• 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.

• 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.