stability, like proline replacements, disulfide bonds, surface loop truncation and ionic pair networks creation. This research evaluated the mechanism of increasing the rigidity of organophosphorus hydrolase enzyme by flexible loop truncation. Bioinformatics analysis revealed that the mutated protein retains its stability after loop truncation (five amino acids deleted). The thermostability of the wild-type (OPH-wt) and mutated (OPH-D5) enzymes were investigated by half-life, ΔGi, and fluorescence and far-UV CD analysis. Results demonstrated an increase half-life and ΔGi in OPH-D5 compared to OPH-wt. These results were confirmed by extrinsic fluorescence and circular dichroism (CD) spectrometry experiments, therefore, as rigidity increased in OPHD5 after loop truncation, half-life and ΔGi also increased. Based on these findings, a strong case is presented for thermostability improvement of OPH enzyme by flexible loop truncation after bioinformatics analysis.
Similar content being viewed by others
References
Abdel-Razek MA-RS, Folch-Mallol JL, Perezgasga-Ciscomani L, Sánchez-Salinas E, Castrejón-Godínez ML and Ortiz-Hernández ML 2013 Optimization of methyl parathion biodegradation and detoxification by cells in suspension or immobilized on tezontle expressing the opd gene. J. Environ. Sci. Health 48 449–461
Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B and Lindahl E 2015 GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2 19–25
Almog O, Gallagher DT, Ladner JE, Strausberg S, Alexander P, Bryan P and Gilliland GL 2002 Structural basis of thermostability analysis of stabilizing mutations in subtilisin BPN. J. Biol. Chem. 277 27553–27558
Amini-Bayat Z, Hosseinkhani S, Jafari R and Khajeh K 2012 Relationship between stability and flexibility in the most flexible region of Photinus pyralis luciferase. Biochim. Biophys. Acta Proteins Proteomics 1824 350–358
Armstrong CD 2007 Elucidating the chemical and thermal unfolding profiles of organophosphorus hydrolase and increasing its operational stability (Texas A&M University)
Ásgeirsson B, Adalbjörnsson BV and Gylfason GA 2007 Engineered disulfide bonds increase active-site local stability and reduce catalytic activity of a cold-adapted alkaline phosphatase. Biochim. Biophys. Acta Proteins Proteomics 1774 679–687
Benning MM, Shim H, Raushel FM and Holden HM 2001 High resolution X-ray structures of different metal-substituted forms of phosphotriesterase from Pseudomonas diminuta. Biochemistry 40 2712–2722
Boone CD, Rasi V, Tu C and McKenna R 2015a Structural and catalytic effects of proline substitution and surface loop deletion in the extended active site of human carbonic anhydrase II. FEBS J. 282 1445–1457
Briseño-Roa L, Oliynyk Z, Timperley CM, Griffiths AD and Fersht AR 2011 Highest paraoxonase turnover rate found in a bacterial phosphotriesterase variant. Protein Eng. Design Selection 24 209–211
Chu X-y, Tian J, Wu N-f and Fan Y-l 2010 An intramolecular disulfide bond is required for the thermostability of methyl parathion hydrolase, OPHC2. Appl. Microbiol. Biotechnol. 88 125–131
D’Amico S, Gerday C and Feller G 2003 Temperature adaptation of proteins: engineering mesophilic-like activity and stability in a cold-adapted alpha-amylase. J. Mol. Biol. 332 981–988
Dagan S, Hagai T, Gavrilov Y, Kapon R, Levy Y, Reich Z 2013 Stabilization of a protein conferred by an increase in folded state entropy. Proc. Nat. Acad. Sci. 110 10628–10633
Damnjanović J, Nakano H and Iwasaki Y 2014 Deletion of a dynamic surface loop improves stability and changes kinetic behavior of phosphatidylinositol‐synthesizing Streptomyces phospholipase D. Biotechnol. Bioeng. 111 674–682
Danson MJ, Hough DW, Russell RJ, Taylor GL and Pearl L 1996 Enzyme thermostability and thermoactivity. Protein Eng. 9 629–630
Ely F, Hadler K, Gahan L, Guddat L, Ollis D and Schenk G 2010 The organophosphate-degrading enzyme from Agrobacterium radiobacter displays mechanistic flexibility for catalysis. Biochem. J. 432 565–573
Farnoosh G and Latifi AM 2014 A review on engineering of organophosphorus hydrolase (OPH) enzyme. J. Appl. Biotechnol. Rep. 1 1–10
Farnoosh G, Khajeh K, Latifi AM and Aghamollaei H 2016 Engineering and introduction of de novo disulphide bridges in organophosphorus hydrolase enzyme for thermostability improvement. J. Biosci. 41 577–588
Froger A and Hall JE 2007 Transformation of plasmid DNA into E. coli using the heat shock method. JoVE 6 253
Ghisaidoobe AB and Chung SJ 2014 Intrinsic tryptophan fluorescence in the detection and analysis of proteins: a focus on Forster resonance energy transfer techniques. Int. J. Mol. Sci. 15 22518–22538
Grote A, Hiller K, Scheer M, Münch R, Nörtemann B, Hempel DC and Jahn D 2005 JCat: a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res. 33 W526–W531
Haghani K, Salmanian AH, Ranjbar B, Zakikhan-Alang K and Khajeh K 2008 Comparative studies of wild type Escherichia coli 5-enolpyruvylshikimate 3-phosphate synthase with three glyphosate-insensitive mutated forms: Activity, stability and structural characterization. Biochim. Biophys. Acta Proteins Proteomics 1784 1167–1175
Halliwell LM 2015 Protein engineering utilising single amino acid deletions within Photinus pyralis firefly luciferase (Cardiff University)
Han Z-l, Han S-Y, Zheng S-P and Lin Y 2009 Enhancing thermostability of a Rhizomucor miehei lipase by engineering a disulfide bond and displaying on the yeast cell surface. Appl. Microbiol. Biotechnol. 85 117–126
Hawwa R, Aikens J, Turner RJ, Santarsiero BD and Mesecar AD 2009 Structural basis for thermostability revealed through the identification and characterization of a highly thermostable phosphotriesterase-like lactonase from Geobacillus stearothermophilus. Arch. Biochem. Biophys. 488 109–120
Imani M, Hosseinkhani S, Ahmadian S and Nazari M 2010 Design and introduction of a disulfide bridge in firefly luciferase: increase of thermostability and decrease of pH sensitivity. Photochem. Photobiol. Sci. 9 1167–1177
Jorgensen WL, Maxwell DS and Tirado-Rives J 1996 Development and testing of the OPLS all-atom force field on conformational energetics and properties of organic liquids. J. Am. Chem. Soc. 118 11225–11236
Kachooei E, Moosavi-Movahedi AA, Khodagholi F, Mozafarian F, Sedeghi P, Hadi-Alijanvand H, Ghasemi A, Saboury AA, Farhadi M and Sheibani N 2014 Inhibition study on insulin fibrillation and cytotoxicity by paclitaxel. J. Biochem. 155 361–373
Kazan D, Ertan H and Erarslan A 1997 Stabilization of Escherichia coli penicillin G acylase against thermal inactivation by cross-linking with dextran dialdehyde polymers. Appl. Microbiol. Biotechnol. 48 191–197
Kim J, Kim S, Yoon S, Hong E and Ryu Y 2015 Improved enantioselectivity of thermostable esterase from Archaeoglobus fulgidus toward (S)-ketoprofen ethyl ester by directed evolution and characterization of mutant esterases. Appl. Microbiol. Biotechnol. 99 6293–6301
Kumar S and Nussinov R 2001 How do thermophilic proteins deal with heat? Cell. Mol. Life Sci. 58 1216–1233
Le QAT, Joo JC, Yoo YJ and Kim YH 2012 Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge. Biotechnol. Bioeng. 109 867–876
Li B, Yang G, Wu L and Feng Y 2012 Role of the NC-loop in catalytic activity and stability in lipase from Fervidobacterium changbaicum. PLoS One 7 e46881
Lobanov MY, Bogatyreva NS and Galzitskaya OV 2008 Radius of gyration as an indicator of protein structure compactness. Mol. Biol. 42 623–628
Paliwal S 2008 Development of enzyme-based biosensors for the detection of organophosphate neurotoxins (ProQuest)
Pawlowski PH and Zielenkiewicz P 2013 Theoretical model explaining the relationship between the molecular mass and the activation energy of the enzyme revealed by a large-scale analysis of bioinformatics data. Acta Biochim. Pol. 60 239–247
Pinkerton TS 2005 The recombinant expression and potential applications of bacterial organophosphate hydrolase in Zea mays L (Texas A&M University)
Sakurai M, Ohzeki M, Miyazaki K, Moriyama H, Sato M, Tanaka N and Oshima T 1996a Structure of a loop-deleted variant of 3-isopropylmalate dehydrogenase from Thermus thermophilus: an internal reprieve tolerance mechanism. Acta Crystallogr. Biol. Crystallogr. 52 124–128
Thompson MJ and Eisenberg D 1999 Transproteomic evidence of a loop-deletion mechanism for enhancing protein thermostability. J. Mol. Biol. 290 595–604
Tian J, Wang P, Gao S, Chu X, Wu N and Fan Y 2010 Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rational engineering of a glycine to proline mutation. FEBS J. 277 4901–4908
Tian J, Wang P, Huang L, Chu X, Wu N and Fan Y 2013 Improving the thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 using a computationally aided method. Appl. Microbiol. Biotechnol. 97 2997–3006
Ugarova N and Koksharov M 2012 Thermostabilization of firefly luciferases using genetic engineering (INTECH Open Access Publisher)
van Mierlo CP and Steensma E 2000 Protein folding and stability investigated by fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy: the flavodoxin story. J. Biotechnol. 79 281–298
Yang H, Carr P, McLoughlin SY, Liu J, Horne I, Qiu X, Jeffries C, Russell R, Oakeshott J and Ollis D 2003 Evolution of an organophosphate-degrading enzyme: a comparison of natural and directed evolution. Protein Eng. 16 135–145
Yin X, Hu D, Li J-F, He Y, Zhu T-D and Wu M-C 2015 Contribution of disulfide bridges to the thermostability of a type A feruloyl esterase from Aspergillus usamii. PLoS One https://doi.org/10.1371/journal.pone.0126864
Yipp BG, Petri B, Salina D, et al. 2012 Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nat. Med. 18 1386–1393
Zuker M 2003 Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31 3406–3415
Acknowledgements
The authors would like to thank the manager and colleagues of Applied Biotechnology Research Centre, Baqiyatallah University of Medical Sciences, Tehran, Iran, for their useful discussions and critical reading of the manuscript. It should be noted that this paper was extracted from dissertation of the research project of Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by BJ RAO.
Corresponding editor: BJ Rao
Rights and permissions
About this article
Cite this article
Farnoosh, G., Khajeh, K., Mohammadi, M. et al. Catalytic and structural effects of flexible loop deletion in organophosphorus hydrolase enzyme: A thermostability improvement mechanism. J Biosci 45, 54 (2020). https://doi.org/10.1007/s12038-020-00026-5
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s12038-020-00026-5