Cloning and Expression of mxaJ and mxaG genes and Their interaction with Methanol dehydrogenase

Abstract

Methylophaga aminosulfidovorans SK1 is a marine methylotrophic bacterium that is capable of growing in the presence of methanol as a sole carbon and energy source. Oxidation of methanol to formaldehyde in M. aminosulfidovorans SK1 is catalyzed by the periplasmic quinoprotein methanol dehydrogenase (MDH) and cytochrome cL as its primary electron acceptor. Genomic DNA fragments, which relating the methanol oxidation (mxaFJGIR genes), were cloned and sequenced fully. I cloned a gene designated as mxaG that encoded cytochrome cL and expressed the recombinant gene in Escherichia coli under anaerobic conditions. During purification of methanol dehydrogenase (MDH) from M. aminosulfidovorans SK1, I obtained two different fractions (MDH I and MDH II) which had MDH activities. MDH I and MDH II fractions showed different biochemical features such as molecular weight and pI. Each α-subunit of MDH I and MDH II was identical except that MDH II fraction had 30kDa unknown protein. Sequence analysis of N-terminal amino acid of the protein represented that 30 kDa unknown peptide may be the product of mxaJ gene. In this study, we compared the characters of the recombinant and wild type cytochrome cL, purified 30 kDa unknown peptide, and studied interaction between mxaJ and methanol dehydrogenase in M. aminosulfidovorans SK1 via yeast two - hybrid system.