• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.4
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• pp.337-343
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• 2006

Trimethylamine dehydrogenase (TMADH, EC 1.5.99.7), an iron-sulfur flavoprotein that catalyzes the oxidative demethylation of trimethylamine to form dimethylamine and formaldehyde, was purified from Methylophaga sp. strain SK1. The active TMADH was purified 12.3-fold through three purification steps. The optimal pH and temperature for enzyme activity was determined to be 8.5 and $55^{\circ}C$, respectively. The $V_{max}\;and\;K_m$ values were 7.9 nmol/min/mg protein and 1.5 mM. A genomic DNA of 2,983 bp from Methylophaga sp. strain SK1 was cloned, and DNA sequencing revealed the open reading frame (ORF) of the gene coding for TMADH. The ORF contained 728 amino acids with extensive identity (82%) to that of Methylophilus methylotrophus $W_3A_1$.

• Journal of Microbiology
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• v.43 no.6
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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.

• Korean Journal of Microbiology
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• v.41 no.1
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• pp.1-7
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• 2005

Methylophaga aminosulfidovorans SK1 (KCTC 10323 BP) can utilize trimethylamine as a sole carbon, nitrogen, and energy source. The bacterial flavin-containing monooxygenase (bFMO) gene was identified in the strain and the recombinant enzyme expressed in E. coli oxidized trimethylamine. To study the function and regulation of the bfmo, over 8,000 nucleotide sequences of the neighboring regions including the bfmo were determined. Three open reading frames proceeding to the bfmo gene encoded analogues to highly conserved nitrate/nitrite sensing two-component system regulators and a methyl accepting protein. Two small open reading frames just downstream of the bfmo gene showed no similar proteins of known functions but the sequences were conserved among other bacteria. Reverse transcription-polymerase chain reaction analysis showed that the six putative genes consisted of three transcription units. The three regulatory genes located upstream of the bfmo gene formed two separate transcription units. The bfmo and the two downstream genes were transcribed from a single promoter.

• Proceedings of the Microbiological Society of Korea Conference
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• 2005.05a
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• pp.223.4-223
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• 2005

• Journal of Microbiology and Biotechnology
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• v.12 no.3
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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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• v.30 no.8
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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.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.413-413
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• 2005

• Proceedings of the Microbiological Society of Korea Conference
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• 2008.05a
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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.

• Proceedings of the Zoological Society Korea Conference
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• 2001.10a
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• pp.244.3-245
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• 2001

No Abstract, See Full Text

• Korean Journal of Microbiology
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• v.46 no.3
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• pp.286-290
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• 2010

C-1 compounds are observed in anaerobic sediment of high salt environments. Thus, surface sediments and waters from these environments are therefore potential habitats for aerobic methylotrophic microorganisms. The soil samples collected from saltern and tidal flat as inoculums and methanol as carbon and energy source was supplied. After subculture depending on the salt concentration, methanol oxidizing bacteria growth condition investigated, the results of methanol oxidizing bacteria can grow in salt conditions, and the maximum concentration was 20%. Analysis based on denaturing gradient gel electrophoresis of 16S rRNA genes indicates that Methelyophaga-like bacteria were dominants of methylotrophs in the enrichment culture. Quantitative PCR showed that archaeal cells were about 1-10% of bacterial cells. Additionally archaea were assumed not to be involved in methanol oxidation since bacterial antibiotics completely blocked the methanol oxidation. Our results suggest that Methelyophaga-like bacteria could be involved in C-1 compounds oxidation in hypersaline environments although those activities are sensitive to salinity above 20%.