[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.

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