Presentation Information
[1ENZ-15]Enzymatic analysis of glycerol oxidation by lanthanide-dependent methanol dehydrogenase from Methylosinus trichosporium OB3b
○Hidehiro Ito1, Motoki Tateiri1, Wataru Shiina1, Toshiaki Kamachi1 (1. Institute of Science Tokyo (Japan))
Keywords:
Methanol dehydrogenase,lanthanide,glycerol,methanol,methanotroph
Methanotrophs, which can grow using methane as their sole carbon and energy source, are applied to the production of useful substances such as methanol and biodegradable plastics from methane. It has been reported that some methanotrophs experience growth inhibition due to glycerol, however, the mechanism remains unclear. Glycerol is an inexpensive carbon source; therefore, constructing methanotrophic bacteria capable of metabolizing both glycerol and methane through genetic engineering could lead to the economical and efficient production of valuable substances. To develop glycerol-metabolizing strains of methanotrophic bacteria, it is essential to elucidate in detail the mechanism of glycerol toxicity.
Methylosinus trichosporium OB3b, an aerobic methanotroph, has two types of methanol dehydrogenases (MDHs): XoxF1 and MxaFI. Previous research in our laboratory revealed that the OB3b ΔmxaF strain, in which the MxaF gene had been disrupted in the genome, exhibited strong growth inhibition by glycerol when expressing XoxF1. In this study, we endeavored to analyze the mechanism of glycerol toxicity mediated by lanthanoid-dependent MDH using both microbial cells and enzymes.
Fluorescence spectra of OB3b cells cultured with cerium ions showed that in glycerol-added cells, tryptophan fluorescence at 280 nm decreased while fluorescence at 330 nm increased. The color of OB3b cells cultured with cerium ions and glycerol changed to brown. These results suggest that the Maillard reaction, induced by reactive carbonyl species, proceeds in OB3b cells cultured in the presence of glycerol. Using OB3b cells treated with the MDH inhibitor cyclopropanol (CP), we examined the glycerol response in the fluorescence spectrum excited at 330 nm derived from Maillard reaction products. The results showed that CP-treated cells did not exhibit an increase in fluorescence intensity, as observed in untreated cells. These results revealed that glycerol toxicity in the OB3b strain is caused by the oxidation of glycerol by XoxF1.
We subcultured the OB3b ΔmxaF strain in the presence of cerium ions and glycerol, and obtained an evolved strain (OB3b ΔmxaF-EvoGlyc strain) that did not exhibit growth inhibition due to glycerol. A mutation occurred in xoxF1 in the OB3b ΔmxaF-EvoGlyc strain, resulting in the substitution of the 412th amino acid residue of XoxF1 from alanine to threonine. Enzyme activity measurement suggested that it reduced the affinity of the mutated XoxF1 for glycerol in the OB3b ΔmxaF-EvoGlyc strain.
Methylosinus trichosporium OB3b, an aerobic methanotroph, has two types of methanol dehydrogenases (MDHs): XoxF1 and MxaFI. Previous research in our laboratory revealed that the OB3b ΔmxaF strain, in which the MxaF gene had been disrupted in the genome, exhibited strong growth inhibition by glycerol when expressing XoxF1. In this study, we endeavored to analyze the mechanism of glycerol toxicity mediated by lanthanoid-dependent MDH using both microbial cells and enzymes.
Fluorescence spectra of OB3b cells cultured with cerium ions showed that in glycerol-added cells, tryptophan fluorescence at 280 nm decreased while fluorescence at 330 nm increased. The color of OB3b cells cultured with cerium ions and glycerol changed to brown. These results suggest that the Maillard reaction, induced by reactive carbonyl species, proceeds in OB3b cells cultured in the presence of glycerol. Using OB3b cells treated with the MDH inhibitor cyclopropanol (CP), we examined the glycerol response in the fluorescence spectrum excited at 330 nm derived from Maillard reaction products. The results showed that CP-treated cells did not exhibit an increase in fluorescence intensity, as observed in untreated cells. These results revealed that glycerol toxicity in the OB3b strain is caused by the oxidation of glycerol by XoxF1.
We subcultured the OB3b ΔmxaF strain in the presence of cerium ions and glycerol, and obtained an evolved strain (OB3b ΔmxaF-EvoGlyc strain) that did not exhibit growth inhibition due to glycerol. A mutation occurred in xoxF1 in the OB3b ΔmxaF-EvoGlyc strain, resulting in the substitution of the 412th amino acid residue of XoxF1 from alanine to threonine. Enzyme activity measurement suggested that it reduced the affinity of the mutated XoxF1 for glycerol in the OB3b ΔmxaF-EvoGlyc strain.
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