α- and β-tubulin from Phytophthora capsici KACC 40483: molecular cloning, biochemical characterization, and antimicrotubule screening

  • Bon-Sung Koo
  • Haechul Park
  • Satish Kalme
  • Hye-Yeon Park
  • Jin Wook Han
  • Yun-Soo Yeo
  • Sang-Hong Yoon
  • Soo-Jin Kim
  • Chang-Muk Lee
  • Moon-Young Yoon
Applied Genetics and Molecular Biotechnology

Abstract

Internal fragments of α- and β-tubulin genes were generated using reverse transcription polymerase chain reaction (RT-PCR), and the termini were isolated using 5′- and 3′-rapid amplification of cDNA ends. Phytophthora capsici α- and β-tubulin specific primers were then used to generate full-length cDNA by RT-PCR. The recombinant α- and β-tubulin genes were expressed in Escherichia coli BL21 (DE3), purified under denaturing conditions, and average yields were 3.38–4.5 mg of α-tubulin and 2.89–4.0 mg of β-tubulin, each from 1-l culture. Optimum conditions were obtained for formation of microtubule-like structures. A value of 0.12 mg/ml was obtained as the critical concentration of polymerization of P. capsici tubulin. Benomyl inhibited polymerization with half-maximal inhibition (IC50) = 468 ± 20 μM. Approximately 18.66 ± 0.13 cysteine residues per tubulin dimer were accessible to 5,5′-dithiobis-(2-nitrobenzoic acid), a quantification reagent of sulfhydryl and 12.43 ± 0.12 residues were accessible in the presence of 200 μM benomyl. The order of preference for accessibility to cysteines was benomyl > colchicine > GTP > taxol, and cysteine accessibility changes conformed that binding sites of these ligands in tubulin were folding correctly. Fluorescence resonance energy transfer technique was used for high throughput screening of chemical library in search of antimitotic agent. There was significant difference in relative fluorescence by 210-O-2 and 210-O-14 as compared to colchicine.

Keywords

Phytophthora capsici Recombinant tubulin Benomyl DTNB FRET 

Notes

Acknowledgment

This study was carried out with the support of “On-Site Cooperative Agriculture Research Project (20070201080024)” and National Institute of Agricultural Biotechnology, RDA, Republic of Korea.

References

  1. Bai R, Choe K, Ewell JB, Nguyen NY, Hamel E (1998) Direct photoaffinity labeling of cysteine-295 of α-tubulin by guanosine 5′-triphosphate bound in the nonexchangeable site. J Biol Chem 273:9894–9897CrossRefGoogle Scholar
  2. Banerjee A, Roach MC, Trcka P, Luduena RF (1992) Preparation of a monoclonal antibody specific for the class IV isotype of β tubulin: purification and assembly of αβII, αβIII and αβIV tubulin dimers from bovine brain. J Biol Chem 267:5625–5630Google Scholar
  3. Basusarkar P, Chandra S, Bhattacharyya B (1997) The colchicine-binding and pyrene-excimer-formation activities of tubulin involve a common cysteine residue in the beta subunit. Eur J Biochem 244:378–383CrossRefGoogle Scholar
  4. Bellocq C, Andrey-Tornare I, Paunier-Doret AM, Maeder B, Paturle L, Job D, Haiech J, Edelstein SJ (1992) Purification of assembly-competent tubulin from Saccharomyces cerevisiae. Eur J Biochem 210:343–349CrossRefGoogle Scholar
  5. Britto PJ, Knipling L, McPhie P, Wolff J (2005) Thiol-disulphide interchange in tubulin: kinetics and the effect on polymerization. Biochem J 389:549–558CrossRefGoogle Scholar
  6. Burke D, Gasdaska P, Hartwell L (1989) Dominant effects of tubulin overexpression in Saccharomyces cerevisiae. Mol Cell Biol 9:1049–1059Google Scholar
  7. Chung S, Kong H, Buyer JS, Lakshman DK, Lydon J, Kim SD, Roberts DP (2008) Isolation and partial characterization of Bacillus subtilis ME488 for suppression of soilborne pathogens of cucumber and pepper. Appl Microbiol Biotechnol 80:115–123CrossRefGoogle Scholar
  8. Daly S, Yacoub A, Dundon W, Mastromei G, Islam K, Lorenzetti R (1997) Isolation and characterization of a gene encoding α-tubulin from Candida albicans. Gene 187:151–158CrossRefGoogle Scholar
  9. Damien H, Allen PM (2005) Turbidity as a probe of tubulin polymerization kinetics: a theoretical and experimental re-examination. Anal Biochem 345:198–213CrossRefGoogle Scholar
  10. Davidse LC, Flach W (1977) Differential binding of methyl benzimidazol-2-yl carbamate to fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of Aspergillus nidulans. J Cell Biol 72:174–193CrossRefGoogle Scholar
  11. Davis A, Sage CR, Wilson L, Farrell KW (1993) Purification and biochemical characterization of tubulin from the budding yeast Saccharomyces cerevisiae. Biochemistry 32:8823–8835CrossRefGoogle Scholar
  12. Eyer P, Worek F, Kiderlen D, Sinko G, Stuglin A, Simeon-Rudolf V, Reiner E (2003) Molar absorption coefficients for the reduced Ellman reagent: reassessment. Anal Biochem 312:224–227CrossRefGoogle Scholar
  13. Ferre F, Clote P (2006) DiANNA 1.1: an extension of the DiANNA web server for ternary cysteine classification. Nucleic Acids Res 34:W182–W185CrossRefGoogle Scholar
  14. French-Monar RD, Jones JB, Ozores-Hampton M, Roberts PD (2007) Survival of inoculum of Phytophthora capsici in soil through time under different soil treatments. Plant Disease 91:593–598CrossRefGoogle Scholar
  15. Hollomon DW, Butters JA, Barker H, Hall L (1998) Fungal β tubulin, expressed as a fusion protein, binds benzimidazole and phenylcarbamate fungicides. Antimicrob Agents Chemother 42:2171–2173Google Scholar
  16. Hoyt MA, Totis L, Roberts BT (1991) Saccharomyces cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell 66:507–517CrossRefGoogle Scholar
  17. Dong H, Li YZ, Hu W (2004) Analysis of purified tubulin in high concentration of glutamate for application in high throughput screening for microtubule-stabilizing agents. Assay Drug Develop Technol 2:621–628CrossRefGoogle Scholar
  18. Hwang BK, Kim CH (1995) Phytophthora blight of pepper and its control in Korea. Plant Dis 79:221–227Google Scholar
  19. Jang MH, Kim J, Kalme S, Han JW, Yoo HS, Kim J, Koo B, Kim SK, Yoon MY (2008) Cloning, purification, and polymerization of Capsicum annuum recombinant α and β tubulin. Biosci Biotech Biochem 72:1048–1055CrossRefGoogle Scholar
  20. Keinath AP (2007) Sensitivity of populations of Phytophthora capsici from South Carolina to mefenoxam, dimethomorph, zoxamide, and cymoxanil. Plant Dis 91:743–748CrossRefGoogle Scholar
  21. Kilmartin JV (1981) Purification of yeast tubulin by self-assembly in vitro. Biochemistry 20:3629–3633CrossRefGoogle Scholar
  22. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T7. Nature 277:680–685CrossRefGoogle Scholar
  23. Li J, Katiyar SK, Edlind TD (1996) Site-directed mutagenesis of Saccharomyces cerevisiae β-tubulin: Interaction between residue 167 and benzimidazole compounds. FEBS Lett 385:7–10CrossRefGoogle Scholar
  24. Liu CH, Chen X, Liu TT, Lian B, Gu Y, Caer V, Xue YR, Wang BT (2007) Study of the antifungal activity of Acinetobacter baumannii LCH001 in vitro and identification of its antifungal components. Appl Microbiol Biotechnol 76:459–466CrossRefGoogle Scholar
  25. Lowe J, Li H, Downing KH, Nogales E (2001) Refined structure of αβ-tubulin at 3.5 Å resolution. J Mol Biol 313:1045–1057CrossRefGoogle Scholar
  26. Luduena RF, Roach MC (1991) Tubulin sulfhydryl groups as probes and targets for antimitotic and antimicrotubule agents. Pharmacol Ther 49:133–152CrossRefGoogle Scholar
  27. MacDonald LM, Armson A, Thompson RC, Reynoldson JA (2001) Expression of Giardia duodenalis β-tubulin as a soluble protein in Escherichia coli. Prot Expr Purif 22:25–30CrossRefGoogle Scholar
  28. MacNeal RK, Purich DL (1978) Stoichiometry and role of GTP hydrolysis in bovine neurotubule assembly. J Biol Chem 253:4683–4687Google Scholar
  29. Marchler-Bauer A, Anderson JB, Derbyshire MK, DeWeese-Scott C, Gonzales NR, Gwadz M, Hao L, He S, Hurwitz DI, Jackson JD, Ke Z, Krylov D, Lanczycki CJ, Liebert CA, Liu C, Lu F, Lu S, Marchler GH, Mullokandov M, Song JS, Thanki N, Yamashita RA, Yin JJ, Zhang D, Bryant SH (2007) CDD: a conserved domain database for interactive domain family analysis. Nucleic Acids Res 35:D237–240CrossRefGoogle Scholar
  30. Mitchison TJ, Kirschner M (1984) Dynamic instability of microtubule growth. Nature 312:237–242CrossRefGoogle Scholar
  31. Neff NF, Thomas JH, Grisafi P, Botstein D (1983) Isolation of the β-tubulin gene from yeast and demonstration of its essential function in vivo. Cell 33:211–219CrossRefGoogle Scholar
  32. O’Brien ET, Salmon ED, Erickson HP (1997) How calcium causes microtubule depolymerization. Cell Motil Cytoskel 36:125–135CrossRefGoogle Scholar
  33. Orbach MJ, Porro EB, Yanofsky C (1986) Cloning and characterization of the gene for β-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker. Mol Cell Biol 6:2452–2461Google Scholar
  34. Oxberry ME, Geary TG, Winterrowd CA, Prichard RK (2001a) Individual expression of recombinant α- and β-tubulin from Haemonchus contortus: polymerization and drug effects. Prot Expr Purif 21:30–39CrossRefGoogle Scholar
  35. Oxberry ME, Geary TG, Prichard RK (2001b) Assessment of benzimidazole binding to individual recombinant tubulin isotypes from Haemonchus contortus. Parasitology 122:683–687CrossRefGoogle Scholar
  36. Pryor W (1962) Mechanisms of sulfur reactions. McGraw-Hill Book Co., New YorkGoogle Scholar
  37. Riddles PW, Blakeley RL, Zerner B (1979) Ellman’s reagent: 5,5′-dithiobis(2-nitrobenzoic acid)-a reexamination. Anal Biochem 94:75–81CrossRefGoogle Scholar
  38. Roychowdhury M, Sarkar N, Manna T, Bhattacharyya S, Sarkar T, Basusarkar P, Roy S, Bhattacharyya B (2000) Sulfhydryls of tubulin. A probe to detect conformational changes of tubulin. Eur J Biochem 267:3469–3476CrossRefGoogle Scholar
  39. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, , nd edn, 2nd edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  40. Shivanna BD, Mejillano MR, Williams TD, Himes RH (1993) Exchangeable GTP binding site of β-tubulin. Identification of cysteine 12 as the major site of cross-linking by direct photoaffinity labeling. J Biol Chem 268:127–132Google Scholar
  41. Sid AA, Ezziyyani M, Pérez-Sánchez C, Candela ME (2003) Effect of chitin on biological control activity of Bacillus sp. and Trichoderma harzianum against root rot disease in pepper (Capsicum annuum) plants. Eur J Plant Pathol 109:418–426Google Scholar
  42. Thomas JH, Neff N, Botstein D (1985) Isolation and characterization of mutations in the β-tubulin gene of Saccharomyces cerevisiae. Genetics 112:715–734Google Scholar
  43. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680CrossRefGoogle Scholar
  44. Vulevic B, Correia JJ (1997) Thermodynamic and structural analysis of microtubule assembly: the role of GTP hydrolysis. Biophys J 72:1357–1375CrossRefGoogle Scholar
  45. Weinstein B, Solomon F (1990) Phenotypic consequences of tubulin overproduction in Saccharomyces cerevisiae: differences between α-tubulin and β-tubulin. Mol Cell Biol 10:5295–5304Google Scholar
  46. Yan K, Dickman MB (1996) Isolation of a β-tubulin gene from Fusarium moniliforme that confers cold-sensitive benomyl resistance. Appl Environ Microbiol 62:3053–3056Google Scholar
  47. Yoon Y, Oakley BR (1995) Purification and characterization of assembly-competent tubulin from Aspergillus nidulans. Biochemistry 34:6373–6381CrossRefGoogle Scholar
  48. Young DH (1991) Effect of zarilamide on microtubules and nuclear division in Phytophthora capsici and tobacco suspension-cultured cells. Pestic Biochem Physiol 40:149–161CrossRefGoogle Scholar
  49. Young DH, Slawecki RA (2001) Mode of action of Zoxamide (RH-7281), a new oomycete fungicide pesticide. Biochem Physiol 69:100–111Google Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Bon-Sung Koo
    • 1
  • Haechul Park
    • 2
  • Satish Kalme
    • 2
    • 3
  • Hye-Yeon Park
    • 2
  • Jin Wook Han
    • 2
  • Yun-Soo Yeo
    • 1
  • Sang-Hong Yoon
    • 1
  • Soo-Jin Kim
    • 1
  • Chang-Muk Lee
    • 1
  • Moon-Young Yoon
    • 2
  1. 1.Microbial Genetics DivisionNational Institute of Agricultural Biotechnology, RDASuwonRepublic of Korea
  2. 2.Department of ChemistryHanyang UniversitySeoulRepublic of Korea
  3. 3.Research Institute for Natural SciencesHanyang UniversitySeoulRepublic of Korea

Personalised recommendations