• Bulletin of the Korean Chemical Society
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• 제26권1호
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• pp.151-156
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• 2005

The conformational dynamics of heme pocket, a small vacant site near the binding site of heme proteins -myoglobin (Mb) and hemoglobin (Hb), was investigated after photolysis of carbon monoxide from MbCO and HbCO in D$_2$O solution at 283 K by probing time-resolved vibrational spectra of photolyzed CO. Two absorption bands, arising from CO in the heme pocket, evolve nonexponentially in time. The band at higher energy side blue shifts and broadens with time and the one at lower energy side narrows significantly with a negligible shift. These spectral evolutions are induced by protein conformational changes following photolysis that modify structure and electric field of heme pocket, and ligand dynamics in it. The conformational changes affecting the spectrum of photolyzed CO in heme pocket likely modulates ligand-binding activity.

• Bulletin of the Korean Chemical Society
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• 제28권2호
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• pp.339-342
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• 2007

• Bulletin of the Korean Chemical Society
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• 제27권11호
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• pp.1825-1831
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• 2006

The influence of solvent viscosity on conformational dynamics of the heme-pocket, a small vacant site near the binding site of myoglobin (Mb) and hemoglobin (Hb), and playing a functionally important role by serving as a station in ligand binding and escape, was studied by probing time-resolved vibrational spectra of CO photodissociated from MbCO and HbCO in $D_2O$, 75 wt% glycerol/$D_2O$, and trehalose at 283 K. Two absorption bands ($B_1$ and $B_2$) of the sample in viscous solvents, arising from CO in the heme pocket, are very similar to those in $D_2O$. Two bands in Mb and Hb under all three solvents exhibit very similar nonexponential spectral evolution ($B_1$ band; blue shifting and broadening, $B_2$ band; narrowing with a negligible shifting), suggesting that in the present experimental time window of 100 ps, the extents of the spectral shift and narrowing is much influenced neither by the viscosity of solvent nor by the quaternary contact of Hb. Spectral evolution can be described by a biexponential function with a fast universal time constant of 0.52 ps and a slow time constant ranging from 13 to 32 ps. For both proteins in all three solvents majority of spectral evolution occurs with the fast universal time constant. The magnitude of the slow rate in the spectral shift of B1 band decreases with increasing solvent viscosity, indicating that it is influenced by global conformational change which is retarded in viscous solvent, thereby serve as a reporter of global conformational change of heme proteins after deligation.

• 구강회복응용과학지
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• 제37권1호
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• pp.31-38
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• 2021

목적: 이 연구의 목적은 치주병원균에 대한 헴의 영향을 살펴보기 위함이다. 연구 재료 및 방법: 치주낭에 존재하는 7종의 혐기성세균을 이용하여 실험을 진행하였다. 세균을 혐기환경에서 배지를 이용하여 hemin의 있고 없음으로 하여 배양을 하였다. 세균의 성장은 매 6시간마다 분광광도계를 이용하여 측정하였다. 결과: 헤민의 존재여부에 따른 성장의 차이는 Porphyromonas gingivalis에서만 관찰되었다. Treponema denticola를 제외한 치주병원균의 성장은 헤민의 농도에 의존적인 것으로 관찰되었다. T. denticola의 성장은 헤민에 의해 방해를 받았다. 결론: 헴은 치은연하 치태의 미생물 생태계에서 미생물분포의 불균형을 유도하여 치주염을 유발하는 환경을 조장할 것이다.

• Journal of Microbiology and Biotechnology
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• 제8권3호
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• pp.295-299
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• 1998

Oligonucleotide primers were designed and successfully applied to amplify DNA fragments of P450 hydroxylase genes from actinomycetes which produce a large variety of medically important metabolites. Primers were designed based on several regions of strong similarities in amino acid sequence of P450 hydroxylases from a variety of actinomycetes, primarily in the regions of an oxygen binding site and a heme ligand pocket. These primers were used to amplify DNA fragments from seven different actinomycetes species producing a variety of different compounds. The deduced amino acid sequences of the isolated fragments revealed significant similarities to known P450 hydroxylase including the product of the suaC or subC genes from Streptomyces griseolus that is capable of metabolizing a number of sulfonylurea herbicides, and to the product of the $P450_{sca2}$ from S. carbophilus that produces a specific HMG-CoA reductase inhibitor. This method should help researchers in cloning the P450 hydroxylase genes involved in the biosynthesis of useful compounds.

• KSBB Journal
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• 제19권4호
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• pp.308-314
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• 2004

모넨신, 니저리신, 사이클로스포린 등을 하이드록실레이션 시키는 균주인 S. benihana에 존재하는 여러 가지 CYP를 클로닝하기 위해, 방선균 CYP의 보존된 부분을 통해서 degenerate primer를 제작하였고, colony hybridization을 통해서 스크리닝 한 결과 총 5 종류의 CYP가 검색되었다. 아미노산 서열의 분석 결과 방선균의 CYP 들과 매우 높은 유사성을 가졌으며, 이들 CYP의 앞 뒤 서열의 검색 결과 이 중 4개의 CYP의 downstream에는 FD 유전자가 존재함을 알 수 있었다. CYP503의 경우 다른 나머지 4개의 CYP의 서열과 차이가 많았으며, 2차 대사산물의 변형과 관련되어 있을 것으로 예상되며, ChoP와 유사성을 보이는 나머지 4개의 CYP는 스테로이드 계열 물질의 하이드록실레이션과 밀접한 연관이 있을 것으로 추정된다.

• Journal of Microbiology
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• 제40권3호
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• pp.211-218
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• 2002

A 4.8-kb DNA fragment encoding the P-450 type hydroxylase and ferredoxin genes was cloned from Pseudonocardia autotrophica IFO 12743 that can convert vitamin D$\_$3/ into its hydroxylated active forms. In order to isolate the P-450 gene cluster in this organism, we designed PCR primers on the basis of the regions of an oxygen binding site and a heme ligand pocket that are general characteristics of the P-450 hydroxylase. Sequencing analysis of the BamHI fragment revealed the presence of four complete and one incomplete ORFs, named PauA, PauB, PauC, and PauD, respectively. As a result of computer-based analyses, PauA and PauB have homology with enoyl-CoA hydratase from several organisms and the positive regulators belonging to the tetR family, respectively. PauC and PauD show similarity with SuaB/C proteins and ferredoxins, respectively, which are composed of P-450 monooxygenase systems for metabolizing two sulfonylurea herbicides in Streptomyces griseolus PauC shows the highest similarity with another CytP-450$\_$Sca2/ protein that is responsible for production of a specific HMG-CoA reductase inhibitor, pravastatin, in S. carbophilus. Cultures of Steptomyces lividans transformant, containing the P-450 gene cluster on the pWHM3 plasmid, was unable to convert vitamin D$\_$3/ to its hydroxylated forms.

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.532-541
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• 2010

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.

• Molecules and Cells
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• 제39권3호
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• pp.211-216
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• 2016

CYP107W1 from Streptomyces avermitilis is a cytochrome P450 enzyme involved in the biosynthesis of macrolide oligomycin A. A previous study reported that CYP107W1 regioselectively hydroxylated C12 of oligomycin C to produce oligomycin A, and the crystal structure of ligand free CYP107W1 was determined. Here, we analyzed the structural properties of the CYP107W1-oligomycin A complex and characterized the functional role of the Trp178 residue in CYP107W1. The crystal structure of the CYP107W1 complex with oligomycin A was determined at a resolution of $2.6{\AA}$. Oligomycin A is bound in the substrate access channel on the upper side of the prosthetic heme mainly by hydrophobic interactions. In particular, the Trp178 residue in the active site intercalates into the large macrolide ring, thereby guiding the substrate into the correct binding orientation for a productive P450 reaction. A Trp178 to Gly mutation resulted in the distortion of binding titration spectra with oligomycin A, whereas binding spectra with azoles were not affected. The Gly178 mutant's catalytic turnover number for the 12-hydroxylation reaction of oligomycin C was highly reduced. These results indicate that Trp178, located in the open pocket of the active site, may be a critical residue for the productive binding conformation of large macrolide substrates.

• 방사선산업학회지
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• 제2권3호
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• pp.97-104
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• 2008

Cinnamate-4-hydroxylase (C4H) is a key enzyme of phenylpropanoid pathway, which leads a variety of secondary metabolites to participate in differentiation and protection of plant against environmental stresses. In this study, we isolated a full-length cDNA of the C4H gene from a black raspberry (Rubus occidentalis L.), using a reverse transcriptase-PCR and rapid amplification of the cDNA ends (RACE)-PCR. The full-length cDNA of the RocC4H gene contained a 1,515 bp open reading frame (ORF) encoding a 504 amino acid protein with a calculated molecular weight of about 57.9 kDa and an isoelectric point (pI) value of 9.1. The genomic DNA analysis revealed that RocC4H gene had three exons and two introns. By multiple sequence alignment, RocC4H protein was highly homologous with other plant C4Hs, and the cytochrome P450-featured motifs, such as the heme-binding domain, the T-containing binding pocket motif (AAIETT), the ERR triad, and the tetrapeptide (PPGP) hinge motif, were highly conserved. Southern blot analysis revealed that RocC4H is a single copy gene in R. occidentalis.