• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2008년도 International Meeting of the Microbiological Society of Korea
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• pp.163-163
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• 2008

Methylophaga aminisulfidivorans $MP^T$, a restricted facultative marine methylotrophic bacterium, was able to utilize methanol as a sole carbon and energy source, and possessed a methanol dehydrogenase (MDH) that is a key enzyme in the process of methanol oxidation. During purification of MDH, three types of MDH (MDH I, II, and III) were obtained in the cell free extracts from $MP^T$ cells grown on methanol. When analyzed by SDS-PAGE and ESI-FT ICR MS, MDH I was confirmed to consist of two subunits and with molecular masses of ~66 and ~10 kDa, respectively, in a form of ${\alpha}_2{\beta}_2$. While MDH II and MDH III contained an additional ~30 kDa protein, designated ${\gamma}$, in a form of ${\alpha}_2{\beta}_2{\gamma}$ and ${\alpha}_2{\beta}_2{\gamma}_2$, respectively. MDH III showed 1.5.2.0 times higher activity than MDH II, while MDH I remained the lowest activity. Based on these observations and experimental data, it seems that the original MDH conformation is ${\alpha}_2{\beta}_2{\gamma}2$ within $MP^T$ growing on methanol, and subunit ${\gamma}$ keeps MDH in an active form, and/or makes MDH easily bind to the substrate, methanol.

• Journal of Microbiology
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• 제34권2호
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• pp.172-178
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• 1996

Methylobacillus sp. strain SK1, which grows only on methanol, was found to grow in the absence of added copper. The doubling time (t$_{d}$ = 1.3 h) of the bacterium growing at the exponential growth phase at 30.deg.C in the absence of copper was the same as that of the cell growing in the presence of copper. The bacterium growing after the exponential phase in the absence of copper, however, grew faster than the cell growing in the presence of copper. Cells harvested after thee arly stationary phase in the presence of copper were found to exhibit no methanol dehydrogenase (MDH) activity, but the amount and subunit structure of the enzyme in the cells were almost the same as that in cells harboring active MDH. Pellets of the cells harvested after the early stationary phase in the presence of copper were pale green. Cell-free extracts prepared from cells harvested at the early stationary phase in the presence of copper were pink and exhibited MDH activity, but it turned dark-green rapidly from the surface under air. The green-colored portions of the extracts showed no MDH activity and contained c-type cytochromes that were oxidized completely. The inactive MDH activity and contained c-type cytochromes that were oxidized completely. The inactive MDH proteins in the green portions were found to have antigenic sites identical to those of the active one as the inactive MDHs in cells grown in the presence of copper. The bacterium was found to accumulate copper actively during the exponential growth phase. MDH prepared from cells grown in the presence or absence of copper was found to be more stable under nitrogen gas than under air. Methanol at 10 mM was found to enhance the stability of the MDH under air.r.

• 한국미생물·생명공학회지
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• 제21권6호
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• pp.577-581
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• 1993

The amino acid threonine was produced from glycine and ethanol in a reaction mixture using cell free extract of the methylotrophic bacterium isolated from soil and identified as mellthylo-bacterium sp. KJ29. Although the isolate could grow on carbon source other than methanol, only the cell free extract from the cells grown on methanol produced threonine. Methanol dehydrogenase (MDH) activity was present only in the cells grown on methanol when compared to the cells grown on heterotrophic substrates.

• Journal of Microbiology and Biotechnology
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• 제12권3호
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• pp.476-482
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• 2002

Methanol dehydrogenase (MDH) and c-type cytochromes from marine methanol-oxidizing bacterium, Methylophaga sp. MP, were purified and characterized. The native MDH had a molecular mass of 148 kDa and its isoelectric point was 5.5. Two c-type cytochromes, $c_L\;and\;c_H$, were found, and their isoelectric points were 3.4 and 8.0, respectively. The purified MDH had higher thermal stability than that of the other soil methylotrophic bacteria. The electron flow rate from MDH to cytochrome $c_L$was higher than that from MDH to cytochrome $c_H$, indicating that the physiological primary electron acceptor for MDH is cytochrome $c_L$. The electron transfer from MDH to phenazine ethosulfate (PES, artificial electron acceptor) in the two dye (PES/DCPIP)-linked assay system was not inhibited by NaCl, whereas the electron flow from MDH to cytochrome $c_L$ in the cytochrome/DCPIP-linked assay system was suppressed significantly by NaCl. Metal chelating agents such as EDTA showed the same effects on the MDH activity.

• Journal of Microbiology and Biotechnology
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• 제12권5호
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• pp.819-825
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• 2002

Methanol dehydrogenase (MDH) is a key enzyme in the process of methanol oxidation in methylotrophic bacteria. However, information on MDH genes from genus Methylobacillus is limited. In this study, a 6.5-kb HindIII DNA fragment of Methylobacillus sp. SK-5 chromosomal DNA was isolated from the genomic library of the strain by using a degenerate oligonucleotide probe that was designed based on JV-terminal amino acid sequence of the MDH $\alpha$ subunit purified from the strain. Molecular analysis of the fragment revealed four tightly clustered genes (mxaFJGI) involved in the methanol oxidation. The first and fourth genes were very similar to mxaF (77% identity for nucleotides an 78% identity for amino acids) and mxaF (67% Identity for nucleotides and 68% Identity for amino acids) genes, respectively, from Methylovorus sp. SSI. Genes mxaF and mxaI encode $\alpha$ and $\beta$ subunits of MDH, respectively. The two subunits were identified from purified MDH from Methylobacillus sp. SK-5. A dendrogram constructed by comparison of amino acid sequences of MDH u subunits suggests that MxaF from Methylobacillus sp. SK-5 belongs to a subfamily cluster of MDH u subunits from $\beta$-subgroup Proteobacteria. The subfamily cluster is separated from the other subfamily that consists of $\beta$- and $\gamma$-subgroup Proteobacteria. This study provided information on mn genes from a methylotrophic bacterium in $\beta$-subgroup Proteobacteria, which would aid to better develop a gene probe to detect one-carbon metabolizing bacteria.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권2호
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• pp.134-139
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• 2006

Methanotrophs are microorganisms that possess the unique ability to utilize methane as their sole source of carbon and energy. A novel culture system, known as the compulsory circulation diffusion system, was developed for rapid growth of methanotrophic bacteria. Methanol dehydrogenase (MDH, EC 1.1.99.8) from Methylomicrobium sp. HG-1, which belongs to the type I group of methanotrophic bacteria, can catalyze the oxidation of methanol directly into formaldehyde. This enzyme was purified 8-fold to electrophoretic homogeneity by means of a 4 step procedure and was found in the soluble fraction. The relative molecular weight of the native enzyme was estimated by gel filtration to be 120 kDa. The enzyme consisted of two identical dimers which, in turn, consisted of large and small subunits in an ${\alpha}_2{\beta}_2$ conformation. The isoelectric point was 5.4. The enzymatic activity of purified MDH was optimum at pH 9.0 and $60^{\circ}C$, and remained stable at that temperature for 20 min. MDH was able to oxidize primary alcohols from methanol to octanol and formaldehyde.

• Journal of Microbiology
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• 제43권6호
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• pp.499-502
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• 2005

Methylophaga sp. strain SK1 is a new restricted facultative methanol-oxidizing bacterium that was isolated from seawater. The aim of this study was to characterize the electron carriers involved in the methanol oxidation process in Methylophaga sp. strain SK1. The gene encoding cytochrome $c_L$ (mxaG) was cloned and the recombinant gene was expressed in Escherichia coli $DH5\alpha$ under strict anaerobic conditions. The recombinant cytochrome $c_L$ had the same molecular weight and absorption spectra as the wild-type cytochrome $c_L$ both in the reduced and oxidized forms. The electron flow rate from methanol dehydrogenase (MDH) to the recombinant cytochrome $c_L$ was similar to that from MDH to the wild-type cytochrome $c_L$. These results suggest that recombinant cytochrome $c_L$ acts as a physiological primary electron acceptor for MDH.

• 미생물학회지
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• 제30권6호
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• pp.533-538
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• 1992

Methylobacterium extorquens AM1 이 pH 5.5 와 7.0 에서 메탄올을 이용하여 성장할 때의 세대 시간은 각각 25 시간과 8.3 시간이었다. pH 7.0 에서 성장한 세균들은 모두 지수성장기 중기에서 가장 높은 methanol dehydrogenase (MDH_) 의 활성을 나타내었고, MDH 함유량은 성장시기에 따른 변화를 보이지 않았다. 세포내 cytochrome c 는 정체기에서 높게 나타났고 pH 7.0 에서 성장한 세균에 더 많이 존재하였다. Putrescine 또는 spermidine 이 첨가된 pH 5.5 의 배지에서 성장한 세균에 더 많이 존재하였다. Putrescine 또는 spermidine 이 첨가된 pH 5.5 의 배지에서 성장한 세균에서는 putrescine 의 함량은 증가하였으나 spermine 의 함량은 감소하였고 spermidine 의 함량에는 변화가 없었다. Spermine 을 첨가한 배지에서 성장한 세균에서는 폴리아민함량에 변화가 없었다. Putrescine 또는 spermidine이 첨가된 배지에서 성장한 세균에서 MDH 와 hydroxypyruvate reductase 의 활성이 증가되었고 MDH 와 cytochrome c 의 함량에는 변화가 없었다. 폴리아민은 invitro 상태에서 MDH 와 hydroxypyruvate reductase 의 활성을 증가시키지 않았다.

• Journal of Microbiology
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• 제35권3호
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• pp.200-205
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• 1997

Bothl $Ca^{2+}$ and Sr$^{2+}$-containing methanol dehydrogenases (MDH) were purified to homogeneity with yields of 48% and 42%, respectively, from Methylabacillus methanolovorus sp. strain SK5. Most of the biochemical and structural properties were similar to each other. However, some differences were found: (1) although the overall shape of the absorption spectrum of Sr$^{2+}$-MDH was very similar to that of $Ca^{2+}$-MDH, the absorption intensity originating from the cofactor in Sr$^{2+}$. MDH was higher than that in $Ca^{2+}$-MDH. Small blue shift of the maximum was also observed. These are probably due to a difference in redox state of the cofactors in $Ca^{2+}$ and Sr$^{2+}$-MDH; (2) Sr$^{2+}$-MDH was more heat-stable than $Ca^{2+}$-MDH above 56$^{\circ}C$; (3) the V$_{max}$ values for the methanol-dependent activities of Sr$^{2+}$- and $Ca^{2+}$-MDH in the presence of 3 mM KCN were 2.038 and 808 nmol/mg protein/min, respectively. In addition, the $K_{m}$ values of Sr$^{2+}$ and $Ca^{2+}$ MDH for methanol were 12 and 21 $\mu$M, respectively; (4) the endogenous activity of $Ca^{2+}$-MDH was more sensitive than that of Sr$^{2+}$-MDH in the presence of cyanide; (5) Diethyl pyrocarbonate treatment increased the enzyme activities of $Ca^{2+}$- and Sr$^{2+}$-MDH 4.2- and 1.4-folds, respectively. These results indicate that Sr$^{2+}$ stabilizes the structural conformation and enhances the activity of MDH more than $Ca^{2+}$.

• Journal of Microbiology and Biotechnology
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• 제30권8호
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• pp.1261-1271
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• 2020

Cytochrome c_L (Cytc_L) is an essential protein in the process of methanol oxidation in methylotrophs. It receives an electron from the pyrroloquinoline quinone (PQQ) cofactor of methanol dehydrogenase (MDH) to produce formaldehyde. The direct electron transfer mechanism between Cytc_L and MDH remains unknown due to the lack of structural information. To help gain a better understanding of the mechanism, we determined the first crystal structure of heme c containing Cytc_L from the aquatic methylotrophic bacterium Methylophaga aminisulfidivorans MP^T at 2.13 Å resolution. The crystal structure of Ma-Cytc_L revealed its unique features compared to those of the terrestrial homologues. Apart from Fe in heme, three additional metal ion binding sites for Na⁺, Ca⁺, and Fe²⁺ were found, wherein the ions mostly formed coordination bonds with the amino acid residues on the loop (G93-Y111) that interacts with heme. Therefore, these ions seemed to enhance the stability of heme insertion by increasing the loop's steadiness. The basic N-terminal end, together with helix α4 and loop (G126 to Y136), contributed positive charge to the region. In contrast, the acidic C-terminal end provided a negatively charged surface, yielding several electrostatic contact points with partner proteins for electron transfer. These exceptional features of Ma-Cytc_L, along with the structural information of MDH, led us to hypothesize the need for an adapter protein bridging MDH to Cytc_L within appropriate proximity for electron transfer. With this knowledge in mind, the methanol oxidation complex reconstitution in vitro could be utilized to produce metabolic intermediates at the industry level.