The experiment was successfully managed a site-directed mutagenesis upon the pirin-like gene which was stored in the pUC18 plasmid. In the expressed protein, the mutation was the shift of Phe56 amino acid residue into Ala. In spite of this achievement, the purpose of getting an enlargement of the protein cavity was thought to be neglected. This assumption was come from the data that the MTF56A enzyme was unable to cleave the quercetin substrate.
89
CONCLUDING REMARKS
The study was able to provide a comparable pirin-like protein gene to the reported Pseudomonas stutzeri Zobell draft genome. The gene was achieved by genome walking and successfully cloned into pUC18 as the finest vector for the pirin-like protein expression. The expressed protein was extracted by sonication for breaking the host cells which is BL21 (DE3). Column chromatography procedure by Ni-NTA and gel filtration have delivered a considerable pure enzyme which has been N-terminal amino acid sequence confirmed to be the pirin-like protein. This enzyme was approved for having quercetinase competence as depside intermediate and carbon monoxide produced in the catalytic quercetin degradation. The enzyme quercetinase activity has an optimum temperature of 40°C and pH of 7.24.
Further analysis of this reliable enzyme has invented three contemporary results. The first was the pirin-like protein metal dependency which was taken by a separated addition of bimetal ion of Fe2+, Cu2+, Ni2+, Zn2+, Co2+ and Mn2+ in which copper was settled to be the most suitable cofactor for the enzyme deoxygenation.
Copper was feasibly bound to the ligand residues of His59, His61, His103 and Glu105 as elucidated by sequence alignment and homology modelling structure regarding to E. coli pirin-like protein. An adjusting type II Cu coordination and the proposed ligand residues were checked by UV-VIS spectrophotometer and EPR.
The second was the substrates specificity against myricetin, kaempferol, fisetin, galangin, taxifolin, morin, and luteolin, in which myricetin shows a higher specificity than quercetin. The last one was managing a site-directed mutagenesis over the pirin gene to expand the expressed enzyme active site cavity but unfortunately could not achieve the intention which was to increase the rate of the pirin-like protein in quercetinase activity.
Finally, this study of pirin-like protein could not gain a clear quercetinase mechanism, but it stipulated a valuable marine bacteria representative of enzymatic degradation of flavonoid.
90 BIBLIOGRAPHY
(1) L.A. Palomares, S. Estrada-Mondaca, O.T. RamírezBalbas, Production of Recombinant Proteins Challenges and Solutions in Recombinant Gene Expression: Reviews and Protocols, 2ndEd., by P. Balbas and A. Lorence, Methods in Mol. Bio. 267(2004) 15 – 43.
(2) J.M. Dunwell, A. Culham, C. E. Carter, C. R. Sosa-Aguirre, P. W.
Goodenough, Evolution of functional diversity in the cupin superfamily, TRENDS in Biochemical Sciences 26 (2001) 740 – 746.
(3) E.I. Solomon, D.E. Heppner, E.M. Johnston, J.W. Ginsbach, J. Cirera, M.
Qayyum, M.T. Kieber-Emmons, C.H. Kjaergaard, R.G. Hadt, L. Tian, Copper Active Site in Biology, Chem. Rev 114 (2014) 3659 – 3853.
(4) S. Tranchimand, P. Brouant, G. Iacazio, The Rutin Catabolic Pathway with Special Emphasis on Quercetinase, Biodegradation 21 (2010) 833 – 859.
(5) J.V. Formica, W. Regelson, Review of the Biology of Quercetin and Related Bioflavonoids, Food Chem. Toxicol 33 (1995) 1061 – 1080.
(6) S.B. Brown, V. Rajananda, J.A. Holroyd, E.G.V. Evans, A study of the mechanism of quercetin oxygenation by 18Q labelling: A comparison of the mechanism with that of haem degradation, Biochem. J. 205 (1982) 205, 239-244.
(7) F. Fusetti, K.H. Schrooter, R.A. Steiner, P.Iv. Noort, T. Pijning, H.J.
Rozeboom, K.H. Kalk, M.R. Egmond, B.W. Dijkstra, Crystal Structure of the Copper-Containing Quercetin 2,3-Dioxygenase from Aspergillus japonicus, Structure 10 (2002), 259 – 268.
(8) S. Tranchimand, G. Ertel, V. Gaydou, C. Gaudin, T. Tron, G. Iacazio, Biochemical and Molecular Characterization of a Quercetinase from Penicillium olsonii, Biochimie 90 (2008) 781 – 789.
(9) H-K. Hund, J. Breuer, F. Lingens, J. Huttermann, R. Kappl, S. Fetzner, Flavonol 2,4-dioxygenase from Aspergillus niger DSM 821, a Type 2 CuII -containing Glycoprotein, Eur. J. Biochem. 263 (1999) 871 – 878.
91
(10) I.M. Kooter, R.A. Steiner, B.W. Dijkstra, P.Iv. Noort, M.R. Egmond, M.
Huber, EPR Characterization of the Mononuclear Cu-containing Aspergillus japonicus Quercetin 2,3-dioxygenase Reveals Dramatic Changes Upon Anaerobic Binding of Substrates, Eur. J. Biochem. 269 (2002) 2971 – 2979.
(11) B. Gopal, L.L. Madan, S.F. Betz, A.A. Kossiakoff, The Crystal Structure of a Quercetin 2,3-Dioxygenase from Bacillus subtilis Suggests
Modulation of Enzyme Activity by a Change in the Metal Ion at the Active Site(s), Biochemistry 44 (2005) 193 – 201.
(12) M.R. Schaab, B.M. Barney, W.A. Francisco, Kinetic and Spectroscopic Studies on the Quercetin 2,3-Dioxygenase from Bacillus subtilis, Biochemistry 45 (2006) 1009 – 1016.
(13) H. Merkens, S. Sielker, K. Rose, S. Fetzner, A New Monocupin
Quercetinase of Streptomyces sp. FLA: Identification and Heterologous Expression of the queD Gene and Activity of the Recombinant Enzyme Towards Different Flavonols, Arch. Microbiol. 187 (2007) 475 – 487.
(14) H. Merkens, R. Kappl, R.P. Jakob, F.X. Schimd, S. Fetzner, Quercetinase QueD of Streptomyces sp. FLA, a Monocupin Dioxygenase with a
Preference for Nickel and Cobalt, Biochemistry 47 (2008) 12185 – 12196.
(15) J.M. Dunwell, Cupins: The Most Functionally Diverse Protein Superfamily?, Phytochemistry 65 (2004) 7 – 17.
(16) T.D.H. Bugg, Dioxygenase Enzymes: Catalytic Mechanisms and Chemical Models, Tetrahedron 59(36) (2003) 7075 – 7101.
(17) S. Fetzner. Ring-Cleaving Dioxygenases with a Cupin Fold. Appl.
Environ. Microbiol. 78 (2012) 2505 – 2514.
(18) H. Pang, M. Batlam, Q. Zeng, H. Miyatake, T. Hisano, K. Miki, L-L.
Wong, G.F. Gao, Z. Rao, Crystal Structure of Human Pirin. J. Biol. Chem.
279 (2004) 1491 – 1498.
(19) W.M.F. Wendler, E. Kremmer, R. Forster, E.L. Winnacker, Identification of Pirin, a Novel Highly Conserved Nuclear Protein, J. Biol. Chem. 272 (1999) 8482 – 8489.
92
(20) M. Adam, Z. Jia, Structural and Biochemical Analysis Reveal Pirins to Possess Quercetinase Activity, J. Biol. Chem. 280 (2005) 28675 – 28682.
(21) D. Orzaez, A.Jd. Jong, E.J. Woltering, A Tomato Homologue of the Human Protein Pirin is Induced during Programmed Cell Death, Plant Mol. Bio.
46 (2001) 459 – 468.
(22) Y.R. Lapik, L.S. Kaufman, The Arabidopsis Cupin Domain Protein AtPirin1 Interacts with the G Protein Alpha-Subunit GPA1 and Regulates Seed Germination and Early Seedling Development, Plant Cell, 15 (2003) 1578 – 1590.
(23) D.A. Orozco-Nunnelly, Ds. Muhammad, R. Mezzich, B-S. Lee, L.
Jayathilaka, L.S. Kaufman, K.M. Warpeha, Pirin1 (PRN1) is a
Multifunctional Protein that Regulates Quercetin, and Impacts Specific Light and UV Responses in the Seed-to-Seedling Transition of
Arabidopsis thaliana, Plosone 9(4) (2014) 1 – 12.
(24) N. Neznanov, A. Kondratova, K.M. Chumakov, L. Neznanova, R.
Kondratov, A.K. Banerjee, A.V. Gudkov, Quercetinase Pirin Makes
Poliovirus Replication Resistant to Flavonoid Quercetin, 27(4) (2008) 191 – 198.
(25) P.G. Pietta, Flavonoids as Antioxidant, J. Nat. Prod. 63 (2000) 1035 – 1042.
(26) A. Plaper, M. Golob, I. Hafner, M. Oblak, T. Solmajer, R. Jerala, Characterization of Quercetin Binding Site on DNA Gyrase, 306 (2) (2003) 530 – 536.
(27) A. Pena, A. Busquets, M. Gomila, R. Bosch, B. Nogales, E. Garcia-Valdes, J. Lalucat, A. Bennasar, Draft Genome of Pseudomonas stutzeri strain Zobell (CCUG 16156), a Marine Isolate and Model Organism for Denitrification Studies, J. Bacteriol. 194(5) (2012) 1277-1278.
(28) J. Lalucat, A. Bennasar, R. Bosch, E. Garcia-Valdes, N.J. Palleroni, Biology of Pseudomonas stutzeri, Microbiol. Mol. Biol. Rev. 70 (2006) 510 – 547.
93
(29) R. H. Garrett and C. M. Grisham, Biochemistry, 3rd Ed, Brooks/Cole Publishing Comp. (2005).
(30) N. Lan, R. Jansen, M. Gerstein, Toward a Systematic Definition of Protein Function That Scales to the Genome Level: Defining Function in Terms of Interactions, Proc. of the IEEE. 90 (12) (2002) 1 – 11.
(31) J. Lodge, P. Lund, S. Minchin, Gene Cloning. Principles and Applications, 1st Ed, Garland Science, Taylor and Francise Group (2006).
(32) A. L. Demain and P. Vaishnav, Production of Recombinant Proteins by Microbes and Higher Organisms, Biotech. Adv. 27 (2009) 297 – 306.
(33) S. Gräslund, P. Nordlund, J. Weigelt, B. M. Hallberg, J. Bray, O. Gileadi, S. Knapp, U. Oppermann, C. Arrowsmith, R. Hui, J.Ming, S.
dhe-Paganon, Hw. Park, A. Savchenko, A. Yee, A. Edwards, R. Vincentelli, C.
Cambillau, R. Kim, S. H. Kim, Z. Rao, Y. Shi, T. C. Terwilliger, C.Y. Kim, L.W. Hung, G. S. Waldo, Y. Peleg, S. Albeck, T. Unger, O. Dym, J.
Prilusky, J. L. Sussman, R. C. Stevens, S. A. Lesley, I. A. Wilson, A.
Joachimiak, F. Collart, I. Dementieva, M. I. Donnelly, W. H. Eschenfeldt, Y. Kim, L. Stols, R. Wu, M. Zhou, S. K. Burley, J. S. Emtage, J. M.
Sauder, D. Thompson, K. Bain, J. Luz, T. Gheyi, F. Zhang, S. Atwell, S. C.
Almo, J. B Bonanno, A. Fiser, S. Swaminathan, F. W. Studier, M. R.
Chance, A. Sali, T. B. Acton, R. Xiao, L. Zhao, L. C. Ma, J. F. Hunt, L.
Tong, K. Cunningham, M. Inouye, S. Anderson, H. Janjua, R. Shastry, C.
K. Ho, D. Wang, H. Wang, M. Jiang, G. T. Montelione, D. I. Stuart, R. J.
Owens, S. Daenke, A. Schütz, U. Heinemann, S. Yokoyama, K. Büssow, K. C. Gunsalus, Protein production and purification, Nat. Methods. 5(2) (2008) 135–146.
(34) T. D. H. Bugg, Introduction to Enzyme and Coenzyme Chemistry, 3rd Ed, John Wiley & Sons, Ltd. (2012).
(35) K. Hirooka, Y. Fujita, Excess Production of Bacillus subtilis Quercetin 2,3-Dioxygenase Affects Cell Viability in the Presence of Quercetin, Biosci. Biotechnol. Biochem. 74 (5) (2010) 1030 – 1038.
94
(36) L. Boowater, S. A. Fairhurst, V. J. Just, S. Bornemann, Bacillus subtilis YxaG is a Novel Fe-Containing Quercetin 2,3-dioxygenase, Febs letter 557 (2004) 45 – 48.
(37) B. M. Barney, M. R. Schaab, R. LoBrutto, W. A. Francisco, Evidence for a New Metal in a Known Active Site: Purification and Characterization of an Iron-Containing Quercetin 2,3-dioxygenase from Bacillus subtilis, Prot.
Exp. and Pure. 35 (2004) 131 – 141.
(38) T. A. Brown, Genomes, 3rd Ed, Garland Science, Taylor and Francise Group (2006).
(39) M. S. Johnson, N. Srinivasan, R. Sowdhamini, T. L. Blundell, Knowledge-based Protein Modeling, Crit. Rev. Biochem. Mol. Biol. 29 (1) (1994) 1 – 68.
(40) W. W.Ward, G. Swiatek, Protein Purification, Cur. Anal. Chem. 5 (2) (2009) 1 – 21.
(41) C. Zhou, Y. Zhang, P. Qin, X. Liu, L. Zhao, S. Yang, Y. Cai, X. Qian, A Method for Rapidly Confirming Protein N-terminal Sequences by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry, Rapid. Commun.
Mass Spectrom. 20 (19) (2006) 2878 – 2884.
(42) V. S. Chedea, S. I. Vicas, C. Socaciu, T. Nagaya, H. J. O. Ogola, K.
Yokota, K. Nishimura, M Jisaka, Lipoxygenase-Quercetin Interaction: A Kinetic Study Through Biochemical and Spectroscopy Approaches in Biochemistry, Genetics and Molecular Biology “Biochemical Testing”, (2012) 151 – 178.
(43) B. W. Matthews, Hydrophobic Interactions in Proteins in Encyclopedia of Life Science, John Wiley and Sons, Nature Publishing Group (2001).
(44) M. M. Ling, B. H. Robinson, Approaches to DNA Mutagenesis: An Overview. Anal. Biochem. 254 (1997) 157- 178.
(45) P. Carter, Review Article: Site-Directed Mutagenesis, Biochem. J. 237 (1986) 1 – 7.
95
(46) M. J. Betts, R. B. Russell, Amino Acid Properties and Consequences of Substitutions in Bioinformatics for Geneticists, edited by M. R. Barnes and I. C. Gray, John Wiley and Sons, (2003) 289 – 316.
(47) R. A. Copeland, Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis, John Wiley and Sons, 2nd Ed. (2001).
(48) J. B. Broderick, Coenzymes and Cofactors in Encyclopedia of Life Science, John Wiley and Sons, Nature Publishing Group (2001).
(49) D.C. Catling, C.R. Glein, K. J. Zahnle, C. P. McKay, Why O2 is Required by Complex Life of Habitable Planets and The Concept of Planetary
‘Oxygenation Time’, Astrobiol. 5 (2005) 415 – 438.
(50) S. Harayama, M. Kok, E.L. Neidle, Functional and Evolutionary Relationships among Diverse Oxygenases, Annu. Rev. Microbiol. 46 (1992) 565 – 601.
(51) J. M. Dunwell, S. Khuri, P. J. Gane, Microbial Relatives of The Seed Storage Proteins of Higher Plants: Conservation of Structure and
Diversification of Function during Evolution of The Cupin Superfamily.
Microbiol. Mol. Biol. Rev .64 (2000) 153–179.
(52) Y. Dai, T. C. Pochapsky, R. H. Abeles, Mechanistic Studies of Two Dioxygenases in the Methionine Salvage Pathway of Klebsiella pneumoniae, Biochem. 40 (2001) 6379–6387.
(53) S. Hassan, U. Mathesius, The Role of Flavonoids in Root-Rhizosphere Signaling: Opportunities and Challenges for Improving Plant-Microbe Interactions, Journal of Exp. Bot. (2012) 1 – 16.
(54) F. J. Simpson, G. Talbot, D. W. S. Westlake, Production of Carbon Monoxide in The Enzymatic Degradation of Rutin, Bioch. and Biophys.
Res. Comm. 2 (1) (1960) 15–18.
(55) H. G. Krishnamurty, F. J. Simpson, Degradation of Rutin by Aspergillus flavus, The J. of Biol. Chem. 245 (6) (1970) 1467 – 1471.
(56) R. A. Steiner, K. H. Kalk, B. W. Dijkstra, Anaerobic Enzyme-Substrate Structures Provide Insight Into The Reaction Mechanism Of The Copper
96
Dependent Quercetin 2,3-Dioxygenase, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 16625–16630.
(57) D. W. Lazinski, A. Camili, Homopolymer Tail-Mediated Ligation PCR: A Streamlined and Highly Efficient Method for DNA Cloning and Library Construction, Biotech. 54(1) (2013) 25–34.
(58) Anonym, DIG High Prime DNA Labelling and Detection Starter Kit I, www.roche-applied-science.com 12 (2012) 1 – 26.
(59) McCreery, T, Digoxigenin Labeling, Mol. Biotech. 7(2) (1997) 121 – 124.
(60) T. A. Brown, Southern Blotting and Related DNA Detection Techniques in Proteins in Encyclopedia of Life Science, John Wiley and Sons, Nature Publishing Group (2001).
(61) J. M. S. Bartlett, D. Stirling, PCR Protocols, Springer Science and Business Media (2003).
(62) C. Leoni, R. Gallerani, L. R. Ceci, A genome walking strategy for the identification of eukaryotic nucleotide sequences adjacent to known regions, BioTechniques 44 (2008) 229-235.
(63) Anonym, DNA Walking SpeedUpTM Premix Kit II User Manual, Seegene 3 (2006) 1– 20.
(64) Mc. Hung, P. C. Wensink, Nucleic Acids Research Different Restriction Enzyme-Generated Sticky DNA Ends can be Joined In Vitro, Nuc. Acids.
Res. 12 (4) (1984) 1863 – 1874.
(65) F. Hçvelmann, O. Seitz, Single Labeled DNA Fit Probes for Avoiding False-Positive Signaling in the Detection of DNA/RNA in qPCR or CellMedia, ChemBioChem 13 (2012) 2072 – 2081.
(66) G. L. Rosano, E. A. Ceccarelli, Recombinant Protein Expression in Escherichia coli: Advances and Challenges, Front. in Microbe. 5 (172) (2014) 1 – 17.
(67) M. N. Joshi, A. Sharma, A. S. Pandit, R. V. Pandya, A. K. Saxena, S. B.
Bagatharia, Draft Genome Sequence of Brevibacillus sp. Strain BAB-2500, a Strain that Might Play an Important Role in Agriculture, Genome Announc. 1(1) (2013) 1 – 2.
97
(68) Terpe, K, Overview of Bacterial Expression Systems for Heterologous Protein Production: from Molecular and Biochemical Fundamentals to Commercial Systems, Appl. Microbiol. Biotechnol 72 ( 2006) 211 – 222.
(69) F. A. Taiwo, Electron Paramagnetic Resonance Spectroscopic Studies of Iron and Copper Proteins, Spectro. 17 (2003) 53 – 63.
(70) W. E. Blumberg and J. Peisach, Paramagnetic Resonance Studies of Copper Proteins, Life. Chem. Rep. 5 (1987) 5 -36.
(71) W. E. Antholine, Low Frequency EPR of Cu2+ in Proteins in Biomedical EPR-Part A: Free Radicals, Metals, Medicine, and Physiology by S. S.
Eaton, G. R. Eaton, L. J. Berliner, BMR. 23 (2005) 417 – 450.
(72) F. Zsila, Z. Bikadi, M. Simonyi, Probing the Binding of the Flavonoid, Quercetin to Human Serum Albumin by Circular Dichroism, Electronic Absorption Spectroscopy and Molecular Modelling Methods, Biochem.
Pharma. 65 (2003) 447 – 456.
(73) W. H. Chan, Y. K. Cheng, M. M. F. Choi, A. W. M. Lee, M. S. Wong, W.
Y. Wong, Application of A Low-Cost Four-LED Based Photometer for Environmental Analysis, Chem. Edu. Jour. 7(2) (2003) 7 – 18.
(74) F. A. Taiwo, Electron Paramagnetic Resonance Spectroscopic Studies of Iron and Copper Proteins, Spectro. 17 (2003) 53 – 63.
(75) A. CR. Martin, C. A. Orengo, E. G. Hutchinson, S. Jones, M.
Karmirantzou, R. A. Laskowski, J. BO. Mitchell, C. Taroni, J. M.
Thornton, Protein Folds and Functions, Struc. 6 (1998) 875 – 884.
(76) R. Raman, C. P. Ptak, CL. Hsieh, R. E. Oswald, YF. Chang, Y. Sharma, The Perturbation of Tryptophan Fluorescence by Phenylalanine to Alanine Mutations Identifies the Hydrophobic Core in a Subset of Bacterial Ig-like Domains, Biochem. 52 (2013) 4589−4591.
(77) R. D. Sieg, J. Kubanek, Chemical Ecology of Marine Angiosperms:
Opportunities at the Interface of Marine and Terrestrial Systems, J. Chem.
Ecol. 39 (2013) 687 – 711.