[논문]Biochemical and Cellular Investigation of Vitreoscilla Hemoglobin (VHb) Variants Possessing Efficient Peroxidase Activity

Isarankura-Na-Ayudhya, Chartchalerm; Tansila, Natta; Worachartcheewan, Apilak; Bulow, Leif; Prachayasittikul, Virapong

doi:10.4014/jmb.0908.08038

[국내논문] Biochemical and Cellular Investigation of Vitreoscilla Hemoglobin (VHb) Variants Possessing Efficient Peroxidase Activity 원문보기

Journal of microbiology and biotechnology, v.20 no.3, 2010년, pp.532 - 541

Isarankura-Na-Ayudhya, Chartchalerm (Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University) , Tansila, Natta (Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University) , Worachartcheewan, Apilak (Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University) , Bulow, Leif (Department of Pure and Applied Biochemistry, Center for Chemistry and Chemical Engineering, Lund University) , Prachayasittikul, Virapong (Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University)

Abstract ▼ AI-Helper

Peroxidase-like activity of Vitreoscilla hemoglobin (VHb) has been recently disclosed. To maximize such activity, two catalytically conserved residues (histidine and arginine) found in the distal pocket of peroxidases have successfully been introduced into that of the VHb. A 15-fold increase in catalytic constant ($k_{cat}$) was obtained in P54R variant,which was presumably attributable to the lower rigidity and higher hydrophilicity of the distal cavity arising from substitution of proline to arginine. None of the modifications altered the affinity towards either $H_2O_2$ or ABTS substrate. Spectroscopic studies revealed that VHb variants harboring the T29H mutation apparently demonstrated a spectral shift in both ferric and ferrous forms (406-408 to 411 nm, and 432 to 424-425 nm, respectively). All VHb proteins in the ferrous state had a $\lambda_{soret}$ peak at ~419 nm following the carbon monoxide (CO) binding. Expression of the P54R mutant mediated the downregulation of iron superoxide dismutase (FeSOD) as identified by two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting (PMF). According to the high peroxidase activity of P54R, it could effectively eliminate autoxidation-derived $H_2O_2$, which is a cause of heme degradation and iron release. This decreased the iron availability and consequently reduced the formation of the $Fe^{2+}$-ferric uptake regulator protein ($Fe^{2+}$-Fur), an inducer of FeSOD expression.

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가설 설정

Some protein spots were picked and identified (numbers of protein spot designated as identified protein shown in Table 2). C. Types of identified proteins were mapped on gels according to their corresponding spot numbers.

제안 방법

Briefly, protein sample was examined using a 2D electrophoresis set purchased from Atto Corporation, Japan(IEF disc gel and mPAGE; Models AES541 and AE-6531). All procedures were done in accordance with tbe mamifecturere recommendation with minor modifications. One hundred μg of cellular proteins was carefully loaded on the top of pre-casting IEF rod gels with pH range of 3-10 (Atto Coporation).
To further investigate whether the high efficiency to catalyze the peroxidase reaction of the VHb variants rendered any alterations or consequent effects on the cellular responses of host cells, total protein extract was analyzed by a proteomic approach. After 2-DE experiment, two areas of each gel displaying protein spots with either down- or upregulation were zoomed, and those of the wild type VHb are displayed in Fig.

대상 데이터

PCR-based site directed mutagenesis of the VHb gene was carried out using pV6h as a template [25]. Mutagenic primers were purchased from MWG Biotech (Ebersberg, Germany). 2, 2-Azino-bis (3-ethylbenzothiazoline- 6-sulfonic acid) (ABTS) and hydrogen peroxide [30%(v/v) H₂O₂, ε230nm=72.

성능/효과

Furthermore, this variant seems to be more applicable in large-scale fermentation, because a high rate of oxygen consumption and cellular metabolism provided by VHb probably makes cells produce more reactive oxygen species (ROS) that impede the productivity of desirable compounds. In conclusion, a highly catalytic variant ofVHb (P54R) possessing -15-fold increase in ecal has successfully been constructed based on mimicking of peroxidases active sites. Replacement of Pro54 with an arginine residue contributes to the relaxed nature and higher hydrophilicity of the active site, which thereby effectively facilitates enzymatic catalysis [34, 42].

후속연구

E, ). The authors would like to acknowledge Dr. Patcharee Panpumthong, Mr. Surasak Jiemsup, and Ms. Sirinuch Banyen for technical assistance in protein identification by mass spectrometry and peptide mass fingerprinting.

참고문헌 (50)

Adams, P. A. 1990. The peroxidasic activity of the haem octapeptide microperoxidase-8(MP-8): The kinetic mechanism of the catalytic reduction of $H_{2}O_{2}$ by MP-8 using 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS) as reducing substrate. J. Chem. Soc. Perkin Trans. 2: 1407-1414.
Allocatelli, C. T., F. Cutruzzola, A. Brancaccio, M. Brunori, J. Qin, and G. N. La Mar. 1993. Structural and functional characterization of sperm whale myoglobin mutants: Role of arginine (E10) in ligand stabilization. Biochemistry 32: 6041-6049.

상세보기
Andersson, C. I., C. Arfvidsson, P. T. Kallio, K. G. Wahlund, and L. Bulow. 2003. Enhanced ribosome and tRNA contents in Escherichia coli expressing a truncated Vitreoscilla hemoglobin mutant analyzed by flow field-flow fractionation. Biotechnol. Lett. 25: 1499-1504.

상세보기
Arseguel, D. and M. Baboulne. 2004. Removal of phenol from coupling of talc and peroxidase: Application for depollution of waste water containing phenolic compounds. J. Chem. Technol. Biotechnol. 61: 331-335.
Ascenzi, P., M. Brunori, M. Coletta, and A. Desideri. 1989. pH effects on the haem iron co-ordination state in the nitric oxide and deoxy derivatives of ferrous horseradish peroxidase and cytochrome c peroxidase. Biochem. J. 258: 473-478.

상세보기
Bauer, F. and H. Sticht. 2007. A proline to glycine mutation in the Lck SH3-domain affects conformational sampling and increases ligand binding affinity. FEBS Lett. 581: 1555-1560.

상세보기
Beale, S. I. 1990. Biosynthesis of the tetrapyrrole pigment precursor, delta-aminolevulinic acid, from glutamate. Plant Physiol. 93: 1273-1279.

상세보기
Berglund, G. I., G. H. Carlsson, A. T. Smith, H. Szoke, A. Henriksen, and J. Hajdu. 2002. The catalytic pathway of horseradish peroxidase at high resolution. Nature 417: 463-468.

상세보기
Bodalo, A., J. L. Gomez, E. Gomez, A. M. Hidalgo, M. Gomez, and A. M. Yelo. 2007. Elimination of 4-chlorophenol by soybean peroxidase and hydrogen peroxide: Kinetic model and intrinsic parameters. Biochem. Eng. J. 34: 242-247.

상세보기
Bolognesi, M., A. Boffi, M. Coletta, A. Mozzarelli, A. Pesce, C. Tarricone, and P. Ascenzi. 1999. Anticooperative ligand binding properties of recombinant ferric Vitreoscilla homodimeric hemoglobin: A thermodynamic, kinetic and X-ray crystallographic study. J. Mol. Biol. 291: 637-650.

상세보기
Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.

상세보기
Delano, W. L. 1998. The PyMOL Molecular Graphic System. Delano Scientific LLC, San Carlos, CA (http://www.pymol.org).
Dubrac, S. and D. Touati. 2002. Fur-mediated transcriptional and post-transcriptional regulation of FeSOD expression in Escherichia coli. Microbiology 148: 147-156.
Fan, C., J. Zhong, R. Guan, and G. Li. 2003. Direct electrochemical characterization of Vitreoscilla sp. hemoglobin entrapped in organic films. Biochim. Biophys. Acta 1649: 123-126.

상세보기
Farres, J. and P. T. Kallio. 2002. Improved cell growth in tobacco suspension cultures expressing Vitreoscilla hemoglobin. Biotechnol. Prog. 18: 229-233.

상세보기
Fee, J. A. 1991. Regulation of sod genes in Escherichia coli: Relevance to superoxide dismutase function. Mol. Microbiol. 5: 2599-2610.

상세보기
Finzel, B. C., T. L. Poulos, and J. Kraut. 1984. Crystal structure of yeast cytochrome c peroxidase refined at 1.7-A resolution. J. Biol. Chem. 259: 13027-13036.
Frey, A. D., B. T. Oberle, J. Farres, and P. T. Kallio. 2004. Expression of Vitreoscilla haemoglobin in tobacco cell cultures relieves nitrosative stress in vivo and protects from NO in vitro. Plant Biotechnol. J. 2: 221-231.

상세보기
Geckil, H., S. Gencer, H. Kahraman, and S. O. Erenler. 2003. Genetic engineering of Enterobacter aerogenes with the Vitreoscilla hemoglobin gene: Cell growth, survival, and antioxidant enzyme status under oxidative stress. Res. Microbiol. 154: 425-431.

상세보기
George, P. 1953. The chemical nature of the second hydrogen peroxide compound formed by cytochrome c peroxidase and horseradish peroxidase. I. Titration with reducing agents. Biochem. J. 54: 267-276.

상세보기
Isarankura-Na-Ayudhya, C., P. Panpumthong, T. Tangkosakul, S. Boonpangrak, and V. Prachayasittikul. 2008. Shedding light on the role of Vitreoscilla hemoglobin on cellular catabolic regulation by proteomic analysis. Int. J. Biol. Sci. 4: 71-80.

상세보기
Kaur, R., R. Pathania, V. Sharma, S. C. Mande, and K. L. Dikshit. 2002. Chimeric Vitreoscilla hemoglobin (VHb) carrying a flavoreductase domain relieves nitrosative stress in Escherichia coli: New insight into the functional role of VHb. Appl. Environ. Microbiol. 68: 152-160.

상세보기
Khosla, C. and J. E. Bailey. 1988. Heterologous expression of a bacterial haemoglobin improves the growth properties of recombinant Escherichia coli. Nature 331: 633-635.

상세보기
Khosla, C., J. E. Curtis, J. DeModena, U. Rinas, and J. E. Bailey. 1990. Expression of intracellular hemoglobin improves protein synthesis in oxygen-limited Escherichia coli. Biotechnology (NY) 8: 849-853.

상세보기
Kvist, M., E. S. Ryabova, E. Nordlander, and L. Bulow. 2007. An investigation of the peroxidase activity of Vitreoscilla hemoglobin. J. Biol. Inorg. Chem. 12: 324-334.

상세보기
Ling, K. Q. and L. M. Sayre. 2005. Horseradish peroxidasemediated aerobic and anaerobic oxidations of 3-alkylindoles. Bioorg. Med. Chem. 13: 3543-3551.

상세보기
Liu, C. Y. and D. A. Webster. 1974. Spectral characteristics and interconversions of the reduced oxidized and oxygenated forms of purified cytochrome o. J. Biol. Chem. 249: 4261-4266.
Matsui, T., S. Ozaki, E. Liong, G. N. Phillips Jr., and Y. Watanabe. 1999. Effects of the location of distal histidine in the reaction of myoglobin with hydrogen peroxide. J. Biol. Chem. 274: 2838-2844.

상세보기
Miessler, G. L. and D. A. Tarr. 2004. Inorganic Chemistry, 3rd Ed. Prentice Hall, Upper Saddle River, New Jersey.
Nagababu, E., F. J. Chrest, and J. M. Rifkind. 2003. Hydrogenperoxide-induced heme degradation in red blood cells: The protective roles of catalase and glutathione peroxidase. Biochim. Biophys. Acta 1620: 211-217.

상세보기
Nagababu, E. and J. M. Rifkind. 2000. Heme degradation during autoxidation of oxyhemoglobin. Biochem. Biophys. Res. Commun. 273: 839-845.

상세보기
Nagababu, E. and J. M. Rifkind. 2000. Reaction of hydrogen peroxide with ferrylhemoglobin: Superoxide production and heme degradation. Biochemistry 39: 12503-12511.

상세보기
Patapas, J., M. M. Al-Ansari, K. E. Taylor, J. K. Bewtra, and N. Biswas. 2007. Removal of dinitrotoluenes from water via reduction with iron and peroxidase-catalyzed oxidative polymerization: A comparison between Arthromyces ramosus peroxidase and soybean peroxidase. Chemosphere 67: 1485-1491.

상세보기
Redaelli, C., E. Monzani, L. Santagostini, L. Casella, A. M. Sanangelantoni, R. Pierattelli, and L. Banci. 2002. Characterization and peroxidase activity of a myoglobin mutant containing a distal arginine. Chembiochem 3: 226-233.

상세보기
Rodriguez-Lopez, J. N., A. T. Smith, and R. N. Thorneley. 1996. Role of arginine 38 in horseradish peroxidase. A critical residue for substrate binding and catalysis. J. Biol. Chem. 271: 4023-4030.

상세보기
Roos, V., C. I. Andersson, C. Arfvidsson, K. G. Wahlund, and L. Bulow. 2002. Expression of double Vitreoscilla hemoglobin enhances growth and alters ribosome and tRNA levels in Escherichia coli. Biotechnol. Prog. 18: 652-656.

상세보기
Schiodt, C. B., N. C. Veitch, and K. G. Welinder. 2007. Roles of distal arginine in activity and stability of Coprinus cinereus peroxidase elucidated by kinetic and NMR analysis of the Arg51Gln, -Asn, -Leu, and -Lys mutants. J. Inorg. Biochem. 101: 336-347.

상세보기
Suwanwong, Y., M. Kvist, C. Isarankura-Na-Ayudhya, N. Tansila, L. Bulow, and V. Prachayasittikul. 2006. Chimeric antibodybinding Vitreoscilla hemoglobin (VHb) mediates redox-catalysis reaction: New insight into the functional role of VHb. Int. J. Biol. Sci. 2: 208-215.

상세보기
Suzuki, T., Y. H. Watanabe, M. Nagasawa, A. Matsuoka, and K. Shikama. 2000. Dual nature of the distal histidine residue in the autoxidation reaction of myoglobin and hemoglobin comparison of the H64 mutants. Eur. J. Biochem. 267: 6166-6174.

상세보기
Tarricone, C., A. Galizzi, A. Coda, P. Ascenzi, and M. Bolognesi. 1997. Unusual structure of the oxygen-binding site in the dimeric bacterial hemoglobin from Vitreoscilla sp. Structure 5: 497-507.

상세보기
Tsuruga, M., A. Matsuoka, A. Hachimori, Y. Sugawara, and K. Shikama. 1998. The molecular mechanism of autoxidation for human oxyhemoglobin. Tilting of the distal histidine causes nonequivalent oxidation in the beta chain. J. Biol. Chem. 273: 8607-8615.

상세보기
Veitch, N. C. 2004. Horseradish peroxidase: A modern view of a classic enzyme. Phytochemistry 65: 249-259.

상세보기
Verma, S., S. Patel, R. Kaur, Y. T. Chung, B. T. Duk, K. L. Dikshit, B. C. Stark, and D. A. Webster. 2005. Mutational study of the bacterial hemoglobin distal heme pocket. Biochem. Biophys. Res. Commun. 326: 290-297.

상세보기
Wakabayashi, S., H. Matsubara, and D. A. Webster. 1986. Primary sequence of a dimeric bacterial haemoglobin from Vitreoscilla. Nature 322: 481-483.

상세보기
Wei, X. X. and G. Q. Chen. 2008. Applications of the VHb gene vgb for improved microbial fermentation processes. Methods Enzymol. 436: 273-287.

상세보기
Winterbourn, C. C. 1985. Free-radical production and oxidative reactions of hemoglobin. Environ. Health Perspect. 64: 321-330.

상세보기
Yang, G., R. Yuan, and Y. Q. Chai. 2008. A high-sensitive amperometric hydrogen peroxide biosensor based on the immobilization of hemoglobin on gold colloid/L-cysteine/gold colloid/nanoparticles Pt-chitosan composite film-modified platinum disk electrode. Colloids Surf. B Biointerfaces 61: 93-100.

상세보기
Zhang, J. and M. Oyama. 2004. A hydrogen peroxide sensor based on the peroxidase activity of hemoglobin immobilized on gold nanoparticles-modified ITO electrode. Electrochim. Acta 50: 85-90.

상세보기
Zhang, K., L. Mao, and R. Cai. 2000. Stopped-flow spectrophotometric determination of hydrogen peroxide with hemoglobin as catalyst. Talanta 51: 179-186.

상세보기
Zhang, L., Y. Li, Z. Wang, Y. Xia, W. Chen, and K. Tang. 2007. Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering. Biotechnol. Adv. 25: 123-136.

상세보기

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